WO2001083782A2 - Nouvelles proteases - Google Patents

Nouvelles proteases Download PDF

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WO2001083782A2
WO2001083782A2 PCT/US2001/014431 US0114431W WO0183782A2 WO 2001083782 A2 WO2001083782 A2 WO 2001083782A2 US 0114431 W US0114431 W US 0114431W WO 0183782 A2 WO0183782 A2 WO 0183782A2
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seq
ofthe
protease
polypeptide
nucleic acid
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PCT/US2001/014431
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WO2001083782A3 (fr
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Gregory D. Plowman
David Whyte
Sucha Sudarsanam
Gerard Manning
Sean Caenepeel
Vilia Ann Payne
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Sugen, Inc.
Pharmacia Corporation
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Priority to JP2001580389A priority Critical patent/JP2004504812A/ja
Priority to US10/275,107 priority patent/US20040063107A1/en
Priority to EP01933003A priority patent/EP1294901A2/fr
Priority to CA002408105A priority patent/CA2408105A1/fr
Priority to AU2001259473A priority patent/AU2001259473A1/en
Publication of WO2001083782A2 publication Critical patent/WO2001083782A2/fr
Publication of WO2001083782A3 publication Critical patent/WO2001083782A3/fr
Priority to HK03106801.3A priority patent/HK1054571A1/zh

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    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)

Definitions

  • the present invention relates to protease polypeptides, nucleotide sequences encoding the protease polypeptides, as well as various products and methods useful for the diagnosis and treatment of various protease-related diseases and conditions.
  • Protease proteinase
  • peptidase are synonymous terms applying to all enzymes that hydrolyse peptide bonds, i.e. proteolytic enzymes. Proteases are an exceptionally important group of enzymes in medical research and biotechnology.
  • Nucleic Acids Res., 1999, 27:325-331) (http://www.babraham.co.uk/Merops/ Merops.htm (Which is incorporated herein by reference in its entirety including any figures, tables, or drawings.)) have classified peptidases into 157 families based on structural similarity at the catalytic core sequence. These families are further classed into 26 clans, based on indications of common evolutionary relationship. Peptidases play key roles in both the normal physiology and disease-related pathways in mammalian cells.
  • Examples include the modulation of apoptosis (caspases), control of blood pressure (renin, angiotensin-converting enzymes), tissue remodeling and tumor invasion (collagenase), the development of Alzheimer's Disease ( ⁇ - secretase), protein turnover and cell-cycle regulation (proteosome), and inflammation (TNF- ⁇ convertase).
  • caspases the modulation of apoptosis
  • renin angiotensin-converting enzymes
  • tissue remodeling and tumor invasion collagenase
  • ⁇ - secretase the development of Alzheimer's Disease
  • proteosome protein turnover and cell-cycle regulation
  • Peptidases are classed as either exopeptidases or endopeptidases.
  • the exopeptidases act only near the ends of polypeptide chains: aminopeptidases act at the free N-terminus and carboxypeptidases at the free C-terminus.
  • the endopeptidases are divided, on the basis of their mechanism of action, into six sub- subclasses: aspartyl endopeptidases (3.4.23), cysteine endopeptidases (3.4.22), metalloendopeptidases (3.4.24), serine endopeptidases (3.4.21), thre ⁇ nine endopeptidases (3.4.25), and a final group that could not be assigned to any ofthe above classes (3.4.99).
  • the catalytic nucleophile is the reactive group of an amino acid side chain, either a hydroxyl group (serine- and threonine-type peptidases) or a sulfhydryl group (cysteine-type peptidases).
  • nucleophile is commonly an activated water molecule.
  • water molecule In aspartic-type peptidases, the water molecule is directly bound by the side chains of aspartate residues.
  • metal ions In metallopeptidases, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions.
  • the metal may be zinc, cobalt or manganese.
  • One metal ion is usually attached to three amino acid ligands. Families of peptidases are referred to by use of the numbering system of Rawlings & Barrett (Rawlings, N. D. & Barrett, A. J. MEROPS: the peptidase database. Nucleic Acids Research 27 (1999) 325-331, which is incorporated herein by reference in its entirety including any figures, tables, or drawings).
  • Aspartyl proteases also known as acid proteases, are a widely distributed family of proteolytic enzymes in vertebrates, fungi, plants, retroviruses and some plant viruses. Aspartate proteases of eukaryotes are monomeric enzymes which consist of two domains. Each domain contains an active site centered on a catalytic aspartyl residue. The two domains most probably evolved from the duplication of an ancestral gene encoding a primordial domain. Enzymes in this class include cathepsin E, renin, presenilin (PS1), and the APP secretases.
  • PS1 presenilin
  • Eukaryotic cysteine proteases are a family of proteolytic enzymes which contain an active site cysteine. Catalysis proceeds through a thioester intermediate and is facilitated by a nearby histidine side chain; an asparagine completes the essential catalytic triad. Peptidases in this family with important roles in disease include the caspases, calpain, hedgehog, ubiquitin hydrolases, and papain.
  • the metalloproteases are a class which includes matrix metalloproteases (MMPs), collagenase, stromelysin, gelatinase, neprylisin, carboxypeptidase, dipeptidase, and membrane-associated metalloproteases, such as those ofthe ADAM family. They require a metal co-factor for activity; frequently the required metal ion is zinc but some metalloproteases utilize cobalt and manganese.
  • MMPs matrix metalloproteases
  • Proteins ofthe extracellular matrix interact directly with cell surface receptors thereby initiating signal transduction pathways and modulating those triggered by growth factors, some of which may require binding to the extracellular matrix for optimal activity. Therefore the extracellular matrix has a profound effect on the cells encased by it and adjacent to it. Remodeling ofthe extracellular matrix requires protease of several families, including metalloproteases (MMPs).
  • MMPs metalloproteases
  • Serine proteases (Prosite PS00134 trypsin-his; PS00135 trvpsin-ser)
  • the catalytic activity ofthe serine proteases from the trypsin family is provided by a charge relay system involving an aspartic acid residue hydrogen- bonded to a histidine, which itself is hydrogen-bonded to a serine.
  • the sequences in the vicinity ofthe active site serine and histidine residues are well conserved in this family of proteases.
  • proteases known to belong to this large and important family include: blood coagulation factors VII, IX, X, XI and XII; thrombin; plasminogen; complement components Clr, Cls, C2; complement factors B, D and I; complement-activating component of RA-reactive factor; elastases 1, 2, 3 A, 3B (protease E); hepatocyte growth factor activator; glandular (tissue) kallikreins including EGF-binding protein types A, B, and C; NGF- ⁇ hain, ⁇ -renin, and prostate specific antigen (PSA); plasma kallikrein; mast cell proteases; myeloblastin (proteinase 3) (Wegener's autoantigen); plasminogen activators (urokinase-type, and tissue-type); and the trypsins I, II, III, and IV. These peptidases play key roles in coagulation, tumorigenesis, control of blood pressure, release of growth
  • Threonine peptidases (Tl) - (Prosite PDOC00326 PDOC00668)
  • Threonine proteases are characterized by their use of a hydroxyl group of a threonine residue in the catalytic site of these enyzmes. Only a few of these enzymes have been characterized thus far, such as the 20S proteasome from the archaebacterium Thermoplasma acidophilum (Seemuller et al, 1995, Science, 268:579-82, and chapter 167 of Barrett et al, Handbook of Proteolytic Enzymes. 1998, Academic Press, San Diego).
  • This invention concerns the isolation and characterization of novel sequences of human proteases. These sequences are obtained via bioinformatics searching strategies on the predicted amino acid translations of new human genetic sequences. These sequences, now identified as proteases, are translated into polypeptides which are further characterized. Additionaly, the nucleic acid sequences of these proteases are used to obtain full-length cDNA clones ofthe proteases. The partial or complete sequences of these proteases are presented here, together with their classification, predicted or deduced protein structure. Modulation of the activities of these proteases will prove useful therapeutically. Additionally, the presence or absence of these proteases or the DNA sequence encoding them will prove useful in diagnosis or prognosis of a variety of diseases. In this regard, Example 8 describes the chromosomal localization of proteases ofthe present invention, and describes diseases mapping to the chromosomal locations ofthe proteases ofthe invention.
  • a first aspect ofthe invention features an identified, isolated, enriched, or purified nucleic acid molecule encoding a protease polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ.ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:
  • isolated in reference to nucleic acid is meant a polymer of 10 (preferably 21, more preferably 39, most preferably 75) or more nucleotides conjugated to each other, including DNA and RNA that is isolated from a natural source or that is synthesized as the sense or complementary antisense strand.
  • nucleic acids are preferred, for example those of 300, 600, 900, 1200, 1500, or more nucleotides and/or those having at least 50%, 60%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 91%, 98%) or 99% identity to a sequence selected from the group consisting of those set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
  • nucleic acid it is meant, without limitation, DNA, RNA or cDNA, and where the nucleic acid is RNA, the thymine (T) will be uracil (U).
  • the isolated nucleic acid ofthe present invention is unique in the sense that it is not found in a pure or separated state in nature. Use ofthe term "isolated” indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment.
  • sequence is the only nucleotide chain present, but that it is essentially free (preferably about 90% pure, more preferably at least about 95% pure) of non- nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes.
  • enriched in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2- to 5 -fold) ofthe total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount ofthe specific DNA or RNA sequence, or by a combination ofthe two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount ofthe sequence of interest has been significantly increased.
  • the term "significant" is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2-fold, more preferably at least 5 -fold, more preferably at least 10-fold or even more.
  • the term also does not imply that there is no DNA or RNA from other sources.
  • the DNA from other sources may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occurring events, such as viral infection, or tumor-type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
  • nucleotide sequence be in purified form.
  • purified in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation). Instead, it represents an indication that the sequence is relatively more pure than in the natural environment (compared to the natural level this level should be at least 2- to 5-fold greater, e.g., in terms of mg/mL).
  • Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA.
  • the cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA).
  • the construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the 01 14431
  • protease polypeptide 32 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids in a polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, S
  • polypeptides of 100, 200, 300, 400, 450, 500, 550, 600, 700, 800, 900 or more amino acids are preferred.
  • the protease polypeptide can be encoded by a full-length nucleic acid sequence or any portion ofthe full-length nucleic acid sequence, so long as a functional activity ofthe polypeptide is retained. It is well known in the art that due to the degeneracy ofthe genetic code numerous different nucleic acid sequences can code for the same amino acid sequence. Equally, it is also well known in the art that conservative changes in amino acid can be made to arrive at a protein or polypeptide which retains the functionality ofthe original. Such substitutions may include the replacement of an amino acid by a residue having similar physicochemical properties, such as substituting one aliphatic residue
  • amino acid sequence of the protease peptide of the invention will be substantially similar to a sequence having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:
  • SEQ ID NO:38 SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,
  • protease polypeptide will have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%o identity to one ofthe aforementioned sequences.
  • identity is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and gaps and multiplying the product by 100. "Gaps" are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100%) identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, for example Gapped BLAST or PSI-BLAST (Altschul, et al. (1997) Nucleic Acids Res.
  • the invention features isolated, enriched, or purified nucleic acid molecules encoding a protease polypeptide comprising a nucleotide sequence that: (a) encodes a polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO: 36, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 36, SEQ ID NO: 36, SEQ ID NO: 36, SEQ ID NO: 36, SEQ ID NO: 36
  • the invention features isolated, enriched or purified nucleic acid molecules comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ED NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ED NO:15, SEQ ED NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ED NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ED NO:25 ⁇ SEQ ED NO:26, SEQ ED NO:27, SEQ ID NO:28, SEQ ED NO:29, SEQ ID NO:30,
  • nucleotide sequence is the complement of another nucleotide sequence if all of the nucleotides of the first sequence are complementary to all ofthe nucleotides ofthe second sequence.
  • conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides, more preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 50 contiguous nucleotides, most preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 100 contiguous nucleotides. In some instances, the conditions may prevent hybridization of nucleic acids having more than 5 mismatches in the full-length sequence.
  • stringent hybridization assay conditions hybridization assay conditions at least as stringent as the following: hybridization in 50% formamide, 5X SSC, 50 mM NaH 2 PO 4 , pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5X Denhardt's solution at 42 °C overnight; washing with 2X SSC, 0.1% SDS at 45 °C; and washing with 0.2X SSC, 0.1% SDS at 45 °C. Under some ofthe most stringent hybridization assay conditions, the second wash can be done with 0.1X SSC at a temperature up to 70 °C (Berger et al.
  • catalytic activity defines the rate at which a protease catalytic domain cleaves a substrate.
  • Catalytic activity can be measured, for example, by determining the amount of a substrate cleaved as a function of time.
  • Catalytic activity can be measured by methods ofthe invention by holding time constant and determining the concentration of a cleaved substrate after a fixed period of time. Cleavage of a substrate occurs at the active site ofthe protease.
  • active site is normally a cavity in which the substrate binds to the protease and is cleaved.
  • substrate refers to a polypeptide or protein which is cleaved by a protease ofthe invention.
  • cleaved refers to the severing of a covalent bond between amino acid residues of the backbone of the polypeptide or protein.
  • Inserts refers to a portion of a protease that is absent from a close homolog. Inserts may or may not -be the product alternative splicing of exons. Inserts can be identified by using a Smith- Waterman sequence alignment of the protein sequence against the non-redundant protein database, or by means of a multiple sequence alignment of homologous sequences using the DNAStar program Megalign (Preferably, the default settings of this program will be used, but those skilled in the art will recognize whether these settings need to be changed and know how to make the changes.). Inserts may play a functional role by presenting a new interface for protein-protein interactions, or by interfering with such interactions.
  • the invention features isolated, enriched, or purified nucleic acid molecules encoding protease polypeptides, further comprising a vector or promoter effective to initiate transcription in a host cell.
  • the invention also features recombinant nucleic acid, preferably in a cell or an organism.
  • the recombinant nucleic acid may contain a sequence selected from the group consisting of those set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ED NO:4, SEQ ID NO:5, SEQ ED NO:6, SEQ ED NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ED NO:10, SEQ ED NO:ll, SEQ ED NO:12, SEQ ID NO:13, SEQ ED NO:14, SEQ ED NO:15, SEQ ID NO:16, SEQ ED NO:17, SEQ ED NO:18, SEQ ID NO:19, SEQ ED NO:20, SEQ ED NO:21, SEQ ED NO:22, SEQ ED NO:23, SEQ ED NO:24, SEQ ID NO:25, SEQ ED NO:26, SEQ ED NO:27, SEQ ED NO:28, SEQ ED NO:29, SEQ ED NO:30, SEQ ED
  • the recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complementary to an RNA sequence encoding a protease polypeptide and a transcriptional termination region functional in a cell.
  • vector relates to a single or double-stranded circular nucleic acid molecule that can be transfected into cells and replicated within or independently of a cell genome.
  • a circular double-stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes.
  • restriction enzymes An assortment of nucleic acid vectors, restriction enzymes, and the knowledge ofthe nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art.
  • a nucleic acid molecule encoding a protease can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
  • transfecting defines a number of methods to insert a nucleic acid vector or other nucleic acid molecules into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, detergent, or DMSO to render the outer membrane or wall of the cells permeable to nucleic acid molecules of interest or use of various viral transduction strategies.
  • promoter refers to nucleic acid sequence needed for gene sequence expression. Promoter regions vary from organism to organism, but are well known to persons skilled in the art for different organisms. For example, in prokaryotes, the promoter region contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5 '-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence selected from the group consisting of those set forth in SEQ ED NO: 1, SEQ ED NO:2, SEQ ED NO:3, SEQ ED NO:4, SEQ ED NO:5, SEQ ED NO:6, SEQ ED NO:7, SEQ ED NO:8, SEQ ED NO:9, SEQ ID NO:10, SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, SEQ ID NO:14, SEQ ED NO:15, SEQ ED NO:16, SEQ ED NO:17, SEQ ED NO:18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ED NO:21, SEQ ED NO:22, SEQ ED NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ED NO:26, SEQ ED NO:27, SEQ ED NO:28, SEQ ID NO:
  • the nucleic acid may be isolated from a natural source by cDNA cloning or by subtractive hybridization.
  • the natural source may be mammalian, preferably human, blood, semen, or tissue, and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer. 01 14431
  • mice refers preferably to such organisms as mice, rats, rabbits, guinea pigs, sheep, and goats, more preferably to cats, dogs, monkeys, and apes, and most preferably to humans.
  • the nucleic acid is a conserved or unique region, for example those useful for: the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, obtaining antibodies to polypeptide regions, and designing antisense oligonucleotides.
  • nucleic acid regions regions present on two or more nucleic acids encoding a protease polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions.
  • lower stringency conditions suitable for screening for nucleic acid encoding protease polypeptides are provided in Wahl et al. Meth. Enzym. 152:399-407 (1987) and in Wahl et al. Meth. Enzym. 152:415-423 (1987), which are hereby incorporated by reference herein in its entirety, including any drawings, figures, or tables.
  • conserved regions differ by no more than 5 out of 20 nucleotides, even more preferably 2 out of 20 nucleotides or most preferably 1 out of 20 nucleotides.
  • nucleic acid region is meant a sequence present in a nucleic acid coding for a protease polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide. Such regions preferably encode 32
  • SEQ ED NO:54 SEQ ED NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ED NO:58,
  • the nucleic acid probe hybridizes to nucleic acid encoding at least 12, 32, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of a full-length sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ED NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ED NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ED NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ED NO:52, SEQ ED NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ED NO:61
  • Methods for using the probes include detecting the presence or amount of protease RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to protease RNA.
  • the nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a protease polypeptide may be used in the identification ofthe sequence ofthe nucleic acid detected (Nelson et al, in
  • Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe. Methods for using the probes also include using these probes to find the full- length clone of each ofthe predicted proteases by techniques known to one skilled in the art. These clones will be useful for screening for small molecule compounds that inhibit the catalytic activity ofthe encoded protease with potential utility in treating cancers, immune-related diseases and disorders, cardiovascular disease, brain or neuronal-associated diseases, and metabolic disorders.
  • disorders including cancers of tissues, blood, or hematopoietic origin, particularly those involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous system diseases and conditions including migraine, pain, sexual dysfunction, mood disorders, attention disorders, cognition disorders, hypotension, and hypertension; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome; neurodegenerative diseases including Alzheimer's, Parkinson's, multiple sclerosis, and amyotrophic lateral sclerosis; viral or non- viral infections caused by HIV-1, HIV-2 or other viral- or prion-agents or fungal- or bacterial- organisms; metabolic disorders including Diabetes and obesity and their related syndromes, among others; cardiovascular disorders including reperfusion restenosis, coronary thrombosis, clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular disease including glaucoma, retin
  • the invention describes a recombinant cell or tissue comprising a nucleic acid molecule encoding a protease polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ED
  • the nucleic acid may be under the control ofthe genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter.
  • exogenous it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the protease polypeptides.
  • the polypeptide is preferably a fragment ofthe protein encoded by a full- length amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ED NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ED NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ED NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ED NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ED NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO
  • fragment an amino acid sequence present in a protease polypeptide.
  • a sequence comprises at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of a full-length sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ED NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ED NO:41, SEQ ID NO:42, SEQ ED NO:43, SEQ ID NO:44, SEQ ED NO:45, SEQ ID NO:46, SEQ ED NO:47, SEQ ID NO:48, SEQ ED NO:49, SEQ ID NO:50, SEQ ED NO:51, SEQ ID NO:52, SEQ ED NO:53, SEQ ID NO:54, SEQ ED NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ED NO:59, SEQ ED NO:
  • the invention features an isolated, enriched, or purified protease polypeptide having the amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ED NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ED NO:44, SEQ ED NO:45, SEQ ED NO:46, SEQ ID NO:47, SEQ ED NO:48, SEQ ED NO:49, SEQ ED NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ TD NO:54, SEQ ED NO:55, SEQ ED NO:56, SEQ ID NO:57, SEQ ED NO:58, SEQ ED NO:59, SEQ ED NO:60, SEQ ED NO:61, SEQ ID NO:62, SEQ ED NO:63, SEQ ED NO
  • isolated in reference to a polypeptide is meant a polymer of 6 (preferably 12, more preferably 18, most preferably 25, 32, 40, or 50) or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized.
  • polypeptides are preferred, such as those with 100, 200, 300, 400, 450, 500, 550, 600, 700, 800, 900 or more contiguous amino acids of a full-length sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ TD NO:38, SEQ ID NO:39, SEQ TD NO:40, SEQ TD NO:41, SEQ ID NO:42, SEQ ED NO:43, SEQ TD NO:44, SEQ TD NO:45, SEQ TD NO:46, SEQ ID NO:47, SEQ TD NO:48, SEQ ED NO:49, SEQ ED NO:50, SEQ ED NO:51, SEQ ID NO:52, SEQ TD NO:53, SEQ TD NO:54, SEQ TD NO:55, SEQ TD NO:56, SEQ ID NO:57, SEQ TD NO:58, SEQ TD NO:59, SEQ TD NO:
  • isolated polypeptides ofthe present invention are unique in the sense that they are not found in a pure or separated state in nature.
  • Use ofthe term "isolated” indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (at least about 90% pure, more preferably at least about 95% pure or more) of non-amino acid-based material naturally associated with it.
  • enriched in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2- to 5- fold) ofthe total amino acid sequences present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acid sequences present, or by a preferential increase in the amount ofthe specific amino acid sequence of interest, or by a combination ofthe two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount ofthe sequence of interest has been significantly increased.
  • the term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acid sequences of about at least 2-fold, more preferably at least 5- to 10-fold or even more.
  • the term also does not imply that there is no amino acid sequence from other sources.
  • the other source of amino acid sequences may, for example, comprise amino acid sequence encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to increase the proportion ofthe desired amino acid sequence.
  • amino acid sequence be in purified form.
  • purified in reference to a polypeptide does' not require 4431
  • absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment. Compared to the natural level this level should be at least 2-to 5-fold greater (e.g., in terms of mg/mL). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
  • the protease polypeptide is a fragment ofthe protein encoded by a full-length amino acid sequence selected from the group consisting of those set forth in SEQ ED NO:36, SEQ ID NO:37, SEQ ED NO:38, SEQ ED NO:39, SEQ TD NO:40, SEQ TD NO:41, SEQ ID NO:42, SEQ ED NO:43, SEQ ID NO:44, SEQ ED NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ED NO:48, SEQ ID NO:49, SEQ ED NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ TD NO:53, SEQ ED NO:54, SEQ ED NO:55, SEQ ED NO:56, SEQ ID NO:57, SEQ ED NO:58, SEQ ED NO:59, SEQ ED NO:60, SEQ TD NO:61, SEQ ID NO:62, SEQ ID NO:63,
  • the protease polypeptide contains at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of a full-length sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ED NO:41, SEQ ED NO:42, SEQ ID NO:43, SEQ TD NO:44, SEQ ID NO:45, SEQ TD NO:46, SEQ TD NO:47, SEQ TD NO:48, SEQ ED NO:49, SEQ ID NO:50, SEQ ED NO:51, SEQ ED NO:52, SEQ ED NO:53, SEQ ED NO:54, SEQ ID NO:55, SEQ TD NO:56, SEQ TD NO:57, SEQ TD NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ED NO:61, SEQ TD NO:
  • the protease polypeptide comprises an amino acid sequence having an amino acid sequence selected from the group consisting of those set forth in SEQ ED NO:36, SEQ ED NO:37, SEQ ED NO:38, SEQ ED NO:39, SEQ ED NO:40, SEQ ED NO:41, SEQ ED NO:42, SEQ ED NO:43, SEQ ED NO:44, SEQ TD NO:45, SEQ ED NO:46, SEQ TD NO:47, SEQ TD NO:48, SEQ TD NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ TD NO:52, SEQ ED NO:53, SEQ TD NO:54, SEQ TD NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ TD NO:58, SEQ TD NO:59, SEQ TD NO:60, SEQ TD NO:61, SEQ TD NO:62, SEQ TD NO:63, SEQ ID NO:
  • the polypeptide can be isolated from a natural source by methods well- known in the art.
  • the natural source may be mammalian, preferably human, blood, semen, or tissue, and the polypeptide may be synthesized using an automated polypeptide synthesizer.
  • the invention includes a recombinant protease polypeptide having (a) an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ TD NO:39, SEQ TD NO:40, SEQ TD NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ TD NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ TD NO:50, SEQ TD NO:51, SEQ TD NO:52, SEQ ID NO:53, SEQ TD NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ TD NO:57, SEQ ID NO:58, SEQ TD NO:59, SEQ ED NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ TD NO:64,
  • recombinant protease polypeptide is meant a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
  • the polypeptides to be expressed in host cells may also be fusion proteins which include regions from heterologous proteins. Such regions may be included to allow, e.g., secretion, improved stability, or facilitated purification ofthe polypeptide.
  • a sequence encoding an appropriate signal peptide can be incorporated into expression vectors.
  • a DNA sequence for a signal peptide secretory leader
  • a signal peptide that is functional in the intended host cell promotes extracellular secretion of the polypeptide.
  • the signal sequence will be cleaved from the polypeptide upon secretion ofthe polypeptide from the cell.
  • preferred fusion proteins can be produced in which the N-terminus of a protease polypeptide is fused to a carrier peptide.
  • the polypeptide comprises a fusion protein which includes a heterologous region used to facilitate purification of the polypeptide.
  • a heterologous region used to facilitate purification of the polypeptide.
  • Many ofthe available peptides used for such a function allow selective binding of the fusion protein to a binding partner.
  • a preferred binding partner includes one or more ofthe IgG binding domains of protein A are easily purified to homogeneity by affinity chromatography on, for example, IgG-coupled Sepharose.
  • many vectors have the advantage of carrying a stretch of histidine residues that can be expressed at the N-terminal or C-terminal end ofthe target protein, and thus the protein of interest can be recovered by metal chelation chromatography.
  • a nucleotide sequence encoding a recognition site for a proteolytic enzyme such as enterokinase, factor X procollagenase or thrombine may immediately precede the sequence for a protease polypeptide to permit cleavage ofthe fusion protein to obtain the mature protease polypeptide.
  • fusion-protein binding partners include, but are not limited to, the yeast I-factor, the honeybee melatin leader in sf9 insect cells, 6-His tag, thioredoxin tag, hemaglutinin tag, GST tag, and OmpA signal sequence tag.
  • the binding partner which recognizes and binds to the peptide may be any ion, molecule or compound including metal ions (e.g., metal affinity columns), antibodies, or fragments thereof, and any protein or peptide which binds the peptide, such as the FLAG tag.
  • the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity to a protease polypeptide or a protease polypeptide domain or fragment where the polypeptide is selected from the group having a sequence at least about 90% identical to an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO: 36, SEQ TD NO:37, SEQ TD NO:38, SEQ TD NO:39, SEQ TD NO:40, SEQ TD NO:41, SEQ ID NO:42, SEQ TD NO:43, SEQ TD NO:44, SEQ ED NO:45, SEQ ED NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ TD NO:49, SEQ TD NO:50, SEQ ED NO:51, SEQ ID NO:52, SEQ ED NO:53, SEQ ED NO:54, SEQ ID NO:55, SEQ ED NO:56, SEQ ID NO:
  • Antibodies or antibody fragments are polypeptides that contain regions that can bind other polypeptides.
  • the term "specific binding affinity” describes an antibody that binds to a protease polypeptide with greater affinity than it binds to other polypeptides under specified conditions.
  • Antibodies can be used to identify an endogenous source of protease polypeptides, to monitor cell cycle regulation, and for immuno-localization of protease polypeptides within the cell.
  • polyclonal refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof.
  • various host animals may be immunized by injection with the antigen.
  • Various adjuvants may be used to increase the immunological response, depending on the host species.
  • Monoclonal antibodies are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art (Kohler et al, Nature, 1975, 256:495-497, and U.S. Patent No. 4,376,110, both of which are hereby incorporated by reference herein in their entirety including any figures, tables, or drawings).
  • An antibody ofthe present invention includes "humanized” monoclonal and polyclonal antibodies. Humanized antibodies are recombinant proteins in which non-human (typically murine) complementarity determining regions of an antibody have been transferred from heavy and light variable chains ofthe non-human (e.g.
  • Humanized antibodies in accordance with this invention are suitable for use in therapeutic methods.
  • General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Orlandi et al, Proc. Nat'lAcad. Sci. USA 86: 3833 (1989).
  • Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al, Nature 321:522 (1986), Riechmann et al, Nature 332:323 (1988), Verhoeyen et al, Science 239:1534 (1988), Carter et al, Proc. Nat'lAcad.
  • antibody fragment refers to a portion of an antibody, often the hypervariable region and portions ofthe surrounding heavy and light chains, that displays specific binding affinity for a particular molecule.
  • a hypervariable region is a portion of an antibody that physically binds to the polypeptide target.
  • An antibody fragment ofthe present invention includes a "single-chain antibody," a phrase used in this description to denote a linear polypeptide that binds antigen with specificity and that comprises variable or hypervariable regions from the heavy and light chain chains of an antibody.
  • single chain antibodies can be produced by conventional methodology.
  • the Vh and VI regions ofthe Fv fragment can be covalently joined and stabilized by the insertion of a disulfide bond. See Glockshuber, et al, Biochemistry 1362 (1990).
  • the Vh and VI regions can be joined by the insertion of a peptide linker.
  • a gene encoding the Vh, VI and peptide linker sequences can be constructed and expressed using a recombinant expression vector.
  • Amino acid sequences comprising hypervariable regions from the Vh and VI antibody chains can also be constructed using disulfide bonds or peptide linkers.
  • Antibodies or antibody fragments having specific binding affinity to a protease polypeptide ofthe invention may be used in methods for detecting the presence and/or amount of protease polypeptide in a sample by probing the sample with the antibody under conditions suitable for protease-antibody immunocomplex formation and detecting the presence and/or amount ofthe antibody conjugated to the protease polypeptide.
  • Diagnostic kits for performing such methods may be constructed to include antibodies or antibody fragments specific for the protease as well as a conjugate of a binding partner ofthe antibodies or the antibodies themselves.
  • An antibody or antibody fragment with specific binding affinity to a protease polypeptide ofthe invention can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art enable production of antibodies or antibody fragments, in both prokaryotic and eukaryotic organisms. Purification, enrichment, and isolation of antibodies, which are polypeptide molecules, are described above.
  • Antibodies having specific binding affinity to a protease polypeptide ofthe invention may be used in methods for detecting the presence and/or amount of protease polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount ofthe antibody conjugated to the protease polypeptide.
  • Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner ofthe antibody and a label, such as, for example, a radioisotope. The diagnostic kit may also include notification of an FDA approved use and instructions therefor.
  • the invention features a hybridoma wliich produces an antibody having specific binding affinity to a protease polypeptide or a protease polypeptide domain, where the polypeptide is selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ED NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ED NO:41, SEQ ED NO:42, SEQ ID NO:43, SEQ ED NO:44, SEQ ID NO:45, SEQ TD NO:46, SEQ TD NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ TD NO:50, SEQ ID NO:51, SEQ ED NO:52, SEQ ID NO:53, SEQ TD NO:54, SEQ TD NO:55, SEQ ED NO:56, SEQ ED NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ TD NO:60, SEQ TD NO:
  • hybrida an immortalized cell line that is capable of secreting an antibody, for example an antibody to a protease ofthe invention.
  • the antibody to the protease comprises a sequence of amino acids that is able to specifically bind a protease polypeptide of the invention.
  • kits comprising antibodies that bind to a polypeptide encoded by any ofthe nucleic acid molecules described above, and a negative control antibody.
  • negative control antibody refers to an antibody derived from similar source as the antibody having specific binding affinity, but where it displays no binding affinity to a polypeptide ofthe invention.
  • the invention features a protease polypeptide binding agent able to bind to a protease polypeptide selected from the group having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ED NO:37, SEQ ED NO:38, SEQ ED NO:39, SEQ ID NO:40, SEQ ED NO:41, SEQ ED NO:42, SEQ ED NO:43, SEQ ED NO:44, SEQ ID NO:45, SEQ TD NO:46, SEQ ED NO:47, SEQ TD NO:48, SEQ ED NO:49, SEQ ID NO:50, SEQ TD NO:51, SEQ ED NO:52, SEQ ED NO:53, SEQ TD NO:54, SEQ ID NO:55, SEQ TD NO:56, SEQ ED NO:57, SEQ ED NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ED NO:61, SEQ ID NO
  • the binding agent is preferably a purified antibody that recognizes an epitope present on a protease polypeptide ofthe invention.
  • Other binding agents include molecules that bind to protease polypeptides and analogous molecules that bind to a protease polypeptide. Such binding agents may be identified by using assays that measure protease binding partner activity, or they may be identified using assays that measure protease activity, such as the release of a fluorogenic or radioactive marker attached to a substrate molecule.
  • the invention also features a method for screening for human cells containing a protease polypeptide ofthe invention or an equivalent sequence.
  • the method involves identifying the novel polypeptide in human cells using techniques that are routine and standard in the art, such as those described herein for identifying the proteases ofthe invention (e.g., cloning, Southern or Northern blot analysis, in situ hybridization, PCR amplification, etc.).
  • the invention features methods for identifying a substance that modulates protease activity comprising the steps of: (a) contacting a protease polypeptide comprising an amino acid substantially identical to a sequence selected from the group consisting of those set forth in SEQ TD NO:36, SEQ ED NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ED NO:40, SEQ TD NO:41, SEQ ED NO:42, SEQ ID NO:43, SEQ TD NO:44, SEQ TD NO:45, SEQ TD NO:46, SEQ ED NO:47, SEQ ED NO:48, SEQ ED NO:49, SEQ ID NO:50, SEQ TD N0.51, SEQ ID NO:52, SEQ ED NO:53, SEQ ED NO:54, SEQ ED NO:55, SEQ TD NO:56, SEQ TD NO:57, SEQ
  • TD NO:63 SEQ TD NO:64, SEQ ED NO:65, SEQ ED NO:66, SEQ TD NO:67, SEQ
  • TD NO:68, SEQ ED NO:69, and SEQ ED NO:70 with a test substance; (b) measuring the activity of said polypeptide; and (c) determining whether said substance modulates the activity of said polypeptide. More preferably the sequence is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the listed sequences.
  • modulates refers to the ability of a compound to alter the function of a protease ofthe invention.
  • a modulator preferably activates or inhibits the activity of a protease ofthe invention depending on the concentration ofthe compound exposed to the protease.
  • modulates also refers to altering the function of proteases ofthe invention by increasing or decreasing the probability that a complex forms between the protease and a natural binding partner.
  • a modulator preferably increases the probability that such a complex forms between the protease and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the protease and the natural binding partner depending on the concentration ofthe compound exposed to the protease, and most preferably decreases the probability that a complex forms between the protease and the natural binding partner.
  • the term “activates” refers to increasing the cellular activity ofthe protease.
  • the term “inhibits” refers to decreasing the cellular activity ofthe protease.
  • complex refers to an assembly of at least two molecules bound to one another.
  • Signal transduction complexes often contain at least two protein molecules bound to one another.
  • a protein tyrosine receptor protein kinase GRB2, SOS, RAF, and RAS assemble to form a signal transduction complex in response to a mitogenic ligand.
  • proteases involved in blood coagulation and their cofactors are known to form macromolecular complexes on cellular membranes.
  • proteases involved in modification ofthe extracellular matrix are known to form complexes with their inhibitors and also with components ofthe extracellular matrix.
  • natural binding partner refers to polypeptides, lipids, small molecules, or nucleic acids that bind to proteases in cells.
  • a change in the interaction between a protease and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of protease/natural binding partner complex.
  • the term "contacting" as used herein refers to mixing a solution comprising the test compound with a liquid medium bathing the cells ofthe methods.
  • the solution comprising the compound may also comprise another component, such as dimethyl sulfoxide (DMSO), which facilitates the uptake ofthe test compound or compounds into the cells ofthe methods.
  • DMSO dimethyl sulfoxide
  • the solution comprising the test compound may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipette-based device or syringe-based device.
  • the invention features methods for identifying a substance that modulates protease activity in a cell comprising the steps of: (a) expressing a protease polypeptide in a cell, wherein said polypeptide is selected from the group having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ TD NO:39, SEQ TD NO:40, SEQ ID NO:41, SEQ ED NO:42, SEQ TD NO:43, SEQ TD NO:44, SEQ ID NO:45, SEQ TD NO:46, SEQ TD NO:47, SEQ TD NO:48, SEQ TD NO:49, SEQ ID NO:50, SEQ TD NO:51, SEQ TD NO:52, SEQ TD NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ TD NO:56, SEQ TD NO:57, SEQ TD NO:58, SEQ ID NO
  • expressing refers to the production of proteases of the invention from a nucleic acid vector containing protease genes within a cell.
  • the nucleic acid vector is transfected into cells using well known techniques in the art as described herein.
  • Another aspect ofthe instant invention is directed to methods of identifying compounds that bind to protease polypeptides ofthe present invention, comprising contacting the protease polypeptides with a compound, and determining whether the compound binds the protease polypeptides.
  • Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel- shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, soiled analysis, ELIS A, and the like, which are described in, for example, Current Protocols in Molecular Biology, 1999, John Wiley & Sons, NY, which is incorporated herein by reference in its entirety.
  • the compounds to be screened include, but are not limited to, compounds of extracellular, intracellular, biological or chemical origin.
  • the methods ofthe invention also embrace compounds that are attached to a label, such as a radiolabel (e.g., 125 1, 35 S, 32 P, 33 P, 3 H), a fluorescence label, a chemiluminescent label, an enzymic label and an immunogenic label.
  • a label such as a radiolabel (e.g., 125 1, 35 S, 32 P, 33 P, 3 H), a fluorescence label, a chemiluminescent label, an enzymic label and an immunogenic label.
  • a label such as a radiolabel (e.g., 125 1, 35 S, 32 P, 33 P, 3 H)
  • fluorescence label e.g., 125 1, 35 S, 32 P, 33 P, 3 H
  • fluorescence label e.g., 125 1, 35 S, 32 P, 33 P, 3 H
  • chemiluminescent label e.g., chemiluminescent label
  • an enzymic label e.g
  • one skilled in the art can examine the diminution in complex formation between a protease polypeptide and its substrate caused by the compound being tested.
  • Other assays can be used to examine enzymatic activity including, but not limited to, photometric, radiometric, HPLC, electrochemical, and the like, which are described in, for example, Enzyme Assays: A Practical Approach, eds. R. Eisenthal and M. J. Danson, 1992, Oxford University Press, which is incorporated herein by reference in its entirety.
  • Another aspect ofthe present invention is directed to methods of identifying compounds which modulate (i.e., increase or decrease) activity of a protease polypeptide comprising contacting the protease polypeptide with a compound, and determining whether the compound modifies activity ofthe protease polypeptide.
  • These compounds are also referred to as "modulators of proteases.”
  • the activity in the presence ofthe test compound is measured to the activity in the absence ofthe test compound. Where the activity of a sample containing the test compound is higher than the activity in a sample lacking the test compound, the compound will have increased the activity. Similarly, where the activity of a sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound will have inhibited the activity.
  • the present invention is particularly useful for screening compounds by using a protease polypeptide in any of a variety of drug screening techniques.
  • the compounds to be screened include, but are not limited to, extracellular, intracellular, biological or chemical origin.
  • the protease polypeptide employed in such a test may be in any form, preferably, free in solution, attached to a solid support, borne on a cell surface or located intracellularly.
  • One skilled in the art can measure the change in rate that a protease ofthe invention cleaves a substrate polypeptide.
  • One skilled in the art can also, for example, measure the formation of complexes between a protease polypeptide and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between a protease polypeptide and its substrate caused by the compound being tested.
  • the activity of protease polypeptides ofthe invention can be determined by, for example, examining the ability to bind or be activated by chemically synthesised peptide ligands.
  • the activity ofthe protease polypeptides can be assayed by examining their ability to bind metal ions such as calcium, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and photons.
  • modulators ofthe protease polypeptide' s activity may alter a protease function, such as a binding property of a protease or an activity such as cleaving protein substrates or polypeptide substrates, or membrane localization.
  • the assay may take the form of a yeast growth assay, an Aequorin assay, a Luciferase assay, a mitogenesis assay, a
  • the invention includes any ofthe serine proteases, cysteine proteases, aspartyl proteases, metalloproteases, threonine proteases, and other proteases.
  • Biological activities of proteases according to the invention include, but are not limited to, the binding of a natural or a synthetic ligand, as well as any one ofthe functional activities of proteases known in the art.
  • Non-limiting examples of protease activities include cleavage of polypeptide chains, processing the pro-form of a polypeptide chain to the active product, transmembrane signaling of various forms, and/or the modification ofthe extraceullar matrix.
  • the modulators ofthe invention exhibit a variety of chemical structures, which can be generally grouped into mimetics of natural protease ligands, and peptide and non-peptide allosteric effectors of proteases.
  • the invention does not restrict the sources for suitable modulators, which may be obtained from natural sources such as plant, animal or mineral extracts, or non-natural sources such as small molecule libraries, including the products of combinatorial chemical approaches to library construction, and peptide libraries.
  • Recombinant proteins are preferred for binding assay HTS because they allow for better specificity (higher relative purity), provide the ability to generate large amounts of receptor material, and can be used in a broad variety of formats (see Hodgson, Bio/Technology, 1992, 10:973-980 which is incorporated herein by reference in its entirety).
  • a variety of heterologous systems is available for functional expression of recombinant proteins that are well known to those skilled in the art. Such systems include bacteria (Strosberg, et al, Trends in Pharmacological Sciences, 1992,
  • An expressed protease can be used for HTS binding assays in conjunction with its defined ligand, in this case the corresponding peptide that activates it.
  • the identified peptide is labeled with a suitable radioisotope, including, but not limited to, I, H, S or P, by methods that are well known to those skilled in the art.
  • the peptides may be labeled by well-known methods with a suitable fluorescent derivative (Baindur, et al, DrugDev. Res., 1994, 33:373-398; Rogers, Drug Discovery Today, 1997, 2: 156- 160).
  • Radioactive ligand specifically bound to the receptor in membrane preparations made from the cell line expressing the recombinant protein can be detected in HTS assays in one of several standard ways, including filtration ofthe receptor-ligand complex to separate bound ligand from unbound ligand (Williams, Med. Res. Rev., 1991, 11:147-184.; Sweetnam, et al, J. Natural Products, 1993, 56:441-455).
  • Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary (Nakayama, Cur. Opinion Drug Disc. Dev., 1998, 1:85-91 Bosse, et al, J. Biomolecular Screening, 1998, 3:285-292.).
  • Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers, Drug Discovery Today, 1997, 2:156-160; Hill, Cur. Opinion Drug Disc. Dev., 1998, 1:92-97).
  • FRET fluorescence energy transfer
  • direct spectrophotofluorometric analysis of bound ligand or fluorescence polarization
  • proteases and natural binding partners required for functional expression of heterologous protease polypeptides can be native constituents ofthe host cell or can be introduced through well-known recombinant technology.
  • the protease polypeptides can be intact or chimeric.
  • the protease activation may result in the stimulation or inhibition of other native proteins, events that can be linked to a measurable response.
  • Examples of such biological responses include, but are not limited to, the following: the ability to survive in the absence of a limiting nutrient in specifically engineered yeast cells (Pausch, Trends in Biotechnology, 1997, 15:487-494); changes in intracellular Ca 2+ concentration as measured by fluorescent dyes (Murphy, et al, Cur. Opinion Drug Disc. Dev., 1998, 1:192-199). Fluorescence changes can also be used to monitor ligand-induced changes in membrane potential or intracellular pH; an automated system suitable for HTS has been described for these purposes (Schroeder, et al., J. Biomolecular Screening, 1996, 1:75-80). Assays are also available for the measurement of common second but these are not generally preferred for HTS.
  • the invention contemplates a multitude of assays to screen and identify inhibitors of ligand binding to protease polypeptides or of substrate cleavage by protease polypeptides.
  • the protease polypeptide is immobilized and interaction with a binding partner or substrate is assessed in the presence and absence of a candidate modulator such as an inhibitor compound.
  • interaction between the protease polypeptide and its binding partner or a substrate is assessed in a solution assay, both in the presence and absence of a candidate inhibitor compound.
  • an inhibitor is identified as a compound that decreases binding between the protease polypeptide and its natural binding partner or the activity of a protease polypeptide in cleaving a substrate molecule.
  • Another contemplated assay involves a variation ofthe di-hybrid assay wherein an inhibitor of protein/protein interactions is identified by detection of a positive signal in a transformed or transfected host cell, as described in PCT publication number WO 95/20652, published August 3, 1995 and is included by reference herein including any figures, tables, or drawings.
  • Candidate modulators contemplated by the invention include compounds selected from libraries of either potential activators or potential inhibitors. There are a number of different libraries used for the identification of small molecule modulators, including: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules. Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as “hits” or “leads” in other drug discovery screens, while others are derived from natural products, and still others arise from non-directed synthetic organic chemistry.
  • Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see, Science 282:63-68 (1998). Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods. Of particular interest are non-peptide combinatorial libraries.
  • Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
  • combinatorial chemistry and libraries created therefrom see, Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). Identification of modulators through use ofthe various libraries described herein permits modification ofthe candidate "hit” (or “lead”) to optimize the capacity ofthe "hit” to modulate activity.
  • binding partners can be designed and include soluble forms of binding partners, as well as such binding partners as chimeric, or fusion, proteins.
  • assays may be used to identify specific peptide ligands of a protease polypeptide, including assays that identify ligands ofthe target protein through measuring direct binding of test ligands to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods.
  • binding interactions are evaluated indirectly using the yeast two- hybrid system described in Fields et al, Nature, 340:245-246 (1989), and Fields et al, Trends in Genetics, 10:286-292 (1994), both of which are incorporated herein by reference.
  • the two-hybrid system is a genetic assay for detecting interactions between two proteins or polypeptides. It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction. Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drugs.
  • the two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast.
  • UAS upstream activation sequence
  • the assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein.
  • the DNA-binding domain targets the first hybrid protein to the UAS ofthe reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription ofthe reporter gene.
  • the second hybrid protein which contains the activation domain, cannot by itself activate expression ofthe reporter gene because it does not bind the UAS. However, when both hybrid proteins are present, the noncovalent interaction ofthe first and second proteins tethers the activation domain to the UAS, activating transcription ofthe reporter gene.
  • this assay can be used to detect agents that interfere with the binding interaction.
  • Expression ofthe reporter gene is monitored as different test agents are added to the system. The presence of an inhibitory agent results in lack of a reporter signal.
  • the yeast two-hybrid assay can also be used to identify proteins that bind to the gene product.
  • a fusion polynucleotide encoding both a protease polypeptide (or fragment) and a UAS binding domain i.e., a first protein
  • a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay.
  • the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein coding region being fused to the activation domain.
  • This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function ofthe second binding protein.
  • the system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.
  • test ligands may be used to search for agents that bind to the target protein.
  • One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Patent No. 5,585,277, incorporated herein by reference. This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states.
  • a test ligand binds to the folded form of a target protein (/. e. , when the test ligand is a ligand ofthe target protein)
  • the target protein molecule bound by the ligand remains in its folded state.
  • the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand.
  • Binding ofthe ligand to the target protein can be determined by any method which distinguishes between the folded and unfolded states ofthe target protein.
  • the function ofthe target protein need not be known in order for this assay to be performed. Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.
  • methods of screening for compounds which modulate protease activity comprise contacting test compounds with protease polypeptides and assaying for the presence of a complex between the compound and the protease polypeptide.
  • the ligand is typically labelled. After suitable incubation, free ligand is separated from that present in bound form, and the amount of free or uncomplexed label is a measure ofthe ability ofthe particular compound to bind to the protease polypeptide.
  • high throughput screening for compounds having suitable binding affinity to protease polypeptides is employed. Briefly, large numbers of different small peptide test compounds are synthesised on a solid substrate.
  • the peptide test compounds are contacted with the protease polypeptide and washed. Bound protease polypeptide is then detected by methods well known in the art.
  • Purified polypeptides ofthe invention can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the protein and immobilize it on the solid support.
  • inventions comprise using competitive screening assays in which neutralizing antibodies capable of binding a polypeptide ofthe invention specifically compete with a test compound for binding to the polypeptide.
  • the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with a protease polypeptide.
  • the invention includes methods for treating a disease or disorder by administering to a patient in need of such treatment a protease polypeptide substantially identical to an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, S
  • a protease polypeptide ofthe invention may also be administered indirectly by via administration of suitable polynucleotide means for in vivo expression ofthe protease polypeptide.
  • the protease polypeptide will have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identity to one ofthe aforementioned sequences.
  • the invention provides methods for treating a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a protease substantially identical to a sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ
  • the disease is selected from the group consisting of cancers, immune-related diseases and disorders, cardiovascular disease, brain or neuronal- associated diseases, and metabolic disorders. More specifically these diseases include cancer of tissues, blood, or hematopoietic origin, particularly those involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous system diseases and conditions including migraine, pain, sexual dysfunction, mood disorders, attention disorders, cognition disorders, hypotension, and hypertension; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourerte's Syndrome; neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple sclerosis, and Amyotrophic lateral sclerosis; viral or non- viral infections caused by HIV-1, HEV-2 or other viral- or prion-agents or fungal- or bacterial- organisms; metabolic disorders including Diabetes and obesity and their related syndromes, among others; cardiovascular disorders including reperfusion reste
  • the invention provides methods for treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a protease polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ TD NO:38, SEQ TD NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ TD NO:42, SEQ TD NO:43, SEQ TD NO:44, SEQ TD NO:45, SEQ TD NO:46, SEQ TD NO:47, SEQ TD NO:48, SEQ TD NO:49, SEQ TD NO:50, SEQ ID NO:36, SEQ ID NO:37, SEQ TD NO:38, SEQ TD NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ TD NO:42, SEQ TD NO:43, SEQ TD NO:44, SEQ TD NO:45, SEQ
  • the disease is selected from the group consisting of cancers, immune- related diseases and disorders, cardiovascular disease, brain or neuronal-associated diseases, and metabolic disorders.
  • these diseases include cancer of tissues, blood, or hematopoietic origin, particularly those involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous system diseases and conditions including migraine, pain, sexual dysfunction, mood disorders, attention disorders, cognition disorders, hypotension, and hypertension; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome; neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple sclerosis, and Amyotrophic lateral sclerosis; viral or non- viral infections caused by HEV-1, HEV-2 or other viral- or prion-agents or fungal- or bacterial- organisms; metabolic disorders including Diabetes and obesity and their related syndromes, among others; cardiovascular disorders including reperfusion restenosis, coronary thrombosis, clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular disease including glaucoma, retin
  • the invention also features methods of treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a protease polypeptide having an amino acid sequence selected from the group consisting those set forth in SEQ ED NO:36, SEQ ED NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ED NO:40, SEQ ED NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ TD NO:44, SEQ ID NO:45, SEQ ED NO:46, SEQ ID
  • the disease is selected from the group consisting of immune-related diseases and disorders, cardiovascular disease, and cancer.
  • the immune-related diseases and disorders are selected from the group consisting of rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplantation.
  • Substances useful for treatment of protease-related disorders or diseases preferably show positive results in one or more in vitro assays for an activity corresponding to treatment ofthe disease or disorder in question (Examples of such assays are provided herein, including Example 7).
  • Examples of substances that can be screened for favorable activity are provided and referenced throughout the specification, including this section (Screening Methods to Identify Substances that Modulate Protease Activity).
  • the substances that modulate the activity ofthe proteases preferably include, but are not limited to, antisense oligonucleotides, ribozymes, and other inhibitors of proteases, as determined by methods and screens referenced this section and in Example 7, below, and any other suitable methods. The use of antisense oligonucleotides and ribozymes are discussed more fully in the Section "Gene Therapy," below.
  • preventing refers to decreasing the probability that an organism contracts or develops an abnormal condition.
  • treating refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.
  • a therapeutic effect refers to the inhibition or activation factors causing or contributing to the abnormal condition.
  • a therapeutic effect relieves to some extent one or more ofthe symptoms ofthe abnormal condition.
  • a therapeutic effect can refer to one or more of the following: (a) an increase or decrease in the proliferation, growth, and/or differentiation of cells; (b) activation or inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more ofthe symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells.
  • Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.
  • abnormal condition refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism.
  • An abnormal condition can relate to cell proliferation, cell differentiation, or cell survival.
  • Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation.
  • Abnormal differentiation conditions include, but are not limited to neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates.
  • Abnormal cell survival conditions relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated.
  • a number of proteases are associated with the apoptosis pathways. Aberrations in the function of any one ofthe proteases could lead to cell immortality or premature cell death.
  • inhibition in conjunction with the function of a protease in a signal transduction process, refers to a protease that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type protease activity, mutated such that it can no longer interact with a natural binding partner, is no longer modified by another protein, or no longer interacts with a natural binding partner.
  • administering relates to a method of incorporating a compound into cells or tissues of an organism.
  • the abnormal condition can be prevented or treated when the cells or tissues ofthe organism exist within the organism or outside ofthe organism.
  • Cells existing outside the organism can be maintained or grown in cell culture dishes.
  • many techniques exist in the art to administer compounds including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.
  • multiple techniques exist in the art to administer the compounds including (but not limited to) cell microinjection techniques, transformation techniques, and carrier techniques.
  • the abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism.
  • the effect of administering a compound on organism function can then be monitored.
  • the organism is preferably a mouse, rat, rabbit, guinea pig, or goat, more preferably a monkey or ape, and most preferably a human.
  • the invention features methods for detection of a protease polypeptide in a sample as a diagnostic tool for diseases or disorders, wherein the method comprises the steps of: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a protease polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ TD NO:37, SEQ ID NO:38, SEQ ED NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ TD NO:43, SEQ ED NO:44, SEQ ED NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ TD NO:50, SEQ TD NO:51, SEQ ID NO:52, SEQ TD NO:53, SEQ TD NO:54, SEQ TD NO:55, SEQ
  • the disease or disorder is selected from the group consisting of rheumatoid arthritis, arteriosclerosis, autoimmune disorders, organ transplantation, myocardial infarction, cardiomyopathies, stroke, renal failure, oxidative stress-related neurodegenerative disorders, and cancer.
  • the disease is selected from the group consisting of cancers, immune- related diseases and disorders, cardiovascular disease, brain or neuronal-associated diseases, and metabolic disorders.
  • these diseases include cancer of tissues, blood, or hematopoietic origin, particularly those involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous system diseases and conditions including migraine, pain, sexual dysfunction, mood disorders, attention disorders, cognition disorders, hypotension, and hypertension; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome; neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple sclerosis, and Amyotrophic lateral sclerosis; viral or non- viral infections caused by HIV-1, HEV-2 or other viral- or prion-agents or fungal- or bacterial- organisms; metabolic disorders including Diabetes and obesity and their related syndromes, among others; cardiovascular disorders including reperfusion restenosis, coronary thrombosis, clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular disease including glaucoma, retin
  • the protease "target region” is the nucleotide base sequence selected from the group consisting of those set forth in SEQ TD NO:l, SEQ TD NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ TD NO:5, SEQ ID NO:6, SEQ TD NO:7, SEQ ED NO:8, SEQ ED NO:9, SEQ ID NO:10, SEQ ED NO:ll, SEQ ID NO:12, SEQ ED NO:13, SEQ ID NO:14, SEQ TD NO:15, SEQ ED NO:16, SEQ ID NO:17, SEQ ED NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ED NO:22, SEQ ID NO:23, SEQ ED NO:24, SEQ ID NO:25, SEQ TD NO:26, SEQ ID NO:27, SEQ TD NO:28, SEQ ID NO:29, SEQ ED NO:30, SEQ ED NO
  • Specific hybridization indicates that in the presence of other nucleic acids the probe only hybridizes detectably with the nucleic acid target region ofthe protease ofthe invention.
  • Putative target regions can be identified by methods well known in the art consisting of alignment and comparison ofthe most closely related sequences in the database.
  • the nucleic acid probe hybridizes to a protease target region encoding at least 6, 12, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of a sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ED NO:37, SEQ ED NO:38, SEQ ED NO:39, SEQ ED NO:40, SEQ ED NO:41, SEQ ED NO:42, SEQ ED NO:43, SEQ ED NO:44, SEQ ID NO:45, SEQ TD NO:46, SEQ TD NO:47, SEQ TD NO:48, SEQ ID NO:49, SEQ ED NO:50, SEQ ED NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ TD NO:57, SEQ TD NO:58, SEQ ID NO:59, SEQ ID NO:
  • Hybridization conditions should be such that hybridization occurs only with the protease genes in the presence of other nucleic acid molecules. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides. Such conditions are defined in Berger et al. (1987) (Guide to Molecular Cloning Techniques pg 421 , hereby incorporated by reference herein in its entirety including any figures, tables, or drawings.).
  • the diseases for which detection ofprotease genes in a sample could be diagnostic include diseases in which protease nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells.
  • amplification is meant increased numbers ofprotease DNA or RNA in a cell compared with normal cells.
  • proteases may be found as single copy genes.
  • the chromosomal location ofthe protease genes may be amplified, resulting in multiple copies ofthe gene, or amplification.
  • Gene amplification can lead to amplification of protease RNA, or protease RNA can be amplified in the absence ofprotease DNA amplification.
  • RNA can be the detectable presence of protease RNA in cells, since in some normal cells there is no basal expression of protease RNA. In other normal cells, a basal level of expression ofprotease exists, therefore in these cases amplification is the detection of at least 1 -2-fold, and preferably more, protease RNA, compared to the basal level.
  • the diseases that could be diagnosed by detection ofprotease nucleic acid in a sample preferably include cancers.
  • the test samples suitable for nucleic acid probing methods ofthe present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
  • the samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.
  • the invention features a method for detection of a protease polypeptide in a sample as a diagnostic tool for a disease or disorder, wherein the method comprises: (a) comparing a nucleic acid target region encoding the protease polypeptide in a sample, where the protease polypeptide has an amino acid sequence selected from the group consisting those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ ED NO:38, SEQ ID NO:39, SEQ ED NO:40, SEQ ED NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ED NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ TD NO:48, SEQ ID NO:49, SEQ ED NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ TD NO:53, SEQ TD NO:54, SEQ TD NO:55, SEQ ED NO:56
  • these diseases include cancer of tissues, blood, or hematopoietic origin, particularly those involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous system diseases and conditions including migraine, pain, sexual dysfunction, mood disorders, attention disorders, cognition disorders, hypotension, and hypertension; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome; neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple sclerosis, and Amyotrophic lateral sclerosis; viral or non- viral infections caused by HIV-1, HIV-2 or other viral- or prion-agents or fungal- or bacterial- organisms; metabolic disorders including Diabetes and obesity and their related syndromes, among others; cardiovascular disorders including reperfusion restenosis, coronary thrombosis, clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular disease including glaucoma, retinopathy
  • comparing refers to identifying discrepancies between the nucleic acid target region isolated from a sample, and the control nucleic acid target region.
  • the discrepancies can be in the nucleotide sequences, e.g. insertions, deletions, or point mutations, or in the amount of a given nucleotide sequence. Methods to determine these discrepancies in sequences are well-known to one of ordinary skill in the art.
  • the "control" nucleic acid target region refers to the sequence or amount ofthe sequence found in normal cells, e.g. cells that are not diseased as discussed previously.
  • domain refers to a region of a polypeptide which serves a particular function.
  • N-terminal or C-terminal domains of signal transduction proteins can serve functions including, but not limited to, binding molecules that localize the signal transduction molecule to different regions ofthe cell or binding other signaling molecules directly responsible for propagating a particular cellular signal.
  • Some domains can be expressed separately from the rest of the protein and function by themselves, while others must remain part of the intact protein to retain function. The latter are termed functional regions of proteins and also relate to domains.
  • proteases can be modulated by signal transduction pathways such as the Ras/MAP kinase signaling pathways. Additionally, the activity of proteases can modulate the activity ofthe MAP kinase signal transduction pathway. Furthermore, proteases can be shown to be instrumental in the communication between disparate signal transduction pathways.
  • signal transduction pathway refers to the molecules that propagate an extracellular signal through the cell membrane to become an intracellular signal. This signal can then stimulate a cellular response.
  • the polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein tyrosine kinases, receptor and non-receptor protein phosphatases, polypeptides containing SRC homology 2 and 3 domains, phosphotyrosine binding proteins (SRC homology 2 (SH2) and phosphotyrosine binding (PTB and PH) domain containing proteins), proline-rich binding proteins (SH3 domain containing proteins), GTPases, phosphodiesterases, phospholipases, prolyl isomerases, proteases, Ca 2+ binding proteins, cAMP binding proteins, guanyl cyclases, adenylyl cyclases, NO generating proteins, nucleotide exchange factors, and transcription factors.
  • Figures 1 A-W shows the partial nucleotide sequences for human proteases oriented in a 5' to 3' direction (SEQ TD NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ TD NO:4, SEQ ID NO:5, SEQ ED NO:6, SEQ TD NO:7, SEQ ID NO:8, SEQ ED NO:9, SEQ ED NO:10, SEQ TD NO:ll, SEQ TD NO:12, SEQ ID NO:13, SEQ TD NO:14, SEQ ED NO:15, SEQ TD NO:16, SEQ ED NO:17, SEQ ID NO:18, SEQ ED NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ED NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ TD NO:27, SEQ ID NO:28, SEQ ED NO:29, SEQ ID NO:30, S
  • Figure 2A-I shows the partial amino acid sequences for the human proteases encoded by SEQ ID No. 1-35 in the direction of translation (SEQ ID NO:36, SEQ ID NO:37, SEQ TD NO:38, SEQ ED NO:39, SEQ TD NO:40, SEQ TD NO:41, SEQ TD NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ TD NO:45, SEQ ED NO:46, SEQ ED NO:47, SEQ ED NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ED NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ED NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ED NO:63, SEQ ED NO:64, SEQ ID NO
  • Proteases are enzymes capable of severing the amino acid backbone of other proteins, and are involved in a large number of diverse processes within the body.
  • Proteases may be classified into several major groups including serine proteases, cysteine proteases, aspartyl proteases, metalloproteases, threonine proteases, and other proteases.
  • Aspartate proteases of eukaryotes are monomeric enzymes which consist of two domains. Each domain contains an active site centered on a catalytic aspartyl residue.
  • Examples of aspartyl protease polypeptides according to the invention include SGPrl40, rl97, r005 and r078 (SEQ ID NOS:l, 2, 3, and 4, respectively). These polypeptides may have one or more ofthe following activites.
  • Cathepsin E is an immunologically discrete aspartic protease found in the gastrointestinal tract (Azuma et al, 1992, J. Biol. Chem., 267:1609-1614). Cathepsin E is an intracellular proteinase that does not appear to be involved in the digestion of dietary protein. It is found in highest concentration in the surface of epithelial mucus-producing cells ofthe stomach. It is the first aspartic proteinase expressed in the fetal stomach and is found in more than half of gastric cancers. It appears, therefore, to be an 'oncofetal' antigen. Its association with stomach cancers suggests it may play a role in the development of this disease.
  • Cathepsin D a lyosomal aspartyl protease
  • Cathepsin D a lyosomal aspartyl protease
  • Renin catalyzes the first step in the activation pathway of angiotensinogen ⁇ a cascade that can result in aldosterone release, vasoconstriction, and increase in blood pressure. Renin cleaves angiotensinogen to form angiotensin I, which is converted to angiotensin II by angiotensin I converting enzyme, an important regulator of blood pressure and electrolyte balance. Renin occurs in other organs than the kidney, e.g., in the brain, where it is implicated in the regulation of numerous activities.
  • Presenilin proteins Alzheimer's disease (AD) patients with an inherited form ofthe disease carry mutations in the presenilin proteins (PSEN1; PSEN2) or the amyloid precursor protein (APP). These disease-linked mutations result in increased production ofthe longer form of amyloid-beta (main component of amyloid deposits found in AD brains) (Saftig et al, Eur. Arch. Psychiartry Clin. Neurosci., 1999, 249:271-79).
  • Presenilins are postulated to regulate APP processing through their effects on ⁇ - secretase, an enzyme that cleaves APP (Cruts et al, 1998, Hum.
  • PS1 gene result in a selective increase in the production ofthe amyloidogenic peptide amyloid-beta (1-42) by proteolytic processing ofthe amyloid precursor protein (APP)(Lemere et al, 1996, Nat. Med. 2(10):1146-50).
  • APP amyloid precursor protein
  • ⁇ -secretase ⁇ -secretase expressed specifically in the brain, is responsible for the proteolytic processing ofthe amyloid precursor protein (APP) associated with APP.
  • APP amyloid precursor protein
  • Alzheimer's disease (Potter et al, 2000, Nat. Biotechnol. 18(2): 125-26). It cleaves at the amino terminus ofthe ⁇ -peptide sequence, between residues 671 and 672 of APP, leading to the generation and extracellular release of ⁇ -cleaved soluble APP, and a carboxyterminal fragment that is later released by ⁇ -secretase (Kimberly et al, 2000, J. Biol. Chem. 275(5):3173-78). Yan et al. (Nature, 1999 402:533-37) identified a new membrane-bound aspartyl protease (Asp2) with ⁇ -secretase activity.
  • Asp2 membrane-bound aspartyl protease
  • the Asp2 gene is expressed widely in brain and other tissues. Decreasing the expression of Asp2 in cells reduces amyloid ⁇ -peptide production and blocks the accumulation ofthe carboxy-terminal APP fragment that is created by ⁇ -secretase cleavage. Asp2 is a new protein target for drugs that are designed to block the production of amyloid ⁇ -peptide peptide and the consequent formation of amyloid plaque in Alzheimer's disease.
  • Two aspartyl proteases involved in human placentation have recently been isolated: decidual aspartyl protease (DAP-1), and DAP-2. (Moses et al, Mol. Hum. Reprod., 1999, 5:983-89)
  • HEV-1 retropepsin Another member ofthe aspartyl peptidase family is HEV-1 retropepsin, from the human immunodeficiency virus type 1. This enzyme is vital for processing of the viral polyprotein and maturation ofthe mature virion.
  • cysteine proteases Another class of proteases which perform a wide variety of functions within the body are the cysteine proteases. Among their roles are the processing of precursor proteins, and intracellular degradation of proteins marked for disposal via the ubiquitin pathway. Catalysis proceeds through a thioester intermediate and is facilitated by a nearby histidine side chain; an asparagine completes the essential catalytic triad. Peptidases in this family with important roles in disease include calpain, the caspases, hedgehog, papain, and Ubiquitin hydrolases.
  • cysteine protease polypeptides ofthe present invention examples include SGPr084, r009, r286, r008, rl98, r210, r290, rll6, r003, r016 (SEQ ID NOS:5, 6, 7, 8, 9, 10, 11, 12, and 13, respectively). These polypeptides may have one or more ofthe following activities. Cysteine proteases are produced by a large number of cells including those ofthe immune system (macrophages, monocytes, etc.). These immune cells exercise their protective role in the body, in part, by migrating to sites of inflammation and secreting molecules, among the secreted molecules are cysteine proteases.
  • cysteine proteases of the immune system can lead to autoimmune diseases such as rheumatoid arthritis.
  • the over-secretion ofthe cysteine protease cathepsin C causes the degradation of elastin, collagen, laminin and other structural proteins found in bones. Bone subjected to this inappropriate digestion is more susceptible to metastasis.
  • Cysteine proteases may also influence vascular permeability through their effect on the kallikrein/kinin pathway, their ability to form complexes with hemagglutinins, their effect in activation of complement components and their ability to destroy serpins.
  • a cascade ofprotease reactions is believed to be responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death.
  • This cascade involves many members ofthe aspartate-specific cysteine proteases of the caspase family, including Caspases 2, 3, 6, 7, 8, and 10 ((Salvesen and Dixit, Cell, 1997, 91 :443-446).
  • Cancer cells that escape apoptotic signals generated by cytotoxic chemotherapeutics or loss of normal cellular survival signals (as in metastatic cells), can go on to develop palpable tumors.
  • Caspase 1 specifically processes the precursors of IL- 1 ⁇ , and IL- 18 (interferon- ⁇ -inducing factor) (Salvesen and Dixit, Cell, 1997).
  • calpain Calcium-dependent cysteine proteases, collectively called calpain, are widely distributed in mammalian cells (Wang, 2000, Trends NeuroscL 23(l):20-26).
  • the calpains are nonlysosomal intracellular cysteine proteases.
  • the mammalian calpains include 2 ubiquitous proteins, CAPNl and CAPN2, as well as 2 stomach-specific proteins, and CAPN3, which is muscle-specific (Herasse et al, 1999, Mol. Cell. Biol. 19(6):4047-55).
  • the ubiquitous enzymes consist of heterodimers with distinct large subunits associated with a common small subunit, all of wliich are encoded by different genes.
  • the large subunits of calpains can be subdivided into 4 domains; domains I and III, whose functions remain unknown, show no homology with known proteins. The former, however, may be important for the regulation ofthe proteolytic activity. Domain II shows similarity with other cysteine proteases, which share histidine, cysteine, and asparagine residues at their active sites. Domain JV is calmodulin-like. CAPN5 and CAPN6 differ from previously identified vertebrate calpains in that they lack a calmodulin-like domain IV (Ohno et al, 1990, Cytogenet. Cell Genet. 53(4):225-29).
  • hh Drosophila gene 'hedgehog' (hh) that encodes inductive signals during embryogenesis
  • 'Hedgehog' encodes a secreted protein that is involved in establishing cell fates at several points during Drosophila development (Marigo et al, 1995, Genomics 28:44-51).
  • hh Sonic hedgehog
  • Ihh Indian hedgehog
  • Dhh desert hedgehog
  • Shh encodes a signal that is instrumental in patterning the early embryo. It is expressed in Hensen's node, the floorplate of the neural tube, the early gut endoderm, the posterior ofthe limb buds, and throughout the notochord (Chiang et al, 1996, Nature 383:407-413). It has been implicated as the key inductive signal in patterning ofthe ventral neural tube, the anterior-posterior limb axis, and the ventral somites. Oro et al.
  • SHH may be a dominant oncogene in multiple human tumors, a mirror ofthe tumor suppressor activity ofthe opposing 'patched' (PTCH) gene (Aszterbaum et al, 1998, J. Invest. Derm. 110:885-888).
  • PTCH tumor suppressor activity ofthe opposing 'patched'
  • Dhh expression is initiated in Sertoli cell precursors shortly after the activation of Sry and persists in the testis into the adult.
  • Bitgood et al. disclose that female mice homozygous for a Dhh-null mutation show no obvious phenotype, whereas males are viable but infertile having a complete absence of mature sperm, demonstrating that Dhh signaling plays an essential role in the regulation of mammalian spermatogenesis.
  • Dhh has also been found to have a role in the and maintenance of protective nerve sheaths endo-, peri- and epineurium.
  • Ubiquitin carboxyl-terminal hydrolases (3.1.2.15) (deubiquitinating enzymes) are thiol proteases that recognize and hydrolyze the peptide bond at the C- terminal glycine of ubiquitin. These enzymes are involved in the processing of poly-ubiquitin precursors as well as that of ubiquinated proteins. In eukaryotic cells, the covalent attachment of ubiquitin to proteins plays a role in a variety of cellular processes. In many cases, ubiquitination leads to protein degradation by the 26S proteasome. Protein ubiquitination is reversible, and the removal of ubiquitin is catalyzed by deubiquitinating enzymes, or DUBs.
  • a defect in these enzymes, catalyzing the removal of ubiquitin from ubiquinated proteins, may be characteristic of neurodegenerative diseases such as Alzheimer's, Parkinson's, progressive supranuclear palsy, and Pick's and Kuf s disease.
  • Cathepsin K a member ofthe papain family of peptidases, is involved in osteoclastic resorption. It plays an important role in extracellular degradation and may have a role in disorders of bone remodeling, such as pyncodysostosis, an autosomal recessive osteochondrodysplasia characterized by osteosclerosis and short stature.
  • Antigen presentation by major histocompatibihty complex (MHC) class II molecules requires the participation of different proteases in the endocytic route to degrade endocytosed antigens as well as the MHC class Il-associated invariant chain. Only cathepsin S, a member ofthe papain family, appears to be essential for complete destruction ofthe invariant chain.
  • MHC major histocompatibihty complex
  • Cathepsin B is overexpressed in tumors ofthe lung, prostate, colon, breast, and stomach.
  • Hughes et al. Proc. Nat. Acad. Sci. 95: 12410-12415, 1998) found an amplicon at 8 ⁇ 23-p22 that resulted in cathepsin B overexpression in esophageal adenocarcinoma.
  • Abundant extracellular expression of CTSB protein was found in 29 of 40 (72.5%) of esophageal adenocarcinoma specimens by use of immunohistochemical analysis. The findings were thought to support an important role for CTSB in esophageal adenocarcinoma and possibly in other tumors.
  • Cathepsin B a lyosomal protease
  • Cathepsin B is being studied as a prognostic marker in various cancers (breast, pulmonary adenocarcinomas).
  • cysteine protease AEP plays another role in the immune functions. It has been implicated in the protease step required for antigen processing in B cells. (Manoury et al. Nature 396:695-699 (1998)) Hepatitis A viral protease (C3E
  • the Hepatitis A genome encodes a cysteine protease required for enzymatic cleavages in vivo to yield mature proteins (Wang, 1999, Prog. Drug Res. 52:197- 219). This enzyme and its homologs in other viruses (such as hepatitis E virus) are potential targets for chemotherapeutic intervention.
  • metalloprotease protease polypeptides examples include SGPrOl ⁇ , r352, r050, r282, r046, r060, r068, r096, rll9, rl43, rl64, r281, r075, r292, r069, r212, r049, r026, r203, rl57, rl54, r088 (SEQ ID
  • Matrix degradation is an essential step in the spread of cancer.
  • the 72- and 92-kD type TV collagenases are members of a group of secreted zinc metalloproteases which, in mammals, degrade the collagens ofthe extracellular matrix.
  • Other members of this group include interstitial collagenase and stromefysin (Nagase et al, 1992, Matrix Suppl 1:421-424).
  • Vu et al Vu et al (Cell, 1998, 934:11-22) created homozygous mice with a null mutation in the MMP9/gelatinase B gene. These mice exhibited an abnormal pattern of skeletal growth plate vascularization and ossification. Growth plates from MMP9-null mice in culture showed a delayed release of an angiogenic activator, establishing a role for this proteinase in controlling angiogenesis.
  • MMP2 (gelatinase A) have been associated with the aggressiveness of human cancers (Chenard et al, 1999, Int. J. Cancer, 82:208-12).
  • BCC basal cell carcinomas
  • SCC squamous cell carcinomas
  • MMP2 and MMP9 have recently been shown to be modulated by the Ras/MAP kinase signaling pathways (Esparza et al, 1999, Blood, 94:2754-66) (see also, Li et al, 1998, Biochim. Biophys. Ada, 1405:110-20).
  • ADAMs (Ml 2) - TNF. inflammation, growth factor processing
  • ADAM peptidases are a family of proteins containing a disintegrin and metalloproteinase (ADAM) domain (Werb and Yan, Science, 1998, 282:1279-1280). Members of this family are cell surface proteins with a unique structure possessing both potential adhesion and protease domains (Primakoff and Myles, Trends in Genet., 2000, 16:83-87). Activity of these proteases can be linked to TNF, inflammation, and/or growth factor processing.
  • ADAM disintegrin and metalloproteinase
  • ADAM proteases have also been characterized as having a pro- and metalloproteinase domain, a disintegrin domain, a cysteine-rich region and an EGF repeat (Blobel, 1997, Cell, 90:589-592 wliich is hereby incorporated herein by reference in its entirety including any figures, tables, or drawings).
  • TNF- ⁇ Tumor Necrosis Factor-
  • kit-ligand TGF ⁇
  • Fas-ligand cytokine receptors
  • adhesion proteins such as L-selectin
  • b amyloid precursor proteins Blobel, 1997, Cell, 90:589-592.
  • Tumor necrosis factor- ⁇ is synthesized as a proinflammatory cytokine from a 233-amino acid precursor. Conversion ofthe membrane-bound precursor to a secreted mature protein is mediated by a protease termed TNF- ⁇ convertase. TNF- ⁇ is involved in a variety of diseases. AD AMI 7, wliich contains a disintegrin and metalloproteinase domains, is also called 'tumor necrosis factor- ⁇ converting enzyme' (TACE) (Black et al, Nature, 1997, 385:729-33).
  • TACE 'tumor necrosis factor- ⁇ converting enzyme'
  • the gene encodes an 824-amino acid polypeptide containing the features ofthe ADAM family: a secretory signal sequence, a disintegrin domain, and a metalloprotease domain. Expression studies showed that the encoded protein cleaves precursor tumor necrosis factor- ⁇ to its mature form. This enzyme may also play a role in the processing of Transforming Growth Factor- ⁇ (TGF- ⁇ ), as mice which lack the gene are similar in phenotype to those that lack TGF- ⁇ (Peschon et al, Science, 282:1281-1284).
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • Neprylisin (Ml 3) Endothelin-converting enzyme
  • Neprylisin a metallopeptidase active in degradation of enkephalins and other bioactive peptides, is a drug target in hypertension and renal disease (Oef er, et al, J. Mol. Biol, 2000, 296:341-49).
  • Carboxypeptidases specifically remove COOH-terminal basic amino acids(arginine or lysine) (Nesheim, 1998, Curr. Opin. Hematol. 5(5):309-13). They have important functions in many biologic processes, including activation, inactivation, or modulation of peptide hormone activity, neurotransmitter processing, and alteration of physical properties of proteins and enzymes (Ostrowska et al, 1998, Rocz. Akad. Med. Bialymst. 43:39-55).
  • Dipeptidase (M2 - ACE Angiotensin I converting enzyme (EC 3.4.15.1 ), or kininase II, is adipeptidyl carboxypeptidase that plays an important role in blood pressure regulation and electrolyte balance by hydrolyzing angiotensin I into angiotensin II, a potent vasopressor, andaldosterone-stimulating peptide. The enzyme is also able to inactivate bradykinin, a potent vasodilator.
  • angiotensin-converting enzyme has been studied primarily in the context of its role in blood pressure regulation, this widely distributed enzyme has many other physiologic functions.
  • ACE Matrix metalloproteases (Ml OB) - tissue remodeling and inflammation
  • MMPs matrix metalloproteases
  • the matrix metalloproteases (MMPs) are a family of related matrix- degrading enzymes that are important in tissue remodeling and repair during development and inflammation (Belotti et al, 1999, Int. J. Biol. Markers 14(4):232- 38). Abnormal expression is associated with various diseases such as tumor invasiveness (Johansson and Kahari, 2000, Histol Histopathol 15(l):225-37), arthritis (Malemud et al, 1999, Front. Biosci. 4:D762-71), and atherosclerosis (Nagase, 1991, Biol. Chem. 378(3-4):151-60). MMP activity may also be related to tobacco-induced pulmonary emphysema (Dhami et al, Am. J. Respir. Cell Mol. Biol, 2000, 22:244-52).
  • SREBPs sterol-regulatory binding proteins
  • metalloprotease processing of growth factors In addition to the processing of TGF- ⁇ described above, metalloproteases have been directly demonstrated to be active in the processing ofthe precursor of other growth factors such as heparin-binding EGF (proHB-EFG) (Izumi et al, EMBOJ, 1998,17:7260-72), and amphiregulin (Brown et al, 1998, J. Biol. Chem., 27:17258-68). Additionally, metalloproteases have recently been shown to be instrumental in the communication whereby stimulation of a GPCR pathway results in stimulation ofthe MAP kinase pathway (Prenzel et al, 1999, Nature, 402:884-888).
  • HB-EGF The growth factor intermediate in the pathway, HB-EGF is released by the cell in a proteolytic step regulated by the GPCR pathway involving an uncharacterized metalloprotease. After release, the HB-EGF is bound by the extracellular matrix and then presented to the EGF receptors on the surface, resulting in the activation ofthe MAP kinase pathway (Prenzel et al, 1999, Nature, 402:884-888).
  • M-CSF macrophage colony-stimulating factor
  • SCF stem cell factor
  • the serine proteases are a class which includes trypsin, kallikrein, chymotrypsin, elastase, thrombin, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasmin (Werb, Cell, 1997, 91:439-442), kallikrein (Clements, Biol. Res., 1998, 31151-59), and cathepsin G (Shamamian et al, Surgery, 2000, 127: 142-47).
  • proteases have in common a well-conserved catalytic triad of amino acid residues in their active site consisting of histidine-57, aspartic acid-102, and serine-195 (using the chymotrypsin numbering system). Serine protease activity has been linked to coagulation and they may have use as tumor markers. Serine proteases can be further subclassified by their specificity in substrates.
  • the elastases prefer to cleave substrates adjacent to small aliphatic residues such as valine, chymases prefer to cleave near large aromatic hydrophobic residures, and tryptases prefer positively charged residues.
  • small aliphatic residues such as valine
  • chymases prefer to cleave near large aromatic hydrophobic residures
  • tryptases prefer positively charged residues.
  • serine protease has been described recently which prefers to cleave adjacent to a proline. This prolyl endopeptidase has been implicated in the progression of memory loss in
  • proteases known to belong to this large and important family include: blood coagulation factors VII, IX, X, XI and XII; thrombin; plasminogen; complement components Clr, Cls, C2; complement factors B, D and I; complement-activating component of RA-reactive factor; elastases 1, 2, 3 A, 3B (protease E); hepatocyte growth factor activator; glandular (tissue) kallikreins including EGF-binding protein types A, B, and C; NGF- ⁇ chain, ⁇ -renin, and prostate specific antigen (PSA); plasma kallikrein; mast cell proteases; myeloblastin (proteinase 3) (Wegener's autoantigen); plasminogen activators (urokinase-type, and tissue-type); and the trypsins I, II, III, and IV. These peptidases play key roles in coagulation, tumorigenesis, control of blood pressure, release of growth factors
  • Threonine peptidases (Tl) - Prosite PDOC00326/PDOC00668) Proteasomal subunits (T1A)
  • the proteasome is a multicatalytic threonine proteinase complex involved in ATP/ubiquitin dependent non-lysosomal proteolysis of cellular substrates. It is responsible for selective elimination of proteins with aberrant structures, as well as naturally occurring short-lived proteins related to metabolic regulation and cell-cycle progression (Momand et al, 2000, Gene 242(1 -2): 15-29, Bochtler et al, 1999, Annu. Rev. Biophys Biomol Struct.28:295-311).
  • the proteasome inhibitor lactacystin reversibly inhibits proliferation of human endothelial cells, suggesting a role for proteasomes in angiogenesis (Kumeda, et al, Anticancer Res.
  • proteasome Another important function ofthe proteasome in higher vertebrates is to generate the peptides presented on MHC-class 1 molecules to circulating lymphocytes (Castelli et al, 1991, Int. J. Clin. Lab. Res. 27(2): 103- 10).
  • the proteasome has a sedimentation coefficient of 26S and is composed of a 20S catalytic core and a 22S regulatory complex.
  • Eukaryotic 20S proteasomes have a molecular mass of 700 to 800 kD and consist of a set of over 15 kinds of polypeptides of 21 to 32 kD.
  • the prenyl-protein specific protease responsible for post-translational processing ofthe Ras proto-oncogene and other prenylated proteins falls into this class.
  • This class also includes several viral peptidases that may play a role in mammalian infection, including cardiovirus endopeptidase 2A (encephalomyocarditis virus) (Molla et al, 1993, J. Virol. 67(8):4688-95), NS2-3 protease (hepatitis C virus) (Blight et al, 1998, Antivir. Ther.
  • a nucleic acid probe ofthe present invention may be used to probe an appropriate chromosomal or cDNA library by usual hybridization methods to obtain other nucleic acid molecules ofthe present invention.
  • a chromosomal DNA or cDNA library may be prepared from appropriate cells according to recognized methods in the art (cf. "Molecular Cloning: A Laboratory Manual", second edition, Cold Spring Harbor Laboratory, Sambrook, Fritsch, & Maniatis, eds., 1989).
  • nucleic acid probes having nucleotide sequences which correspond to N-terminal and C-terminal portions ofthe amino acid sequence ofthe polypeptide of interest.
  • the synthesized nucleic acid probes may be used as primers in a polymerase chain reaction (PCR) carried out in accordance with recognized PCR techniques, essentially according to PCR Protocols, "A Guide to Methods and Applications", Academic Press, Michael, et al, eds., 1990, utilizing the appropriate chromosomal or cDNA library to obtain the fragment ofthe present invention.
  • PCR polymerase chain reaction
  • hybridization probes ofthe present invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemiluminescence, and the like. After hybridization, the probes may be visualized using known methods.
  • the nucleic acid probes ofthe present invention include RNA, as well as DNA probes, such probes being generated using techniques known in the art.
  • the nucleic acid probe may be immobilized on a solid support.
  • solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such solid supports are well known in the art.
  • the test samples suitable for nucleic acid probing methods ofthe present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
  • samples used in the above-described methods will vary based on the assay format, the detection method and the nature ofthe tissues, cells or extracts to be assayed.
  • Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized.
  • One method of detecting the presence of nucleic acids ofthe invention in a sample comprises (a) contacting said sample with the above-described nucleic acid probe under conditions such that hybridization occurs, and (b) detecting the presence of said probe bound to said nucleic acid molecule.
  • One skilled in the art would select the nucleic acid probe according to techniques known in the art as described above. Samples to be tested include but should not be limited to RNA samples of human tissue.
  • a kit for detecting the presence of nucleic acids ofthe invention in a sample comprises at least one container means having disposed therein the above-described nucleic acid probe.
  • the kit may further comprise other containers comprising one or more ofthe following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe.
  • detection reagents include, but are not limited to radiolabelled probes, enzymatic labeled probes (horseradish peroxidase, alkaline phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin).
  • the kit further comprises instructions for use.
  • a compartmentalized kit includes any kit in which reagents are contained in separate containers.
  • Such containers include small glass containers, plastic containers or strips of plastic or paper.
  • Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and the like), and containers which contain the reagents used to detect the hybridized probe, bound antibody, amplified product, or the like.
  • wash reagents such as phosphate buffered saline, Tris-buffers, and the like
  • the present invention also relates to a recombinant DNA molecule comprising, 5' to 3', a promoter effective to initiate transcription in a host cell and the above-described nucleic acid molecules.
  • the present invention relates to a recombinant DNA molecule comprising a vector and an above-described nucleic acid molecule.
  • the present invention also relates to a nucleic acid molecule comprising a transcriptional region functional in a cell, a sequence complementary to an RNA sequence encoding an amino acid sequence corresponding to the above- described polypeptide, and a transcriptional termination region functional in said cell.
  • the above-described molecules may be isolated and/or purified DNA molecules.
  • the present invention also relates to a cell or organism that contains an above-described nucleic acid molecule and thereby is capable of expressing a polypeptide.
  • the polypeptide may be purified from cells which have been altered to express the polypeptide.
  • a cell is said to be "altered to express a desired polypeptide" when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce or which the cell normally produces at lower levels.
  • One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA, or synthetic sequences into either eukaryotic or prokaryotic cells.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression.
  • the precise nature ofthe regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation.
  • Such regions will normally include those 5'- non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the sequence encoding a protease of the invention may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3 '-region naturally contiguous to the DNA sequence encoding a protease ofthe invention, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences allows the protease sequence to be transcribed, i.e., where the linkage does not (1) result in the introduction of a frame- shift mutation, (2) interfere with the ability ofthe promoter region sequence to direct the transcription of a gene sequence encoding a protease ofthe invention, or (3) interfere with the ability ofthe gene sequence of a protease ofthe invention to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the present invention encompasses the expression of a gene encoding a protease ofthe invention (or a functional derivative thereof) in either prokaryotic or eukaryotic cells.
  • Prokaryotic hosts are, generally, very efficient and convenient for the production of recombinant proteins and are, therefore, one type of preferred expression system for proteases ofthe invention.
  • Prokaryotes most frequently are represented by various strains of E. coli. However, other microbial strains may also be used, including other bacterial strains.
  • plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host may be used.
  • suitable plasmid vectors may include pBR322, pUCl 18, pUCl 19 and the like; suitable phage or bacteriophage vectors may include ⁇ gtlO, ⁇ gtl 1 and the like; and suitable virus vectors may include pMAM-neo, pKRC and the like.
  • the selected vector ofthe present invention has the capacity to replicate in the selected host cell.
  • Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the polypeptide will not be glycosylated.
  • the prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • a protease ofthe invention (or a functional derivative thereof) in a prokaryotic cell, it is necessary to operably link the sequence encoding the protease ofthe invention to a functional prokaryotic promoter.
  • Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
  • constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter ofthe ⁇ -lactamase gene sequence of pBR322, and the cat promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like.
  • inducible prokaryotic promoters include the major right and left promoters of bacteriophage ⁇ (P and P R ), the trp, recA, ⁇ acZ, ⁇ acl, and gal promoters of E. coli, the ⁇ -amylase (Ulmanen et al, J. Bacteriol. 162:176-182,
  • Proper expression in a prokaryotic cell may also require the presence of a ribosome-binding site upstream ofthe gene sequence-encoding sequence.
  • ribosome-binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404, 1981).
  • the selection of control sequences, expression vectors, transformation methods, and the like, are dependent on the type of host cell used to express the gene.
  • “cell”, “cell line”, and “cell culture” may be used interchangeably and all such designations include progeny.
  • the words “transformants” or “transformed cells” include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that ofthe originally transformed cell.
  • Host cells which may be used in the expression systems ofthe present invention are not strictly limited, provided that they are suitable for use in the expression ofthe protease polypeptide of interest. Suitable hosts may often include eukaryotic cells. Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as EMR 332, which may provide better capacities for correct post-translational processing.
  • eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or
  • plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, and nopaline synthase promoter and polyadenylation signal sequences.
  • Another preferred host is an insect cell, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science 240:1453-1459, 1988).
  • baculovirus vectors can be engineered to express large amounts of proteases ofthe invention in insect cells (Jasny, Science 238:1653, 1987; Miller et al, in: Genetic Engineering, Vol. 8, Plenum, Setlow et al, eds., pp.
  • yeast expression systems Any of a series of yeast expression systems can be utilized which incorporate promoter and termination elements from the actively expressed sequences coding for glycolytic enzymes that are produced in large quantities when yeast are grown in mediums rich in glucose. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. Yeast provides substantial advantages in that it can also carry out post-translational modifications. A number of recombinant DNA strategies exist utilizing strong promoter sequences and high copy number plasmids which can be utilized for production ofthe desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian genes and secretes peptides bearing leader sequences (i.e., pre-peptides). Several possible vector systems are available for the expression of proteases ofthe invention in a mammalian host.
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature ofthe host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, and the like, may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression ofthe gene sequences can be modulated.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.
  • eukaryotic regulatory regions Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include, for example, the promoter ofthe mouse metallothionein I gene sequence (Hamer et al, J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al, Nature (London) 290:304-31, 1981); and the yeast gal4 gene sequence promoter (Johnston et al, Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982; Silver et al, Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).
  • eukaryotic mRNA Translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes a protease ofthe invention (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in the formation of a fusion protein (if the AUG codon is in the same reading frame as the protease ofthe invention coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the protease ofthe invention coding sequence).
  • a nucleic acid molecule encoding a protease ofthe invention and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a nonreplicating DNA or RNA molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the gene may occur through the transient expression ofthe introduced sequence. Alternatively, permanent expression may occur through the integration ofthe introduced DNA sequence into the host chromosome.
  • a vector may be employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
  • cDNA expression vectors incorporating such elements include those described by Okayama (Mol. Cell. Biol. 3 :280-289, 1983).
  • the introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies ofthe vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, ⁇ BR322, Col ⁇ l, pSC 101 , p ACYC 184, ⁇ VX; "Molecular Cloning: A Laboratory Manual", 1989, supra).
  • Bacillus plasmids include pC194, pC221, pT127, and the like (Gryczan, In: The Molecular Biology of the Bacilli. Academic Press, NY, pp. 307-329, 1982).
  • Suitable Streptomyces plasmids include plJlOl (Kendall et al, J. Bacteriol.
  • Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2- micron circle, and the like, or their derivatives.
  • Such plasmids are well known in the art (Botstein et al, Miami Wntr. Symp. 19:265-274, 1982; Broach, In: The Molecular Biology ofthe Yeast Saccharomyces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach, Cell 28:203-204, 1982; T ollon et al., J. Clin. Hematol. Oncol. 10:39-48, 1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608, 1980).
  • the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like.
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression ofthe cloned gene(s) results in the production of a protease ofthe invention, or fragments thereof.
  • the present invention relates to an antibody having binding affinity to a protease ofthe invention.
  • the protease polypeptide may have the amino acid sequence selected from the group consisting of those set forth in SEQ TD NO:36, SEQ TD NO:37, SEQ ID NO:38, SEQ ED NO:39, SEQ ID NO:40, SEQ ED NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ED NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ED NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ TD NO:51, SEQ TD NO:52, SEQ TD NO:53, SEQ TD NO:54, SEQ ID NO:55, SEQ TD NO:56, SEQ ED NO:57, SEQ ID NO:58, SEQ ED NO:59, SEQ ID NO:60, SEQ ED NO:61, SEQ ID NO:62, SEQ
  • the present invention also relates to an antibody having specific binding affinity to a protease ofthe invention.
  • an antibody may be isolated by comparing its binding affinity to a protease ofthe invention with its binding affinity to other polypeptides.
  • Those which bind selectively to a protease ofthe invention would be chosen for use in methods requiring a distinction between a protease ofthe invention and other polypeptides.
  • Such methods could include, but should not be limited to, the analysis of altered protease expression in tissue containing other polypeptides.
  • the proteases ofthe present invention can be used in a variety of procedures and methods, such as for the generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA protein interaction.
  • the proteases ofthe present invention can be used to produce antibodies or hybridomas.
  • One skilled in the art will recognize that if an antibody is desired, such a peptide could be generated as described herein and used as an immunogen.
  • the antibodies ofthe present invention include monoclonal and polyclonal antibodies, as well fragments of these antibodies, and humanized forms. Humanized forms ofthe antibodies ofthe present invention may be generated using one ofthe procedures known in the art such as chimerization or CDR grafting.
  • the present invention also relates to a hybridoma which produces the above- described monoclonal antibody, or binding fragment thereof.
  • a hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
  • the polypeptide may be modified or administered in an adjuvant in order to increase the peptide antigenicity.
  • Methods of increasing the antigenicity of a polypeptide are well known in the art. Such procedures include coupling the antigen with a heterologous protein (such as globulin or ⁇ -galactosidase) or through the inclusion of an adjuvant during immunization.
  • a heterologous protein such as globulin or ⁇ -galactosidase
  • spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Agl4 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
  • myeloma cells such as SP2/0-Agl4 myeloma cells
  • Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELIS A assay, western blot analysis, or radioimmunoassay (L tz et al., Exp. Cell Res. 175:109-124, 1988).
  • Hybridomas secreting the desired antibodies are cloned and the class and subclass are determined using procedures known in the art (Campbell, "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology", supra, 1984).
  • antibody-containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one ofthe above-described procedures.
  • the above-described antibodies may be detectably labeled.
  • Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, and the like), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, and the like) fluorescent labels (such as FITC or rhodamine, and the like), paramagnetic atoms, and the like. Procedures for accomplishing such labeling are well-known in the art, for example, see Stemberger et al, J.
  • the antibodies ofthe present invention may be indirectly labelled by the use of secondary labelled antibodies, such as labelled anti- rabbit antibodies.
  • the labeled antibodies ofthe present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues wliich express a specific peptide.
  • the above-described antibodies may also be immobilized on a solid support.
  • solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al, "Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10, 1986; Jacoby et al, Meth. Enzym. 34, Academic Press, N.Y., 1974).
  • the immobilized antibodies ofthe present invention can be used for in vitro, in vivo, and in situ assays as well as in immunochromotography.
  • Anti-peptide peptides can be generated by replacing the basic amino acid residues found in the peptide sequences ofthe proteases ofthe invention with acidic residues, while maintaining hydrophobic and uncharged polar groups. For example, lysine, arginine, and/or histidine residues are replaced with aspartic acid or glutamic acid and glutamic acid residues are replaced by lysine, arginine or histidine.
  • the present invention also encompasses a method of detecting a protease polypeptide in a sample, comprising: (a) contacting the sample with an above- described antibody, under conditions such that immunocomplexes form, and (b) detecting the presence of said antibody bound to the polypeptide.
  • the methods comprise incubating a test sample with one or more ofthe antibodies ofthe present invention and assaying whether the antibody binds to the test sample. Altered levels of a protease ofthe invention in a sample as compared to normal levels may indicate disease.
  • Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature ofthe antibody used in the assay.
  • immunological assay formats such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion-based Ouchterlony, or rocket immunofluorescent assays
  • Examples of such assays can be found in Chard ("An Introduction to Radioimmunoassav and Related
  • the immunological assay test samples ofthe present invention include cells, protein or membrane extracts of cells, or biological fluids such as blood, serum, plasma, or urine.
  • the test samples used in the above-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can readily be adapted in order to obtain a sample which is testable with the system utilized.
  • kits contains all the necessary reagents to carry out the previously described methods of detection.
  • the kit may comprise: (i) a first container means containing an above-described antibody, and (ii) second container means containing a conjugate comprising a binding partner ofthe antibody and a label.
  • the kit further comprises one or more other containers comprising one or more ofthe following: wash reagents and reagents capable of detecting the presence of bound antibodies.
  • detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.
  • the compartmentalized kit may be as described above for nucleic acid probe kits.
  • the antibodies described in the present invention can readily be incorporated into one ofthe established kit formats which are well known in the art.
  • the present invention also relates to a method of detecting a compound capable of binding to a protease ofthe invention comprising incubating the compound with a protease ofthe invention and detecting the presence ofthe compound bound to the protease.
  • the compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts.
  • the present invention also relates to a method of detecting an agonist or antagonist ofprotease activity or protease binding partner activity comprising incubating cells that produce a protease ofthe invention in the presence of a compound and detecting changes in the level ofprotease activity or protease binding partner activity.
  • the compounds thus identified would produce a change in activity indicative ofthe presence ofthe compound.
  • the compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts. Once the compound is identified it can be isolated using techniques well known in the art.
  • the present invention also encompasses a method modulating protease associated activity in a mammal comprising administering to said mammal an agonist or antagonist to a protease ofthe invention in an amount sufficient to effect said modulation.
  • a method of treating diseases in a mammal with an agonist or antagonist of the activity of one of the proteases of the invention comprising administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize protease-associated functions is also encompassed in the present application.
  • PMSF phenylmethylsulfonyl fluoride
  • DFP diisopropylfluorophosphate
  • protease inhibitors include, but are not limited to, aprotinin, amastatin, antipain, calcineurin autoinhibitory fragment, and histatin 5 (Id.).
  • these inhibitors will have molecular weights from 100 to 200 daltons, from 200 to 300 daltons, from 300 to 400 daltons, from 400 to 600 daltons, from 600 to 1000 daltons, from 1000 to 2000 daltons, from 2000 to 4000 daltons, and from 4000 to 8000 daltons.
  • Compounds that can traverse cell membranes and are resistant to acid hydrolysis are potentially advantageous as therapeutics as they can become highly bioavailable after being administered orally to patients.
  • many of these protease inhibitors only weakly inhibit the function of proteases.
  • many inhibit a variety of proteases and will therefore cause multiple side-effects as therapeutics for diseases.
  • DNA can be injected into the pronucleus of a fertilized egg before fusion ofthe male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g., the nucleus of a two-cell embryo) following the initiation of cell division (Brinster et al, Proc. Nat. Acad. Sci. USA 82:4438-4442, 1985).
  • Embryos can be infected with viruses, especially retroviruses, modified to carry inorganic-ion receptor nucleotide sequences ofthe invention.
  • Pluripotent stem cells derived from the inner cell mass ofthe embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences ofthe invention.
  • a transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapolis, IN), etc.
  • transgenic mouse female mice are induced to superovulate. Females are placed with males, and the mated females are sacrificed by CO 2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts. Surrounding cumulus cells are removed. Pronuclear embryos are then washed and stored until the time of injection. Randomly cycling adult female mice are paired with vasectomized males. Recipient females are mated at the same time as donor females. Embryos then are transferred surgically. The procedure for generating transgenic rats is similar to that of mice (Hammer et al, Cell 63:1099-1112, 1990).
  • a clone containing the sequence(s) ofthe invention is co-transfected with a gene encoding resistance.
  • the gene encoding neomycin resistance is physically linked to the sequence(s) ofthe invention.
  • Transfection and isolation of desired clones are carried out by any one of several methods well known to those of ordinary skill in the art (E.J. Robertson, supra).
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombina-tion (Capecchi, Science 244:1288-1292, 1989).
  • the invention provides transgenic, nonhuman mammals containing a transgene encoding a protease ofthe invention or a gene affecting the expression of the protease.
  • transgenic nonhuman mammals are particularly useful as an in vivo test system for studying the effects of introduction of a protease, or regulating the expression of a protease (i.e., through the introduction of additional genes, antisense nucleic acids, or ribozymes).
  • transgenic animal is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part ofthe genome ofthe animal which develops from that cell.
  • Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats.
  • the transgenic DNA may encode human proteases. Native expression in an animal may be reduced by providing an amount of antisense RNA or DNA effective to reduce expression ofthe receptor.
  • an expression vector containing a protease coding sequence is inserted into cells, the cells are grown in vitro and then infused in large numbers into patients.
  • a DNA segment containing a promoter of choice (for example a strong promoter) is transferred into cells containing an endogenous gene encoding proteases ofthe invention in such a manner that the promoter segment enhances expression ofthe endogenous protease gene (for example, the promoter segment is transferred to the cell such that it becomes directly linked to the endogenous protease gene).
  • the gene therapy may involve the use of an adenovirus containing protease cDNA targeted to a tumor, systemic protease increase by implantation of engineered cells, injection with protease-encoding virus, or injection of naked protease DNA into appropriate tissues.
  • Target cell populations may be modified by introducing altered forms of one or more components ofthe protein complexes in order to modulate the activity of such complexes. For example, by reducing or inhibiting a complex component activity within target cells, an abnormal signal transduction event(s) leading to a condition may be decreased, inhibited, or reversed. Deletion or missense mutants of a component, that retain the ability to interact with other components ofthe protein complexes but cannot function in signal transduction, may be used to inhibit an abnormal, deleterious signal transduction event.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, several RNA viruses, or bovine papilloma virus, may be used for delivery of nucleotide sequences (e.g., cDNA) encod-ing recombinant protease ofthe invention protein into the targeted cell population (e.g., tumor cells).
  • nucleotide sequences e.g., cDNA
  • Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing coding sequences (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989; Ausubel et al, Current Protocols in Molecular Biology.
  • nucleic acid molecules encoding protein sequences can be used as naked DNA or in a reconstituted system e.g., liposomes or other lipid systems for delivery to target cells (e.g., Feigner et al, Nature 337:387-8, 1989).
  • a reconstituted system e.g., liposomes or other lipid systems for delivery to target cells (e.g., Feigner et al, Nature 337:387-8, 1989).
  • gene transfer can be performed by simply injecting minute amounts of DNA into the nucleus of a cell, through a process of microinjection (Capecchi, Cell 22:479-88, 1980). Once recombinant genes are introduced into a cell, they can be recognized by the cell's normal mechanisms for transcription and translation, and a gene product will be expressed. Other methods have also been attempted for introducing DNA into larger numbers of cells. These methods include: transfection, wherein DNA is precipitated with calcium phosphate and taken into cells by pinocytosis (Chen et al, Mol Cell Biol.
  • adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells.
  • the admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression ofthe recombinant gene (Curiel et al, Am. J. Respir. Cell. Mol. Biol, 6:247-52, 1992).
  • Gene transfer means the process of introducing a foreign nucleic acid molecule into a cell. Gene transfer is commonly performed to enable the expression of a particular product encoded by the gene.
  • the product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA.
  • Gene transfer can be performed in cultured cells or by direct administration into animals. Generally gene transfer involves the process of nucleic acid contact with a target cell by non-specific or receptor mediated interactions, uptake of nucleic acid into the cell through the membrane or by endocytosis, and release of nucleic acid into the cyto-plasm from the plasma membrane or endosome. Expression may require, in addition, movement ofthe nucleic acid into the nucleus ofthe cell and binding to appropriate nuclear factors for transcription.
  • gene therapy is a form of gene transfer and is included within the definition of gene transfer as used herein and specifically refers to gene transfer to express a therapeutic product from a cell in vivo or in vitro. Gene transfer can be performed ex vivo on cells which are then transplanted into a patient, or can be performed by direct administration ofthe nucleic acid or nucleic acid-protein complex into the patient.
  • a vector having nucleic acid sequences encoding a protease polypeptide is provided in which the nucleic acid sequence is expressed only in specific tissue. Methods of achieving tissue-specific gene expression are set forth in International Publication No. WO 93/09236, filed November 3, 1992 and published May 13, 1993.
  • the nucleic acid sequence contained in the vector may include additions, deletions or modifications to some or all ofthe sequence ofthe nucleic acid, as defined above.
  • Expression, including over-expression, of a protease polypeptide of the invention can be inhibited by administration of an antisense molecule that binds to and inhibits expression ofthe mRNA encoding the polypeptide.
  • expression can be inhibited in an analogous manner using a ribozyme that cleaves the mRNA.
  • Ribozyme refers to an RNA structure of one or more RNAs having catalytic properties. Ribozymes generally exhibit endonuclease, ligase or polymerase activity. Ribozymes are structural RNA molecules which mediate a number of RNA self-cleavage reactions. Various types of trans-acting ribozymes, including “hammerhead” and “hairpin” types, which have different secondary structures, have been identified. A variety of ribozymes have been characterized.
  • antisense refers of nucleic acid molecules or their derivatives which specifically hybridize, e.g., bind, under cellular conditions, with the genomic DNA and/or cellular mRNA encoding a protease polypeptide of the invention, so as to inhibit expression of that protein, for example, by inhibiting transcription and/or translation.
  • the binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove ofthe double helix.
  • the antisense construct is an nucleic acid which is generated ex vivo and that, when introduced into the cell, can inhibit gene expression by, without limitation, hybridizing with the mRNA and/or genomic sequences of a protease polynucleotide ofthe invention.
  • Antisense approaches can involve the design of oligonucleotides (either
  • DNA or RNA that are complementary to protease polypeptide mRNA and are based on the protease polynucleotides of the invention, including SEQ TD NO:l, SEQ TD NO:2, SEQ ED NO:3, SEQ ED NO:4, SEQ ID NO:5, SEQ TD NO:6, SEQ TD NO:7, SEQ TD NO:8, SEQ TD NO:9, SEQ ID NO:10, SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, SEQ ED NO:14, SEQ ED NO:15, SEQ TD NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ TD NO:19, SEQ TD NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ED NO:23, SEQ ED NO:24, SEQ ED NO:25, SEQ ED NO:26, SEQ ID NO:27, SEQ TD NO:28, SEQ
  • the antisense oligonucleotides will bind to the protease polypeptide mRNA transcripts and prevent translation. Although absolute complementarity is preferred, it is not required.
  • a sequence "complementary" to a portion of an RNA means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid.
  • the longer the hybridizing nucleic acid the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to detennine the melting point ofthe hybridized complex.
  • oligonucleotides that are complementary to the 5' end of the message e.g., the 5' untranslated sequence up to and including the AUG initiation codon
  • sequences complementary to the 3' untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well.
  • Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5', 3' or coding region of the protease polypeptide mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably less than about 100 and more preferably less than about 50 or 30 nucleotides in length. Typically they should be between 10 and 25 nucleotides in length.
  • the antisense sequence is selected from an oligonucleotide sequence that comprises, consists of, or consists essentially of about 10-30, and more preferably 15-25, contiguous nucleotide bases of a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ TD NO:3, SEQ ID NO:4, SEQ TD NO:5, SEQ TD NO:6, SEQ TD NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ TD NO:10, SEQ TD NO:ll, SEQ ED NO:12, SEQ TD NO:13, SEQ TD NO:14, SEQ ED NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ TD NO:18, SEQ TD NO:19, SEQ TD NO:20, SEQ ID NO:21, SEQ TD NO:22, SEQ TD NO:15, SEQ ID NO:16, SEQ ID NO:21, SEQ TD
  • the invention includes an isolated, enriched or purified nucleic acid molecule comprising, consisting of or consisting essentially of about 10-30, and more preferably 15-25 contiguous nucleotide bases of a nucleic acid sequence that encodes a polypeptide that selected from the group consisting of SEQ ID NO:36, SEQ TD NO:37, SEQ TD NO:38, SEQ TD NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ TD NO:43, SEQ TD NO:44, SEQ ED NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ED NO:48, SEQ ID NO:49, SEQ ED NO:50, SEQ ED NO:51, SEQ ED NO:52, SEQ ED NO:53, SEQ ED NO:54, SEQ ID NO:55, SEQ TD NO:56, SEQ TD NO:57, SEQ TD NO:58, SEQEQ ID NO:
  • antisense oligonucleotides can be designed. Such antisense oligonucleotides would be administered to cells expressing the target protease and the levels of the target RNA or protein with that of an internal control RNA or protein would be compared. Results obtained using the antisense oligonucleotide would also be compared with those qbtained using a suitable control oligonucleotide.
  • a preferred control oligonucleotide is an oligonucleotide of approximately the same length as the test oligonucleotide. Those antisense oligonucleotides resulting in a reduction in levels of target RNA or protein would be selected.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability ofthe molecule, hybridization, etc.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl Acad. Sci. U.S.A. 86:6553- 6556; Lemaitre et al.
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization- triggered cleavage agent, etc.
  • the antisense oligonucleotide may comprise at least one modified base moiety wliich is selected from moieties such as 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, and 5- (carboxyhydroxyethyl) uracil.
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof, (see also U.S. Pat. Nos.
  • the antisense oligonucleotide is an ⁇ -anomeric oligonucleotide.
  • An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al. (1987) Nucl. Acids Res. 15:6625-
  • the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al. (1987) Nucl.
  • peptidyl nucleic acids which are polypeptides such as polyserine, polythreonine, etc. including copolymers containing various amino acids, which are substituted at side-chain positions with nucleic acids (T,A,G,C,U).
  • an antisense construct of the present invention can be delivered, for example, as an expression plasmid or vector that, when transcribed in the cell, produces RNA complementary to at least a unique portion of the cellular mRNA which encodes a protease polypeptide of the invention.
  • antisense nucleotides complementary to the protease polypeptide coding region sequence can be used, those complementary to the transcribed untranslated region are most preferred.
  • Gene replacement as used herein means supplying a nucleic acid sequence which is capable of being expressed in vivo in an animal and thereby providing or augmenting the function of an endogenous gene which is missing or defective in the animal.
  • protease polypeptides ofthe invention can be administered to a human patient per se, or in pharmaceutical compositions where it is mixed with other active ingredients, as in combination therapy, or suitable carriers or excipient(s).
  • suitable carriers or excipient(s) suitable carriers or excipient(s).
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • composition/Formulation may be administered in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody.
  • the liposomes will be targeted to and taken up selectively by the tumor.
  • compositions ofthe present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing ofthe active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the agents ofthe invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Suitable carriers include excipients such as, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum fragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents maybe added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g-. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix ofthe compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. Additionally, suspensions ofthe active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility ofthe compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for the hydrophobic compounds ofthe invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water- miscible organic polymer, and an aqueous phase.
  • the cosolvent system may be the VPD co-solvent system.
  • VPD is a solution of 3% w/v benzyl alcohol, 8% w/v ofthe nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art.
  • Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • additional strategies for protein stabilization may be employed.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • protease modulating compounds ofthe invention may be provided as salts with pharmaceutically compatible counterions.
  • Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • compositions suitable for use in the present invention include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival ofthe subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light ofthe detailed disclosure provided herein.
  • the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition ofthe protease activity). Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy ofthe compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% ofthe population) and the ED 50 (the dose therapeutically effective in 50% ofthe population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD 50 and ED 50 .
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view ofthe patient's condition. (See e.g., Fingl et al, 1975, in The Pharmacological Basis of Therapeutic
  • Dosage amount and interval may be adjusted individually to provide plasma levels ofthe active moiety which are sufficient to maintain the protease modulating effects, or minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition ofthe protease using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value.
  • Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% ofthe time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration ofthe drug may not be related to plasma concentration.
  • compositions administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity ofthe affliction, the manner of administration and the judgment ofthe prescribing physician.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency ofthe form ofthe polynucleotide for human or veterinary administration.
  • Such notice for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • compositions comprising a compound ofthe invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
  • a functional derivative is meant a “chemical derivative,” “fragment,” or “variant,” ofthe polypeptide or nucleic acid ofthe invention, which terms are defined below.
  • a functional derivative retains at least a portion ofthe function ofthe protein, for example reactivity with an antibody specific for the protein, enzymatic activity or binding activity mediated through noncatalytic domains, which permits its utility in accordance with the present invention. It is well known in the art that due to the degeneracy ofthe genetic code numerous different nucleic acid sequences can code for the same amino acid sequence. Equally, it is also well known in the art that conservative changes in amino acid can be made to arrive at a protein or polypeptide that retains the functionality ofthe original. In both cases, all permutations are intended to be covered by this disclosure.
  • nucleic acid molecules Included within the scope of this invention are the functional equivalents of the herein-described isolated nucleic acid molecules.
  • the degeneracy ofthe genetic code permits substitution of certain codons by other codons that specify the same amino acid and hence would give rise to the same protein.
  • the nucleic acid sequence can vary substantially since, with the exception of methionine and tryptophan, the known amino acids can be coded for by more than one codon.
  • portions or all ofthe genes ofthe invention could be synthesized to give a nucleic acid sequence significantly different from one selected from the group consisting of those set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ED NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ED NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ TD NO:ll, SEQ TD NO:12, SEQ TD NO:13, SEQ TD NO:14, SEQ ID NO:15, SEQ ED NO:16, SEQ ED NO:17, SEQ ED NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ TD NO:21, SEQ TD NO:22, SEQ TD NO:23, SEQ ED NO:24, SEQ ID NO:25, SEQ TD NO:26, SEQ ID NO:27, SEQ TD NO:28, SEQ TD NO:29, SEQ
  • the nucleic acid sequence may comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5'-end and/or the 3'-end ofthe nucleic acid formula selected from the group consisting of those set forth in SEQ TD NO:l, SEQ TD NO:2, SEQ ED NO:3, SEQ ED NO:4, SEQ ED NO:5, SEQ ED NO:6, SEQ ID NO:7, SEQ ED NO:8, SEQ ED NO:9, SEQ ED NO:10, SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, SEQ ID NO:14, SEQ TD NO:15, SEQ TD NO:16, SEQ TD NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ TD NO:20, SEQ TD NO:21, SEQ TD NO:22, SEQ ED NO:23, SEQ ID NO:24, SEQ ED NO:
  • nucleotide or polynucleotide may be used in this regard, provided that its addition, deletion or substitution does not alter the amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:36, SEQ ID NO:37, SEQ TD NO:38, SEQ TD NO:39, SEQ TD NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ TD NO:43, SEQ ID NO:44, SEQ ED NO:45, SEQ TD NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ED NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ED NO:53, SEQ ID NO:54, SEQ ED NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ED NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ED NO:63, S
  • the present invention is intended to include any nucleic acid sequence resulting from the addition of ATG as an initiation codon at the 5'-end ofthe inventive nucleic acid sequence or its derivative, or from the addition of TTA, TAG or TGA as a termination codon at the 3 '-end ofthe inventive nucleotide sequence or its derivative.
  • the nucleic acid molecule ofthe present invention may, as necessary, have restriction endonuclease recognition sites added to its 5 '-end and/or 3'-end.
  • nucleic acid sequence Such functional alterations of a given nucleic acid sequence afford an opportunity to promote secretion and/or processing of heterologous proteins encoded by foreign nucleic acid sequences fused thereto. All variations ofthe nucleotide sequence ofthe protease genes ofthe invention and fragments thereof permitted by the genetic code are, therefore, included in this invention.
  • the two polypeptides are functionally equivalent, as are the two nucleic acid molecules that give rise to their production, even though the differences between the nucleic acid molecules are not related to the degeneracy ofthe genetic code.
  • a "chemical derivative" ofthe complex contains additional chemical moieties not normally a part ofthe protein. Covalent modifications ofthe protein or peptides are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues ofthe peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues, as described below.
  • Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2- oxa-l,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge ofthe lysinyl residues.
  • Other suitable reagents for derivatizing primary amine containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase- catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because ofthe high pKa ofthe guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine ⁇ -amino group. Tyrosyl residues are well-known targets of modification for introduction of spectral labels by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Carboxyl side groups are selectively modified by reaction with carbodiimide (R'-N-C-N-R') such as l-cyclohexyl-3-(2- morpholinyl(4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
  • carbodiimide R'-N-C-N-R'
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • Derivatization with bifunctional agents is useful, for example, for cross- linking the component peptides ofthe protein to each other or to other proteins in a complex to a water-insoluble support matrix or to other macromolecular carriers.
  • cross-linking agents include, for example, l,l-bis(diazoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-l,8-octane.
  • Derivatizing agents such as methyl-3-[p- azidophenyl) dithiolpropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water- insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • Such derivatized moieties may improve the stability, solubility, absorption, biological half life, and the like.
  • the moieties may alternatively eliminate or attenuate any undesirable side effect ofthe protein complex and the like.
  • Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990).
  • fragment is used to indicate a polypeptide derived from the amino acid sequence ofthe proteins, ofthe complexes having a length less than the full-length polypeptide from which it has been derived.
  • a fragment may, for example, be produced by proteolytic cleavage ofthe full-length protein.
  • the fragment is obtained recombinantly by appropriately modifying the DNA sequence encoding the proteins to delete one or more amino acids at one or more sites ofthe C-terminus, N-terminus, and/or within the native sequence.
  • Fragments of a protein are useful for screening for substances that act to modulate signal transduction, as described herein. It is understood that such fragments may retain one or more characterizing portions ofthe native complex.
  • variant polypeptide which either lacks one or more amino acids or contains additional or substituted amino acids relative to the native polypeptide.
  • the variant may be derived from a naturally occurring complex component by appropriately modifying the protein DNA coding sequence to add, remove, and/or to modify codons for one or more amino acids at one or more sites ofthe C-terminus, N-terminus, and/or within the native sequence.
  • a functional derivative of a protein with deleted, inserted and/or substituted amino acid residues may be prepared using standard techniques well-known to those of ordinary skill in the art.
  • the modified components ofthe functional derivatives may be produced using site-directed mutagenesis techniques (as exemplified by Adelman et al, 1983, DNA 2:183) wherein nucleotides in the DNA coding the sequence are modified such that a modified coding sequence is modified, and thereafter expressing this recombinant DNA in a prokaryotic or eukaryotic host cell, using techniques such as those described above.
  • proteins with amino acid deletions, insertions and/or substitutions may be conveniently prepared by direct chemical synthesis, using methods well-known in the art.
  • the functional derivatives ofthe proteins typically exhibit the same qualitative biological activity as the native proteins.
  • Table 1 documents the name of each gene, the classification of each gene, the positions ofthe open reading frames within the sequence, and the length ofthe corresponding peptide. From left to right the data presented is as follows: “Gene Name”, “ED#na”, “ED#aa”, “FL/Cat”, “Superfamily”, “Group”, “Family”, “NA ength”, “ORF Start”, “ORF End”, “ORF Length”, and “AA ength”.
  • “Gene name” refers to name given the sequence encoding the protease enzyme.
  • Each gene is represented by "SGPr” designation followed by an arbitrary number. The SGPr name usually represents multiple overlapping sequences built into a single contiguous sequence (a “contig”).
  • the "ID#na” and “ID#aa” refer to the identification numbers given each nucleic acid and amino acid sequence in this patent application.
  • FL/Cat refers to the length ofthe gene, with FL indicating full length, and “Cat' indicating that only the catalytic domain is presented.
  • Partial in this column indicates that the sequence encodes a partial catalytic domain.
  • Superfamily identifies whether the gene is a protease.
  • Group and “Family” refer to the protease classification defined by sequence homology.
  • NA_length refers to the length in nucleotides ofthe corresponding nucleic acid sequence.
  • ORF start refers to the beginning nucleotide ofthe open reading frame.
  • ORF end refers to the last nucleotide ofthe open reading frame, including the stop codon.
  • ORF length refers to the length in nucleotides ofthe open reading frame
  • AA length refers to the length in amino acids ofthe peptide encoded in the corresponding nucleic acid sequence.
  • Table 2 lists the following features ofthe genes described in this patent application: chromosomal localization, single nucleotide polymorphisms (SNPs), representation in dbEST, and repeat regions. From left to right the data presented is as follows: “Gene Name”, “ID#na”, “ID#aa”, “FL/Cat”, “Superfamily”, “Group”, “Family”, “Chromosome”, “SNPs”, “dbEST jiits”, & “Repeats”. The contents of the first 7 columns (i.e.,. "Gene Name”, "ED#na”, “ED#aa”, “FL/Cat”, “Superfamily", “Group”, “Family”) are as described above for Table 1.
  • SNPs single nucleotide polymorphisms
  • NCBI http://www.iicbi.nlm.nih.gov/SNP/snpblastBvClir.htmD.
  • dbEST hits lists accession numbers of entries in the public database of ESTs (dbEST, http://www.ncbi.nlm.nih.gov/dbEST/index.html) that contain at least 150 bp of 100% identity to the corresponding gene. These ESTs were identified by blastn of dbEST. "Repeats” contains information about the location of short sequences, approximately 20 bp in length, that are of low complexity and that are present in several distinct genes.
  • Table 3 lists the extent and the boundaries ofthe protease catalytic domains, and other protein domains.
  • the column headings are: “Gene Name”, “ED#na”, “ED#aa”, “FL/Cat”, “Profile_start”, “Profile_end”, “Protease_start”, “Protease_end”, “Profile”, and “Other Domains”.
  • the contents ofthe first 7 columns i.e.,. "Gene Name”, “ED#na”, “ID#aa”, “FL/Cat”, “Superfamily”, “Group”, “Family" are as described above for Table 1.
  • “Profile Start”, “Profile End”, “Protease Start” and “Protease End” refer to data obtained using a Hidden-Markov Model to define catalytic range boundaries. The boundaries ofthe catalytic domain within the overall protein are noted in the “Protease Start” and “Protease End” columns.
  • Profile indicates whether the HMMR search was done with a complete (“Global”) or Smith Waterman ("Local”) model, as described below.
  • Global complete
  • Smith Waterman Long Term Evolution
  • Two hidden Markov models were built. One of them allows for partial matches to the catalytic domain; this is a "local” HMM, similar to Smith- Waterman alignments in sequence matching.
  • the other model allows matches only to the complete catalytic domain; this is a "global” HMM similar to Needleman-Wunsch alignments in sequence matching.
  • the Smith Waterman local model is more specific, allowing for fragmentary matches to the catalytic domain whereas the global "complete” model is more sensitive, allowing for remote homologue identification.
  • the "Other domains” column lists the names and positions of domains within the protein sequence in addition to the protein protease domain. These domains were identified using PFAM (http://pfam.wustl.edu/hmmsearch.shtml) models, a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains. Version 5.5 of Pfam (Sept 2000) contains alignments and models for 2478 protein families (http://pfam.wustl.edu/faq.shtml). The PFAM alignments were downloaded from http://pfam.wustl.edu/hmmsearch.shtml and the HMMr searches were run locally on a Timelogic computer (TimeLogic Corporation, Incline Village, NV).
  • PFAM http://pfam.wustl.edu/hmmsearch.shtml
  • Table 4 describes the results of Smith Waterman similarity searches (Matrix: PamlOO; gap open/extension penalties 12/2) ofthe amino acid sequences against the NCBI database of non-redundant protein sequences (http ://www .ncbi .nlm.nih. gov/Entrez/protein.htmD .
  • the column headings are: "Gene Name”, “ED#na”, “ID#aa”, “FL/Cat”, “Superfamily”, “Group”, “Family”, “Pscore”, “aa ength”, “aa_E _match”, “%Identity”, “%Similar”, “ACC#_nraa_match", and “Description”.
  • the contents ofthe first 7 columns i.e.,.
  • HMMR identified sequences were searched using BLASTN and BLASTX against a nucleotide and amino acid
  • Another method for defining DNA extensions from genomic sequence used iterative searches of genomic databases through the Genscan program to predict exon splicing [Burge and Karlin, JMB (1997) 268(l):78-94)]. These predicted genes were then assessed to see if they represented "real" extensions ofthe partial genes based on homology to related proteases.
  • Genewise requires two inputs, the homologous protein, and genomic DNA containing the gene of interest.
  • the genomic DNA was identified by blastn searches of Celera and Human Genome Project databases.
  • the orthologs were identified by blastp searches ofthe NCBI non-redundant protein database (NRAA).
  • NRAA NCBI non-redundant protein database
  • the sources for the sequence information used to identify genes are listed below.
  • the accession numbers ofthe protein ortholog and the genomic DNA are given. (Genewise uses the ortholog to assemble the coding sequence ofthe target gene from the genomic sequence).
  • the amino acid sequences for the orthologs were obtained from the NCBI non-redundant database of proteins .(ht ⁇ ://www.ncbi.nlm.nih.gov/Entrez/protein.html).
  • the genomic DNA came from two sources: Celera and NCBI-NRNA, as indicated below. cDNA sources are also listed below.
  • Genomic DNA source Celera Assembly 5h contig 90000640151915 Homologs used for Genewise: gi_l 2731929, gb_AAA60062.1 , gi_999902
  • Genomic DNA source Celera Assembly 5h contig 90000642234645
  • Celera_Asm5h where it aligned with contig 90000642234645 (4157978 bp) in the anti-sense orientation. 200 kb ofthe contig was used for genewise/genscan/ sym4 predictions. Genewise was run with human pepsinogen C (gi
  • LGTemplatesMAR2001 AAA41827.1 g206083 pepsinogen 0
  • LGcompseqsMAR2001: 825016H1 4.
  • LGflMAR2001: 7477287CBl 5.
  • LGflMAR2001n g8546678_edit_02 6.
  • LGflMAR2001n 825016Hl_edit_l 7.
  • LGflftAPR2001n 7477287CB1
  • the LGcompseqsMAR2001 EST 7477287CB1 contains an ORF of 1173 bp or 390 aa.
  • NCBI_nonredundant 7477287CB1 shares 62% identity over 372 aa to pepsinogen C o ⁇ Rhinolophus ferrumequinum.
  • 7477287CB1 has 3 conflicts and 4 inserts/deletions.
  • the second conflict occurs at nucleotide 379 of 7477287CB1.
  • nucleotide 379 is a "G” while in the SGPr005 genewise prediction the corresponding nucleotide is a "A”.
  • the nucleotide conflict gives rise to an aa change of D (7477287CB1) to N (SGPr005).
  • SGPr005 genewise sequence Celera_Asm5h contig 90000642234645
  • the third conflict occurs at nucleotide 745 of 7477287CB1.
  • nucleotide 745 is a "G” while in the SGPr005 genewise prediction the corresponding nucleotide is a "T”.
  • the nucleotide conflict gives rise to an aa acid conflict of E (7477287CB1) to STOP (SGPr005).
  • SGPr005 genewise sequence which gives rise to the stop codon.
  • SGPr005 genewise sequence Celera_Asm5h contig 90000642234645 and
  • the first two inserts occurs at nucleotide 214 ofthe SGPr005 genewise predicted sequence and nucleotide 297 of 7477287CB1.
  • 7477287CB1 TTCCTAGTC TCTTTGATACGGGTTCCTCCAATCTGTAGCCTGCCCTC
  • the genomic data from Celera_Asm5h contig 90000642234645 supports the SGPr005 genewise sequence while the HGP_s contig gi
  • the third insert occurs at nucleotide 706 of 7477287CB1.
  • the fourth insert occurs at nucleotide 873 of 7477287CB 1. 7477287CB1:
  • Genomic DNA source Public genomic contig: gi
  • Genomic DNA source Celera Assembly 5h contig 90000636191372 Homologs used for Genewise: gb_AAD31927.1, sp_O43323, ref_NP_031883.1
  • the original HMM hit was blast against Celera_Asm5h where it aligned with contig 90000642045264 (8,329,407bp) in the sense orientation.
  • Nucleotides 14,659 to 111,952 ofthe contig were used for genewise/genscan/sym4 predictions.
  • Genewise was run with human caspase 4 (gi
  • the SGPr009 genewise prediction shares homology (62% identity over 274 aa) with human caspase 4 (gi
  • One EST of importance was LGcompseqsMAR2001 747825 ICBl which overlaps with the SGPr009 genewise prediction and extends the prediction in the 5' direction through the start codon. To correct for sequencing errors in the extended 747825 ICBl sequence, the EST was blastn vs.
  • nucleotide 391 and 393 were changed from A to G based on HGP_s and Celera contigs
  • nucleotide 1041 was changed from A to T based on HGP_s and Celera contigs.
  • Genomic DNA source Celera Assembly 5h contig 90000628729589 Homologs used for Genewise: ref_NP_036246.1, gi_6753280
  • the genomic sequence containing the original HMM hit was blast against Celera_Asm5h where it aligned with contig 90000628729589 (1,488,284 bp) in the anti-sense orientation. 200 kb ofthe contig was used for genewise/genscan sym4 predictions. Genewise was run with human caspase 14 (gi
  • Genomic DNA source Celera Assembly 5h contig 301714258
  • Genomic DNA source Celera Assembly 5h contigs: 9802310, 90000642810957 Homologs used for Genewise: gb_AAF99682.1, gb_AAG22771.1, gi_12722673
  • Genomic DNA source Celera Assembly 5h contig 301714258 Homologs used for Genewise: gb_AAD34600.1, gb_AAD51699.1, gb_AAD56236.1
  • Genomic DNA source Celera Assembly 5h contig 90000627067487
  • Genomic DNA source 90000640081635
  • Genomic DNA source Celera Assembly 5h contig 90000642821147 Homologs used for Genewise: gi_l 079470, ref_NP_055052.1 Notes: Genomic region may be misassembled, predicted protein may have gaps in the middle. Used incyte template 094916.1 to extend genewise prediction
  • SGPr352 SEQ ID NOS:15, 50 Genomic DNA source: Celera Assembly 5h contig 90000628457498
  • Genomic DNA source Celera Assembly 5h contig 90000626814267 Homologs used for Genewise: ref_NP_055087.1, gb_AAG35563.1
  • Clones were expressed in urinary tract (9), respiratory system (3), female genitalia (2), nervous system (2) and connective, exocrine, digestive and musculoskeletal systems (one each)
  • Genomic DNA source Celera Assembly 5h contig 90000641115460
  • Genomic DNA source Celera Assembly 5h contig 92000004436076 Homologs used for Genewise: ref_NP_055087.1, gb_AAG35563.1 Also used Incyte sequences 207915.2 , 207915.5, 207915.11 , 207915.4, 7478405CB1, 9123702. Resolved differences between genomic and EST sequence by blasting against Celera raw reads, public and Incyte ESTs and HGP genomic contigs.
  • Genomic DNA source Celera Assembly 5h contig 90000642001297 Homologs used for Genewise: gb_AAG35563.1, ref_NM_022122.1, ref_NP_l 12217.1
  • 6837795 were used to extend and verify the genewise prediction.
  • Genomic DNA source Celera Assembly 5h contig 90000624770881
  • Incyte sequence 7477386CB1, 1719204CB1 also used. Sequence from 3062-3172 in the mRNA is missing in incyte sequence 7477386CB 1 , leading to the replacement ofthe peptide "GNHQNSTVRADVWELGTPEGQWVPQSEPLHPiNKISST" with "A" in the predicted protein.
  • 7477386CB1 there are two 3nt inserts at splice sites, and a 5 nt insert followed shortly by a 1 nt insert, none of which are found in any genomic sequences, and so may be the result of atypical splicing.
  • Incyte template 196583.5 uses an alternative splice acceptor site in one intron, inserting the sequence "CTCCCCATCTCCCCTCAG" at position 2420 ofthe mRNA and inserting the sequence PISPQA into the protein.
  • Genomic DNA source Celera Assembly 5h contig 90000637859600
  • Genomic DNA source Celera Assembly 5h contig 90000642194924 Homologs used for Genewise: dbj_BAA92550.1, ref_NP_064634.1 Public sequence gi
  • Genomic DNA source Celera Assembly 5h contig 92000004763172 Homologs used for Genewise: emb_AL523577.1
  • nnn in NA sequence and X in peptide sequence represents a probable missing exon; the gene may also be incomplete at either end. Based on searches of all human DNA databases, this gene is likely to be a fragment ofthe ortholog ofthe rat gene used as genewise homolog.
  • Genomic DNA source Celera Assembly 5h contig 90000641768196 Homologs used for Genewise: gb _AAH02631.1, ref_NP_077278.1, gb_AAC21447.1
  • the following polymorphisms are seen: C->T at 572, T->A at 591, T->A at 593, C- >T at 981, deletion of A at 1720.
  • the first is seen in some ESTs and public genomic sources and changes an A to a V in the protein; the second and third are seen in ESTs and a single public genomic sequence, and change a V to an E in the protein.
  • the third is seen only in ESTs and is a synonymous substitution.
  • the fourth is seen in ESTs and public genomic data and is in the 3' UTR ofthe gene.
  • a 3nt deletion at 701-703 is seen in Incyte template 1510368.1, resulting in deletion ofthe D at position 558 ofthe peptide.
  • Genomic DNA source Celera Assembly 5h contig 113000081526387 and public genomic contig gi
  • 9368836 encodes an alternative splice form, missing one exon, and with another exon extended. It predicts a truncated protein product, with AA 1-230 ofthe main form, followed by EPGVG*.
  • a splice variant is created by use of an alternative splice acceptor that eliminates from 1526-1558 in ESTs such as Incyte cDNA 1868183CA2, resulting in the removal ofthe peptide LEFERWLNATG from the protein.
  • An intron within the final exon is seen in Incyte template 1398043.12, which eliminates sequence from 2027- 2079 in the mRNA, a region within the 3' UTR.
  • Genomic DNA source Celera Assembly 5h contig 90000625988051
  • Genomic DNA source Public genomic contigs: gi_9798229, gi_9798027
  • Genomic DNA source Celera Assembly 5h contig 90000625988051 Homologs used for Genewise: dbj_BAB22991.1, ref_NP_060705.1, gi_7023108 Notes: Alternative splice forms are predicted by Incyte EST template 997089.29, public sequence gi_10440455, and Sugen-built clusters of public ESTs: cluster2209_- 1 ljticbi, cluster2209_- 15_ncbi and cluster2209_- 14_ncbi. All result in truncated proteins with short unique C-termini.
  • SGPrl40, SEQ ID NO:l, SEQ ID NO:36 is 1140 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1140, giving an ORF length of 1140 nucleotides.
  • the predicted protein is 379 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Aspartyl, PepsinAI. This gene maps to chromosomal position Ipl3-p33.
  • This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:A969042, AA411567.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 295 tgggtgccctctgtctactgc 315.
  • SGPrl97, SEQ ID NO:2, SEQ ID NO:37 is 1500 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1500, giving an ORF length of 1500 nucleotides.
  • the predicted protein is 499 amino acids long. This sequence codes for a full length protein.
  • SGPr005, SEQ ID NO:3, SEQ ID NO:38 is 1173 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1173, giving an ORF length of 1173 nucleotides.
  • the predicted protein is 390 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Aspartyl, PepsinAI. This gene maps to chromosomal position lp33. This sequence is represented in the database of public ESTs (dbEST) by the following ESTs: none.
  • SGPr078, SEQ ID NO:4, SEQ ID NO:39 is 1239 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1239, giving an ORF length of 1239 nucleotides.
  • the predicted protein is 412 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Aspartyl, PepsinAI. This gene maps to chromosomal position 1 lpl5.
  • This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:BG260401, BF025894, BF793219.
  • SGPr084, SEQ ID NO:5, SEQ ID NO:40 is 1191 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1191, giving an ORF length of 1191 nucleotides.
  • the predicted protein is 396 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Cysteine, HH. This gene maps to chromosomal position 12ql 1.
  • SGPr009, SEQ ID NO:6, SEQ ID NO:41 is 1137 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1137, giving an ORF length of 1137 nucleotides.
  • the predicted protein is 378 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family):
  • Protease, Cysteine, ICEplO This gene maps to chromosomal position 1 lq22
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 900 cttcattgctttcaaatcttcc 921; 77 ttgatgatttgatggaaaat 96.
  • SGPr286, SEQ ID NO:7, SEQ ID NO:42 is 705 nucleotides long. THe open reading frame starts at position 1 and ends at position 705, giving an ORF length of 705 nucleotides.
  • the predicted protein is 234 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease,
  • Cysteine ICEp20. This gene maps to chromosomal position na.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 574 ctggagctgctgactgagg 592; 388 gtggggcccacagctctcc 406.
  • SGPr008, SEQ ID NO:8, SEQ ID NO:43 is 2010 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2010, giving an ORF length of 2010 nucleotides.
  • the predicted protein is 669 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Cysteine, PepC2.
  • This gene maps to chromosomal position 2p23
  • This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:BE075751.
  • SGPrl98, SEQ ID NO:9, SEQ ID NO:44 is 2112 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2112, giving an ORF length of 2112 nucleotides.
  • the predicted protein is 703 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Cysteine, PepC2. This gene maps to chromosomal position lq42.11.This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:BE047777, AW339160.
  • SGPr210, SEQ JX> NO:10, SEQ ID NO:45 is 2127 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2127, giving an ORF length of
  • the predicted protein is 708 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Cysteine, PepC2. This gene maps to chromosomal position 19ql3.2.
  • This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:BE872274.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 1180 gaggaggatgacgaggatgagg 1201.
  • SGPr290, SEQ ID NO:l 1, SEQ ID NO:46 is 2136 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2136, giving an ORF length of 2136 nucleotides.
  • the predicted protein is 711 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Cysteine, PepC2. This gene maps to chromosomal position 2p23.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 1835 agcagctgcacgctgccatg 1854.
  • SGPrl 16 SEQ ID NO:12, SEQ ID NO:47 is 2109 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2109, giving an ORF length of 2109 nucleotides.
  • the predicted protein is 702 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Cysteine, PepC2. This gene maps to chromosomal position 6pl2.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 1003 ctggagatctgcaacctcac 1022.
  • SGPr003, SEQ ID NO:13, SEQ ID NO:48 is 1542 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1542, giving an ORF length of
  • the predicted protein is 513 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Cysteine, PepC2. This gene maps to chromosomal position 2q37. This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:AL526645, BG475966, AL529373. The nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 1520 gctgctgcaggagccgctgctg 1541.
  • SGPr016, SEQ ID NO: 14, SEQ ID NO:49 is 846 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 846, giving an ORF length of 846 nucleotides.
  • the predicted protein is 281 amino acids long.
  • This sequence codes for a partial protein.lt is classified as (superfamily/group/family): Protease, Metalloprotease, ADAM.
  • This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:AW589885, AI024863.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 710 ttaaatatatttcttctcataa 731.
  • SGPr352 SEQ ID NO:15, SEQ ID NO:50 is 3312 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 3312, giving an ORF length of 3312 nucleotides.
  • the predicted protein is 1103 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, ADAM. This gene maps to chromosomal position 19pl3.3. This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:AW027573, AI131032, AI193804.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 1335 agactcgggcctggggctct 1354.
  • SGPr050, SEQ ID NO: 16, SEQ ID NO:51 is 3675 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 3675, giving an ORF length of
  • the predicted protein is 1224 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family):
  • Protease, Metalloprotease, ADAM This gene maps to chromosomal position 5ql5.3.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 2067 tttcttcttttcrttgtcaa 2086; 2061 atttgatttcttcttttctt 2080.
  • SGPr282 SEQ ID NO:17, SEQ ID NO:52 is 2196 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2196, giving an ORF length of 2196 nucleotides.
  • the predicted protein is 731 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, ADAM. This gene maps to chromosomal position 16pl2.3.
  • SGPr046, SEQ ID NO: 18, SEQ ID NO:53 is 2805 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2805, giving an ORF length of 2805 nucleotides.
  • the predicted protein is 934 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, ADAM. This gene maps to chromosomal position 16q23The nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 2353 gtgaggaagagggagatgaagt 2374.
  • SGPr060, SEQ ID NO:19, SEQ ID NO:54 is 4287 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 4287, giving an ORF length of 4287 nucleotides.
  • the predicted protein is 1428 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, ADAM. This gene maps to chromosomal position 15q26. This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:AW575922, AW341169.
  • SGPr068, SEQ ID NO:20, SEQ ID NO:55 is 3561 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 3561, giving an ORF length of 3561 nucleotides.
  • the predicted protein is 1186 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, ADAM. This gene maps to chromosomal position 10q22.This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:AJ403134, .
  • SGPr096, SEQ ID NO:21, SEQ ID NO:56 is 5808 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 5808, giving an ORF length of 5808 nucleotides.
  • the predicted protein is 1935 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, ADAM. This gene maps to chromosomal position 3pl4. This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:BE164543, AW995949, BF842288.
  • SGPrl 19 SEQ ID NO:22, SEQ ID NO:57 is 4518 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 4518, giving an ORF length of 4518 nucleotides.
  • the predicted protein is 1505 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, ADAM.
  • This gene maps to chromosomal position 12ql l-ql2. This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:AU132053, .
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 1257 taaagaaatgaaagttacaaa 1277.
  • SGPrl43, SEQ ID NO:23, SEQ ID NO:58 is 2649 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2649, giving an ORF length of 2649 nucleotides.
  • the predicted protein is 882 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, ADAM. This gene maps to chromosomal position 20pl3.
  • This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:AA442551.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 2212 tgccactgtgctccaggctg 2231.
  • This protein is predicted to have a transmembrane helix between amino acids 78 and 100.
  • TSHMM Hidden Markov Model based transmenbrane prediction program, Sonnhammer, et al Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 AAAI Press, 1998.
  • SGPrl 64, SEQ ID NO:24, SEQ ID NO:59 is 2937 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2937, giving an ORF length of
  • SGPr281, SEQ ID NO:25, SEQ ID NO:60 is 3285 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 3285, giving an ORF length of 3285 nucleotides.
  • the predicted protein is 1094 amino acids long. This sequence codes for a nearly full length protein, with just the amino terminus missing.lt is classified as (superfamily/group/family): Protease, Metalloprotease, ADAM. This gene maps to chromosomal position 5q31.
  • SGPr075, SEQ ID NO:26, SEQ ID NO:61 is 375 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 375, giving an ORF length of 375 nucleotides.
  • the predicted protein is 125 amino acids long. This sequence codes for a partial protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, ADAM. This gene maps to chromosomal position na.
  • SGPr292 SEQ ID NO:27, SEQ ID NO:62 is 1710 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1710, giving an ORF length of 1710 nucleotides.
  • the predicted protein is 569 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, PepMlO. This gene maps to chromosomal position
  • the sequence is represented in the database of public ESTs (dbEST) by the following ESTs:AW665196.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 52 gctccctggcccacccagcc 71; 959 aagcaattcaaagctgtatg 979.
  • SGPr069, SEQ ID NO:28, SEQ ID NO:63 is 2232 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2232, giving an ORF length of 2232 nucleotides.
  • the predicted protein is 743 amino acids long. This sequence codes for a full length protein.
  • SGPr049, SEQ ID NO:30, SEQ ID NO:65 is 2973 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2973, giving an ORF length of 2973 nucleotides.
  • the predicted protein is 990 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, PepMl. This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:AI222989.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 2269 aatttaatatggaatatttat 2289.
  • SGPr026, SEQ ID NO:31, SEQ ID NO:66 is 1953 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1953, giving an ORF length of 1953 nucleotides.
  • the predicted protein is 650 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, PepMl..
  • SGPr203, SEQ ID NO:32, SEQ ID NO:67 is 2175 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 2175, giving an ORF length of 2175 nucleotides.
  • the predicted protein is 724 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, PepMl. This gene maps to chromosomal position 2q37.
  • This sequence is represented in the database of public ESTs (dbEST) by the following ESTs: AU132908, BE735172, BE563549 (many).
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 83 tggacgtggcctcggcctcca 103.
  • SGPrl 57, SEQ ID NO:33, SEQ ID NO:68 is 1524 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1524, giving an ORF length of 1524 nucleotides.
  • the predicted protein is 507 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, PepM20. This gene maps to chromosomal position 18q22.3. This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:BE386438, BE386547, BF920454 (many).
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 614 ccctggaggaacttgtggaa 633; 561 tcctgtgaatatcaaattca 580.
  • SGPrl54, SEQ ID NO:34, SEQ ID NO:69 is 1422 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1422, giving an ORF length of 1422 nucleotides.
  • the predicted protein is 473 amino acids long. This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, PepM20.
  • the nucleic acid contains short repetitive sequence (the position and sequence ofthe repeat): 806 tccttgcagctgctgtcagc 825.
  • SGPr088, SEQ ID NO:35, SEQ ID NO:70 is 1428 nucleotides long.
  • the open reading frame starts at position 1 and ends at position 1428, giving an ORF length of 1428 nucleotides.
  • the predicted protein is 475 amino acids long.
  • This sequence codes for a full length protein. It is classified as (superfamily/group/family): Protease, Metalloprotease, PepM20. This gene maps to chromosomal position 18q23. This sequence is represented in the database of public ESTs (dbEST) by the following ESTs:AL541127, AL542184, AL529661 (many).
  • EXAMPLE 2 Expression Analysis of Mammalian Proteases Materials and Methods Quantitative PCR Analysis
  • RNA is isolated from a variety of normal human tissues and cell lines. Single stranded cDNA is synthesized from 10 ⁇ g of each RNA as described above using the Superscript Preamplification System (GibcoBRL). These single strand templates are then linearly amplified with a pair of specific primers in a real time PCR reaction on a Light Cycler (Roche Molecular Biochemical). Graphical readout can provide quantitative analysis ofthe relative abundance ofthe targeted gene in the total RNA preparation.
  • DNA-free RNA is isolated from a variety of normal human tissues, cryostat sections, and cell lines.
  • Single stranded cDNA is synthesized from lOug RNA or lug mRNA using a modification ofthe SMART PCR cDNA synthesis technique (Clontech). The procedure can be modified to allow asymmetric labeling ofthe 5' and 3' ends of each transcript with a unique oligonucleotide sequence.
  • the resulting sscDNAs are then linearly amplified using Advantage long-range PCR (Clontech) on a Light Cycler PCR machine. Reactions are halted when the graphical real-time display demonstrates the products have begun to plateau.
  • the double stranded cDNA products are purified using Millipore DNA purification matrix, dried, resuspended, quantified, and analyzed on an agarose gel. The resulting elements are referred to as "tissue cDNAs".
  • Tissue cDNAs are spotted onto GAPS coated glass slides (Corning) using a Genetic Microsystems (GMS) arrayer at 500 ng/ul.
  • GAPS coated glass slides Corning
  • GMS Genetic Microsystems
  • Fluorescent labeled oligonucleotides are synthesized to each novel exon, ensuring they contained internal mismatches with the closest known homologue.
  • oligos are 45 nucleotides long, labeled on the 5' end with Cy5.
  • Exon-specific Cy5-labeled oligos are hybridized to the tissue cDNAs arrayed onto glass slides, and washed using standard buffers and conditions. Hybridizing signals are then quantified using a GMS Scanner.
  • tissue cDNAs are manually spotted onto Nylon membranes using a 384 pin replicator, and hybridized to 32 P-end labeled oligo probes.
  • Tissue cDNAs are generated from multiple RNA templates selected to provide information of relevance to the disease areas of interest and to reflect the biological mechanism of action for each protease.
  • These templates include: human tumor cell lines, cryostat sections of primary human tumors and 32 normal human tissues to identify cancer-related genes; sections of normal, Alzheimer's, Parkinson's, and Schizophrenia brain regions for CNS-related genes; nonnal and diabetic or obese skeletal muscle, adipose, or liver for metabolic-related genes; and purified hematopoeitic cells, and lymphoid tissues for immune-related genes.
  • tissue cDNAs are generated to reflect angiogenesis (cultured endothelial cells treated with VEGF ligand, anti-angiogenic drugs, or hypoxia), motility (A549 cells stimulated with HGF ligand, orthotopic metastases, primary tumors with matched metastatic tumors), cell cycle (Hela, HI 299, and other cell lines synchronized by drug block and harvested at various times in the cell cycle), checkpoint integrity and DNA repair (p53 normal or defective cells treated with ⁇ -radiation, UV, cis-platinum, or oxidative stress), and cell survival (cells induced to differentiate or at various stages of apoptosis).
  • angiogenesis cultured endothelial cells treated with VEGF ligand, anti-angiogenic drugs, or hypoxia
  • motility A549 cells stimulated with HGF ligand, orthotopic metastases, primary tumors with matched metastatic tumors
  • cell cycle Hela, HI 299, and other cell lines synchronized by drug block and harvested at
  • SGPrl40, SEQ ID NO:l, SEQ ID NO:36 encodes a protein that is 379 amino acids long. It is classified as an Aspartylprotease, ofthe PepsinAI family.
  • the protease domain in this protein matches the hidden Markov profile for a Eukaryotic aspartyl protease, from amino acid 65 to amino acid 378.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 356.
  • Other domains identified within this protein are: none.
  • SGPrl 97 SEQ ID NO:2, SEQ ID NO:37 encodes a protein that is 499 amino acids long. It is classified as an Aspartylprotease, ofthe PepsinAI family.
  • the protease domain in this protein matches the hidden Markov profile for a Ubiquitin carboxyl- terminal hydrolases family 2, from amino acid 199 to amino acid 230.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 32.
  • Other domains identified within this protein are: Zn-finger in ubiquitin-hydrolases (amino acid 26 to amino acid 96)
  • P_Score 5.6e-025.
  • protease domain in this protein matches the hidden Markov profile for a Eukaryotic aspartyl protease, from amino acid 65 to amino acid 389.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 356.
  • Other domains identified within this protein are: none.
  • SGPr078, SEQ ID NO:4, SEQ ID NO:39 encodes a protein that is 412 amino acids long. It is classified as an Aspartylprotease, of the PepsinAI family.
  • the protease domain in this protein matches the hidden Markov profile for a Eukaryotic aspartyl protease, from amino acid 70 to amino acid 409.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 356.
  • Other domains identified within this protein are: none.
  • SGPr084, SEQ ID NO:5, SEQ ID NO:40 encodes a protein that is 396 amino acids long. It is classified as a Cysteineprotease, ofthe HH family.
  • the protease domain in this protein matches the hidden Markov profile for a Hedgehog amino-terminal signaling domain, from amino acid 23 to amino acid 185.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 163.
  • SGPr009 SEQ ID NO:6, SEQ ID NO:41 encodes a protein that is 378 amino acids long. It is classified as a Cysteineprotease, ofthe ICEplO family. The protease domain in this protein matches the hidden Markov profile for a ICE-Iike protease
  • caspase p20 domain, from amino acid 131 to amino acid 264.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 141.
  • Other domains identified within this protein are: ICE-Iike protease (caspase) plO domain, amino acids 291-376; profile from 1-95: Caspase recruitment domain from amino acids 2-91.
  • SGPr286, SEQ ID NO:7, SEQ ID NO:42 encodes a protein that is 234 amino acids long. It is classified as a Cysteineprotease, ofthe ICEp20 family.
  • the protease domain in this protein matches the hidden Markov profile for a ICE-Iike protease (caspase) p20 domain, from amino acid 19 to amino acid 58.
  • the positions within the HMMR profile that match the protein sequence are from profile position 22 to profile position 61.
  • Other domains identified within this protein are: ICE-Iike protease (caspase) plO domain, amino acids 144-202; profile from 1-61. .
  • SGPrOO ⁇ SEQ ID NO:8, SEQ ID NO:43 encodes a protein that is 669 amino acids long. It is classified as a Cysteineprotease, ofthe PepC2 family.
  • the protease domain in this protein matches the hidden Markov profile for a Calpain family cysteine protease; Peptidase_C2, from amino acid 35 to amino acid 333.
  • the positions within the HMMR profile that match the protein sequence are from profile position 2 to profile position 344.
  • SGPrl98, SEQ TD NO:9, SEQ ID NO:44 encodes a protein that is 703 amino acids long. It is classified as a Cysteineprotease, ofthe PepC2 family.
  • the protease domain in this protein matches the hidden Markov profile for a Calpain family cysteine protease; Peptidase_C2, from amino acid 45 to amino acid 344.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 344.
  • Other domains identified within this protein are: Calpain large subunit, domain III, amino acids 355-512, profile from 1-163.
  • SGPr210, SEQ ID NO:10, SEQ ID NO:45 encodes a protein that is 708 amino acids long. It is classified as a Cysteineprotease, ofthe PepC2 family.
  • the protease domain in this protein matches the hidden Markov profile for a Calpain family cysteine protease; Peptidase_C2, from amino acid 45 to amino acid 341.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 344.
  • Other domains identified within this protein are: Calpain large subunit, domain III, amino acids 353-499, profile from 1-163.
  • SGPr290 SEQ ID NO: 11, SEQ ID NO:46 encodes a protein that is 711 amino acids long. It is classified as a Cysteineprotease, ofthe PepC2 family.
  • the protease domain in this protein matches the hidden Markov profile for a Calpain family cysteine protease; Peptidase_C2, from amino acid 43 to amino acid 346.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 344.
  • Other domains identified within this protein are: Calpain large subunit, domain III, amino acids 347-490, profile from 1-163.
  • two EF hand motifs at amino acids 561-593 and 595-622; EF hands match from 1- 26 of profile. .
  • SGPrl 16 encodes a protein that is 702 amino acids long. It is classified as a Cysteineprotease, ofthe PepC2 family.
  • the protease domain in this protein matches the hidden Markov profile for a Calpain family cysteine protease; Peptidase_C2, from amino acid 42 to amino acid 341.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 344.
  • Other domains identified within this protein are: Calpain large subunit, domain III, amino acids 352-510, profile from 1-163.
  • two EF hand motifs at amino acids 577-605 and 607-635; EF hands match from 1- 26 of profile. .
  • SGPr003, SEQ ID NO: 13, SEQ ID NO:48 encodes a protein that is 513 amino acids long. It is classified as a Cysteineprotease, ofthe PepC2 family.
  • the protease domain in this protein matches the hidden Markov profile for a Calpain family cysteine protease; Peptidase_C2, from amino acid 13 to amino acid 322.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 344.
  • Other domains identified within this protein are: Calpain large subunit, domain III, amino acids 338-494, profile from 3-163..
  • SGPr016, SEQ ID NO: 14, SEQ ID NO:49 encodes a protein that is 281 amino acids long. It is classified as a Metalloprotease, ofthe ADAM family.
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin family propeptide, Pep_M12B_propep, from amino acid 58 to amino acid 175.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 119.
  • Other domains identified within this protein are: none.
  • SGPr352 encodes a protein that is 1103 amino acids long. It is classified as a Metalloprotease, ofthe ADAM family.
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin (M12B) family zinc metalloprotease, from amino acid 239 to amino acid 457.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 203.
  • Other domains identified within this protein are: Reprolysin family propeptide, from amino acids 90-201, matching profile from 1- 119. Also five Thrombospondin type 1 domains from 551-601, 829-884, 888-944, 946-1002, 1007-1057.
  • SGPr050, SEQ ID NO: 16, SEQ ID NO:51 encodes a protein that is 1224 amino acids long. It is classified as a Metalloprotease, ofthe ADAM family.
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin (M12B) family zinc metalloprotease, from amino acid 292 to amino acid 495.
  • the positions within the HMMR profile that match the protein sequence are from profile position 3 to profile position 203.
  • Other domains identified within this protein are: Reprolysin family propeptide from 111-235, matching profile from 1-119. Also has five Thrombospondin type 1 domains from 590-640, 930-986, 990-1047, 1055- 1101, 1128-1180..
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin family propeptide, Pep_M12B_propep, from amino acid 75 to amino acid 190.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 119.
  • Other domains identified within this protein are: Disintegrin domain at amino acids 415-487; matches profile from 4-86. Also EGF- like domain at amino acids 633-661..
  • SGPr046, SEQ ID NO: 18, SEQ ID NO:53 encodes a protein that is 934 amino acids long. It is classified as a Metalloprotease, ofthe ADAM family.
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin (M12B) family zinc metalloprotease, from amino acid 1 to amino acid 194.
  • the positions within the HMMR profile that match the protein sequence are from profile position 13 to profile position 203.
  • Other domains identified within this protein are: Six Thrombospondin type 1 domains at 289-339, 569-627, 634-687, 689-736, 769-828, 844-890..
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin (M12B) family zinc metalloprotease, from amino acid 639 to amino acid 860.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 203.
  • Other domains identified within this protein are: Reprolysin family propeptide, Pep_M12B_propep from amino acids 502-615. Matches profile from 1-119. Also has one thrombospondin type 1 domain from 954- 1004, matching profile from 1 -54..
  • SGPr068, SEQ ID NO:20, SEQ ID NO:55 encodes a protein that is 1186 amino acids long. It is classified as a Metalloprotease, ofthe ADAM family.
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin (M12B) family zinc metalloprotease, from amino acid 261 to amino acid 460.
  • the positions within the HMMR profile that match the protein sequence are from profile position
  • SGPr096, SEQ ID NO:21, SEQ ID NO:56 encodes a protein that is 1935 amino acids long. It is classified as a Metalloprotease, ofthe ADAM family.
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin (M12B) family zinc metalloprotease, from amino acid 293 to amino acid 499.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 203.
  • Other domains identified within this protein are:
  • Reprolysin family propeptide Pep_M12B_propep from amino acids 112-242, matching profile from 1-119. Also has 13 thrombospondin type 1 domains between 589 - 1733..
  • PAMIOO Smith Waterman search
  • NRAA public database of amino acid sequences
  • SGPrll9, SEQ ID NO:22, SEQ ID NO:57 encodes a protein that is 1505 amino acids long. It is classified as a Metalloprotease, ofthe ADAM family.
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin (M12B) family zinc metalloprotease, from amino acid 259 to amino acid 467.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 203.
  • Other domains identified within this protein are: Reprolysin family propeptide, Pep_M12B_propep from amino acids 92-215, matching profile from 1-119. Also has eight thrombospondin type 1 domains between 561 - 1416..
  • SGPrl43, SEQ TD NO:23, SEQ ID NO:58 encodes a protein that is 882 amino acids long. It is classified as a Metalloprotease, ofthe ADAM family.
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin (M12B) family zinc metalloprotease, from amino acid 275 to amino acid 478.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 203.
  • Other domains identified within this protein are: Reprolysin family propeptide, Pep_M12B_propep from amino acids 145-263, matching profile from 1-119. Also has Disintegrin motif 495-570..
  • SGPrl 64, SEQ ID NO:24, SEQ ID NO:59 encodes a protein that is 978 amino acids long. It is classified as a Metalloprotease, ofthe ADAM family.
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin (M12B) family zinc metalloprotease, from amino acid 243 to amino acid 452.
  • the positions within the HMMR profile that match the protein sequence are from profile position
  • Reprolysin family propeptide Pep_M12B_propep from amino acids 92-206, matching profile from 1-119. Also has three Thrombospondin type 1' domains from amino acids 545 to 978. Also has Glucose-6-phosphate dehydrogenase motif at 855- 878..
  • SGPr281, SEQ ID NO:25, SEQ ID NO:60 encodes a protein that is 1094 amino acids long. It is classified as a Metalloprotease, ofthe ADAM family.
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin (M12B) family zinc metalloprotease, from amino acid 317 to amino acid 432.
  • the positions within the HMMR profile that match the protein sequence are from profile position 89 to profile position 203.
  • Other domains identified within this protein are: Six Thrombospondin type 1 domains from amino acid 346 to 1030..
  • SGPr075, SEQ ID NO:26, SEQ ID NO:61 encodes a protein that is 125 amino acids long. It is classified as a Metalloprotease, ofthe ADAM family.
  • the protease domain in this protein matches the hidden Markov profile for a Reprolysin (M12B) family zinc metalloprotease, from amino acid 1 to amino acid 123.
  • the positions within the HMMR profile that match the protein sequence are from profile position 14 to profile position 203.
  • Other domains identified within this protein are: none.
  • SGPr292 SEQ ID NO:27, SEQ ID NO:62 encodes a protein that is 569 amino acids long. It is classified as a Metalloprotease, ofthe PepMl 0 family.
  • the protease domain in this protein matches the hidden Markov profile for a Peptidase_Ml 0, Matrixin, from amino acid 56 to amino acid 267.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 171.
  • Other domains identified within this protein are: Also has four Hemopexin domains at amino acids 333-391, 394-449, 451-499, 506-549..
  • SGPr069, SEQ ID NO:28, SEQ ID NO:63 encodes a protein that is 743 amino acids long. It is classified as a Metalloprotease, ofthe PepM13 family.
  • the protease domain in this protein matches the hidden Markov profile for a Peptidase family M13, from amino acid 535 to amino acid 742.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 225.
  • Other domains identified within this protein are: None.
  • SGPr212, SEQ ID NO:29, SEQ ID NO:64 encodes a protein that is 909 amino acids long. It is classified as a Metalloprotease, ofthe PepMl family.
  • the protease domain in this protein matches the hidden Markov profile for a Peptidase family Ml, from amino acid 275 to amino acid 306.
  • the positions within the HMMR profile that match the protein sequence are from profile position 343 to profile position 374.
  • Other domains identified within this protein are: None.
  • SGPr049, SEQ ID NO:30, SEQ ID NO:65 encodes a protein that is 990 amino acids long. It is classified as a Metalloprotease, ofthe PepMl family.
  • the protease domain in this protein matches the hidden Markov profile for a Peptidase family Ml, from amino acid 98 to amino acid 506.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 441. Other domains identified within this protein are: None.
  • SGPr026, SEQ ID NO:31, SEQ ID NO:66 encodes a protein that is 650 amino acids long. It is classified as a Metalloprotease, ofthe PepMl family. The protease domain in this protein matches the hidden Markov profile for a Peptidase family
  • SGPr203, SEQ ID NO:32, SEQ ID NO:67 encodes a protein that is 724 amino acids long. It is classified as a Metalloprotease, ofthe PepMl family.
  • the protease domain in this protein matches the hidden Markov profile for a Peptidase family Ml, from amino acid 194 to amino acid 444.
  • the positions within the HMMR profile that match the protein sequence are from profile position 161 to profile position 441.
  • Other domains identified within this protein are: None.
  • SGPrl 57, SEQ ID NO:33, SEQ ID NO:68 encodes a protein that is 507 amino acids long. It is classified as a Metalloprotease, ofthe PepM20 family.
  • the protease domain in this protein matches the hidden Markov profile for a Peptidase_M20 , from amino acid 106 to amino acid 450.
  • the positions within the HMMR profile that match the protein sequence are from profile position 42 to profile position 368. Other domains identified within this protein are: None.
  • SGPrl 54, SEQ ID NO:34, SEQ ID NO:69 encodes a protein that is 473 amino acids long. It is classified as a Metalloprotease ofthe PepM20 family.
  • the protease domain in this protein matches the hidden Markov profile for a Peptidase_M20, from amino acid 55 to amino acid 286.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 247. Other domains identified within this protein are: None.
  • SGPr088, SEQ ID NO:35, SEQ ID NO:70 encodes a protein that is 475 amino acids long. It is classified as a Metalloprotease ofthe PepM20 family.
  • the protease domain in this protein matches the hidden Markov profile for a Peptidase_M20, from amino acid 22 to amino acid 417.
  • the positions within the HMMR profile that match the protein sequence are from profile position 1 to profile position 368. Other domains identified within this protein are: None.
  • RNAs are isolated using the Guanidine Salts/Phenol extraction protocol of Chomczynski and Sacchi (P. Chomczynski and N. Sacchi, Anal.
  • RNAs are used to generate single-stranded cDNA using the Superscript Preamplification System
  • a typical reaction uses 10 ⁇ g total RNA with 1.5 ⁇ g oligo(dT) 12 - ⁇ 8 in a reaction volume of 60 ⁇ L.
  • the product is treated with RNaseH and diluted to 100 ⁇ L with H 2 0.
  • 1-4 ⁇ L of this sscDNA is used in each reaction.
  • PCR reactions are performed using degenerate primers applied to multiple single-stranded cDNAs.
  • the primers are added at a final concentration of 5 ⁇ M each to a mixture containing 10 mM TrisHCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 200 ⁇ M each deoxynucleoside triphosphate, 0.001% gelatin, 1.5 U AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and 1-4 ⁇ L cDNA.
  • the cycling conditions are 94 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min 45 s for 35 cycles.
  • PCR fragments migrating between 300-350 bp are isolated from 2% agarose gels using the GeneClean Kit (Biol 01), and T-A cloned into the pCRII vector (Invitrogen Corp. U.S.A.) according to the manufacturer's protocol. Colonies are selected for mini plasmid DNA-preparations using Qiagen columns and the plasmid DNA is sequenced using a cycle sequencing dye- terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City, CA). Sequencing reaction products are run on an ABI Prism 377 DNA Sequencer, and analyzed using the BLAST alignment algorithm (Altschul, S.F. et al, J.Mol.Biol. 215: 403-10).
  • Probes are 32 P-labeled by random priming and used at 2xl0 6 cpm mL following standard techniques for library screening. Pre- hybridization (3 h) and hybridization (overnight) are conducted at 42 °C in 5X SSC, 5X Denhart's solution, 2.5% dextran sulfate, 50 mM Na 2 PO 4 /NaHPO 4 , pH 7.0, 50% formamide with 100 mg/mL denatured salmon sperm DNA. Stringent washes are performed at 65 °C in 0.1X SSC and 0.1% SDS. DNA sequencing is carried out on both strands using a cycle sequencing dye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City, CA). Sequencing reaction products are run on an ABI Prism 377 DNA Sequencer.
  • FS AmpliTaq DNA Polymerase
  • EXAMPLE 4 Expression Analysis of Mammalian Proteases Materials and Methods Northern blot analysis
  • Northern blots are prepared by running 10 ⁇ g total RNA isolated from 60 human tumor cell lines (such as HOP-92, EKVX, NCI-H23, NCI-H226, NCI- H322M, NCI-H460, NCI-H522, A549, HOP-62, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, IGROV1, SK-OV-3, SNB-19, SNB-75, U251, SF-268, SF-295, SF-539, CCRF-CEM, K-562, MOLT-4, HL-60, RPMI 8226, SR, DU-145, PC-3, HT-29, HCC-2998, HCT-116, SW620, Colo 205, HTC15, KM-12, UO-31, SN12C, A498, CaKil, RXF-393, ACHN, 786-0, TK-10, LOX DVIVI, Malme-3M, SK-MEL
  • Filters are hybridized with random primed [ ⁇ 32 P]dCTP-labeled probes synthesized from the inserts of several ofthe protease genes. Hybridization is performed at 42 °C overnight in 6X SSC, 0.1% SDS, IX Denhardt's solution, 100 ⁇ g/mL denatured herring sperm DNA with 1-2 x 10 6 cpm/mL of 32 P-labeled DNA probes. The filters are washed in 0. IX SSC/0.1 % SDS, 65 °C, and exposed on a Molecular Dynamics phosphorimager.
  • RNA is isolated from a variety of normal human tissues and cell lines. Single stranded cDNA is synthesized from 10 ⁇ g of each RNA as described above using the Superscript Preamplification System (GibcoBRL). These single strand templates are then used in a 25 cycle PCR reaction with primers specific to each clone. Reaction products are electrophoresed on 2% agarose gels, stained with ethidium bromide and photographed on a UV light box. The relative intensity ofthe STK-specific bands were estimated for each sample.
  • Plasmid DNA array blots are prepared by loading 0.5 ⁇ g denatured plasmid for each protease on a nylon membrane.
  • the [ ⁇ 32 P]dCTP labeled single stranded DNA probes are synthesized from the total RNA isolated from several human immune tissue sources or tumor cells (such as thymus, dendrocytes, mast cells, monocytes, B cells (primary, Jurkat, RPMI8226, SR), T cells (CD8/CD4+, TH1, TH2, CEM, MOLT4), K562 (megakaryocytes).
  • Hybridization is performed at 42 °C for 16 hours in 6X SSC, 0.1% SDS, IX Denhardt's solution, 100 ⁇ g/mL denatured herring sperm DNA with 10 6 cpm/mL of [ ⁇ 32 P]dCTP labeled single stranded probe.
  • the filters are washed in 0.1X SSC/0.1% SDS, 65 °C, and exposed for quantitative analysis on a Molecular Dynamics phosphorimager.
  • EXAMPLE 5 Protease Gene Expression
  • Expression constructs are generated for some ofthe human cDNAs including: a) full-length clones in a pCDNA expression vector; and b) a GST-fusion construct containing the catalytic domain ofthe novel protease fused to the C- terminal end of a GST expression cassette; and c) a full-length clone containing a mutation within the predicted polypeptide cleaving site within the protease domain. inserted in the pCDNA vector.
  • mutants ofthe protease might function as dominant negative constructs, and will be used to elucidate the function of these novel proteases.
  • the various immune sera are first tested for reactivity and selectivity to recombinant protein, prior to testing for endogenous sources.
  • the washing buffer is PBST (standard phosphate-buffered saline pH 7.4 + 0.1% Triton
  • Blocking and antibody incubation buffer is PBST +5% milk. Antibody dilutions are varied from 1 : 1000 to 1 :2000.
  • EXAMPLE 7 Recombinant Expression and Biological Assays for Proteases Materials and Methods
  • the pcDNA expression plasmids (10 ⁇ g DNA/100 mm plate) containing the protease constructs are introduced into 293 cells with lipofectamine (Gibco BRL). After 72 hours, the cells are harvested in 0.5 mL solubilization buffer (20 mM HEPES, pH 7.35, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2 , 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ⁇ g/mL aprotinin).
  • Sample aliquots are resolved by SDS polyacrylamide gel electrophoresis (PAGE) on 6%> acrylamide/0.5% bis-acrylamide gels and electrophoretically transferred to nitrocellulose.
  • Non-specific binding is blocked by preincubating blots in Blotto (phosphate buffered saline containing 5%> w/v non-fat dried milk and 0.2% v/v nonidet P-40 (Sigma)), and recombinant protein is detected using the various anti- peptide or anti-GST-fusion specific antisera.
  • Blotto phosphate buffered saline containing 5%> w/v non-fat dried milk and 0.2% v/v nonidet P-40 (Sigma)
  • Assays are carried out using a spectrofluorometer, such as Perkin-Elrner 204S.
  • the standard reaction mixtures 100 ⁇ l contains 200 mM Tris-HCl, pH8.5, and 200 ⁇ M fluorogenic peptide substrate. After enzyme addition, reaction mixtures are incubated at 37 °C for 30 min and terminated by addition of 1.9 ml of 125 mM
  • Calpain activity is measured by the rate of generation ofthe fluorescent product, AMC, from intracellular thiol-conjugated Boc-Leu-Met-CMAC (Rosser, B. G., Powers, S. P., and Gores, G. J. (1993) J. Biol. Chem. 268, 23593-23600).
  • Cells are dispersed, grown on glass coverslips, continuously superfused with physiologic saline solution at 37 °C, and sequentially imaged with a quantitative fluorescence imaging system.
  • Boc-Leu-Met-CMAC (10 ⁇ M, Molecular Probes) is introduced into the superfusion solution, and mean fluorescence intensity (excitation 350 nm, emission 470 nm) of individual cells is measured at 60-s intervals.
  • TNF- (30 ng/ml) is added to the superfusion solution with 10 ⁇ M Boc-Leu- Met-CMAC.
  • the slope ofthe fluorescence change with respect to time represents the intracellular calpain activity (Rosser, et al, 1993, J. Biol. Chem. 268:23593- 23600).
  • the proteolytic activity of enzymes is measured using a commercially available assay system (Athena Environmental Sciences, Inc.).
  • the assay employs a universal substrate of a dye-protein conjugate cross linked to a matrix.
  • Protease activity is determined specfrophotometrically by measuring the absorbance ofthe dye released from the matrix to the supernatant.
  • Reaction vials containing the enzyme and substrate are incubated for 3 h at 37 °C. The activity is measured at different incubation times, and reactions are terminated by adding 500 ⁇ l of 0.2 N NaOH to each vial.
  • the absorbance ofthe supernatant in each reaction vial is measured at 450 nm.
  • the proteolytic activity is monitored using 10 ⁇ l
  • Unlabeled protease is mixed with radiolabeled substrate-containing membranes in buffer (100 mM HEPES, 100 mM NaCl, 125 ⁇ M magnesium acetate, 125 ⁇ M zinc acetate, pH 7.5) and incubated at 30 °C. Typically, each reaction had a final volume of 80-100 ⁇ l. Each reaction is normalized to the same final concentration of lysis buffer components (25 mM Tris, 0.1 M sorbitol, 0.5 mM EDTA, 0.01% NaN 3 , pH 7.5) because the amount of membranes added to each reaction is varied.
  • reactions are assembled without substrate and pretreated with 1.125 mM 1,10-orthophenanthroline for 20 min on ice. Subsequently, metal ions and substrate-containing membranes are added, and reactions are initiated by incubation at 30 °C; the additions result in dilution ofthe 1,10-orthophenanthroline to a final concentration of 1 mM.
  • the metal ions are added in the form of acetate salts from 25-100 mM stock solutions (Zn 2+ ,
  • samples are solubilized by SDS (final concentration, 0.5%), boiled for 3 min, and directly immunoprecipitated after dilution with immunoprecipitation buffer.
  • Immunoprecipitates are subjected to SDS- polyacrylamide gel electrophoresis as described, fixed for 7 min with 20% trichloroacetic acid, dried, and exposed to a Phosphorimager screen for detection and quantitation (Molecular Dynamics, Sunnyvale, CA). All ofthe above reagents can be purchased from Sigma. (Details of this assay can be found in: Schmidt, et al, 2000, JBiol Chem, 275:6227-6233 which is incorporated herein by reference in its entirety including any figures, tables, or drawings). Variation of this assay to apply to substrate not bound to membrane is straightforward.
  • the chromosomal regions for mapped genes are listed Table 2.
  • the chromosomal positions were cross-checked with the Online Mendelian Inheritance in Man database (OMIM, littp ://www.ncbi.nlm.nih. ov/htbin-post/Omim . , which tracks genetic information for many human diseases, including cancer. References for association ofthe mapped sites with chromosomal abnormalities found in human cancer can be found in: Knuutila, et al., Am J Pathol, 1998, 152: 1107-1123. A third source of information on mapped positions was searching published literature (at NCBI, http://www.ncbi.nlm.nih. gov/entrez/query.fcgi) for documented association ofthe mapped position with human disease.
  • protease polynucleotides ofthe present invention can be used to identify individuals who have, or are at risk for developing, relevant diseases. As discussed elsewhere in this application, the polypeptides and polynucleotides ofthe present invention are useful in identifying compounds that modulate protease activity, and in turn ameliorate various diseases.
  • Chromosome 1 alterations in breast cancer allelic loss on lp and lq is related to lymphogenic metastases and poor prognosis. (Genes Chromosomes Cancer. 1992 Nov;5(4):311-20.).
  • SGPrl97 SEQ ID NO:2 6p21.1 Genetic imbalances with impact on survival in head and neck cancer patients. (Am J Pathol. 2000 Aug;157(2):369-75.). Systematic screening ofthe LDL-PLA2 gene for polymorphic variants and case-control analysis in schizophrenia. (Biochem Biophys Res Commun. 1997 Dec 29;241(3):630-5.)
  • a microdeletion in 19ql3.2 associated with mental retardation, skeletal malformations, and Diamond-Blackfan anaemia suggests a novel contiguous gene syndrome (J Med Genet. 2000 Feb;37(2): 128-31.).
  • Familial patent ductus arteriosus and bicuspid aortic valve with hand anomalies a novel heart-hand syndrome.
  • Char syndrome an inherited disorder with patent ductus arteriosus, maps to chromosome 6pl2-p21.
  • Am J Ophthalmol. 1998 Jan;125(l):64-70. Linkage analysis of candidate regions for coeliac disease genes. (Hum Mol Genet. 1997 Aug;6(8): 1335-9.).
  • SGPr003 SEQ ID NO: 13 2q37 The expression of fragile sites in lymphocytes of patients with rectum cancer and their first-degree relatives. (Cancer Lett. 2000 May l;152(2):201-9.). Anterior chamber eye anomalies, redundant skin and syndactyly ⁇ a new syndrome associated with breakpoints at 2q37.2 and 7q36.3. (Clin Dysmorphol. 1999 Jul;8(3):157-63.). Wilms' tumor and gonadal dysgenesis in a child with the 2q37.1 deletion syndrome. (Clin Genet. 1998 Apr;53(4):278-80.).
  • SGPr050 SEQ ID NO:16 5ql5.3 Mucolipidosis type IV: Novel MCOLN1 mutations in Jewish and non- Jewish patients and the frequency ofthe disease in the Ashkenazi Jewish population. (Hum Mutat. 2001 May;17(5):397-402.) Myocarditis, a Rare but Severe manifestation of Q Fever: Report of 8 Cases and Review ofthe Literature. (Clin Infect Dis. 2001 May 15;32(10):1440-1447.)
  • Interleukin-5 is at 5q31 and is deleted in the 5q- syndrome. (Blood. 1988
  • Am J Med Genet. 1999 Jan 29;82(3):249-53 Partial monosomy of chromosome lp36.3: characterization ofthe critical region and delineation of a syndrome.
  • Am J Med Genet. 1995 Dec 4;59(4):467-75 Consistent association of lp loss of heterozygosity with pheochramocytomas from patients with multiple endocrine neoplasia type 2 syndromes. (Cancer Res. 1992 Feb 15;52(4):770-4.).
  • Chromosome 9 deletions and recurrence of superficial bladder cancer identification of four regions of prognostic interest. (Oncogene. 2000 Dec 14;19(54):6317-23). Exclusion of NFIL3 as the gene causing hereditary sensory neuropathy type I by mutation analysis. (Hum Genet. 2000 Jun; 106(6): 594-6). Chromosomal imbalances are associated with a high risk of progression in early invasive (pTl) urinary bladder cancer (Cancer Res. 1999 Nov 15;59(22):5687-91). Brachydactyly type B: linkage to chromosome 9q22 and evidence for genetic heterogeneity. (Am J Hum Genet. 1999 Feb;64(2):578-85).
  • SGPr203 SEQ ID NO:32 2q37 The expression of fragile sites in lymphocytes of patients with rectum cancer and their first-degree relatives.
  • Cancer Lett. 2000 May l;152(2):201-9. Anterior chamber eye anomalies, redundant skin and syndactyly-a new syndrome associated with breakpoints at 2q37.2 and 7q36.3.
  • Clin Dysmorphol. 1999 Jul;8(3):157-63. Wilms' tumor and gonadal dysgenesis in a child with the 2q37.1 deletion syndrome.
  • Albright hereditary osteodysfrophy and del(2) (q37.3) in four unrelated individuals.
  • SNPs single nucleotide polymorphisms
  • Candidate SNPs for the genes in this patent were identified by blastn searching the nucleic acid sequences against the public database of sequences containing documented SNPs (dbSNP, at NCBI, http://www.ncbi.nlm.nih.gov/SNP/snpblastpretty.html). dbSNP accession numbers for the SNP-containing sequences are given.
  • SNPs were also identified by comparing several databases of expressed genes (dbEST, NRNA) and genomic sequence (i.e., NRNA) for single basepair mismatches. The results are shown in Table 1, in the column labeled "SNPs". These are candidate SNPs - their actual frequency in the human population was not determined. The code below is standard for representing DNA sequence:
  • R G or A, puRine
  • V A, C or G (i.e., not T)
  • N A,C,GorT,aNy
  • SNPs may be important in identifying heritable traits associated with a gene.
  • Nylon membranes are purchased from Boehringer Mannheim. Denaturing solution contains 0.4 M NaOH and 0.6 M NaCl. Neutralization solution contains 0.5 M Tris-HCL, pH 7.5 and 1.5 M NaCl. Hybridization solution contains 50% formamide, 6X SSPE, 2.5X Denhardt's solution, 0.2 mg/mL denatured salmon DNA, 0.1 mg/mL yeast tRNA, and 0.2 % sodium dodecyl sulfate. Restriction enzymes are purchased from Boehringer Mannheim. Radiolabeled probes are prepared using the Prime-it ⁇ kit by Sfratagene. The ⁇ -actin DNA fragment used for a probe template is purchased from Clontech. Genomic DNA is isolated from a variety of tumor cell lines (such as MCF-7,
  • MDA-MB-231 Calu-6, A549, HCT-15, HT-29, Colo 205, LS-180, DLD-1, HCT- 116, PC3, CAPAN-2, MIA-PaCa-2, PANC-1, AsPc-1, BxPC-3, OVCAR-3, SKOV3, SW 626 and PA-1, and from two normal cell lines.
  • a 10 ⁇ g aliquot of each genomic DNA sample is digested with EcoR I restriction enzyme and a separate 10 ⁇ g sample is digested with Hind III restriction enzyme.
  • the restriction-digested DNA samples are loaded onto a 0.7% agarose gel and, following electrophoretic separation, the DNA is capillary-transferred to a nylon membrane by standard methods (Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory).
  • Phage display provides a method for isolating molecular interactions based on affinity for a desired bait. cDNA fragments cloned as fusions to phage coat proteins are displayed on the surface ofthe phage. Phage(s) interacting with a bait are enriched by affinity purification and the insert DNA from individual clones is analyzed.
  • Protein domains to be used as baits are generated as C-terminal fusions to GST and expressed in E. coli.
  • Peptides are chemically synthesized and biotinylated at the N-terminus using a long chain spacer biotin reagent. Selection
  • nucleic acids also encode the claimed peptides and proteins ofthe invention.
  • all four nucleic acid sequences GCT, GCC, GCA, and GCG encode the amino acid alanine. Therefore, if for an amino acid there exists an average of three codons, a polypeptide of 100 amino acids in length will, on average, be encoded by 3100, or 5 x 1047, nucleic acid sequences.
  • a nucleic acid sequence can be modified to form a second nucleic acid sequence, encoding the same polypeptide as encoded by the first nucleic acid sequences, using routine procedures and without undue experimentation.
  • nucleic acids that encode the claimed peptides and proteins are also fully described herein, as if all were written out in full taking into account the codon usage, especially that preferred in humans.
  • changes in the amino acid sequences of polypeptides, or in the corresponding nucleic acid sequence encoding such polypeptide may be designed or selected to take place in an area ofthe sequence where the significant activity ofthe polypeptide remains unchanged. For example, an amino acid change may take place within a ⁇ -turn, away from the active site ofthe polypeptide.
  • changes such as deletions (e.g. removal of a segment ofthe polypeptide, or in the corresponding nucleic acid sequence encoding such polypeptide, which does not affect the active site) and additions (e.g.

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Abstract

La présente invention concerne des polypeptides protéases, des séquences nucléotidiques codant lesdits polypeptides protéases, ainsi que divers produits et méthodes utiles pour le diagnostic et le traitement de diverses maladies et affections liées aux protéases. L'utilisation d'une technique bio-informatique a permis d'identifier des membres mammifères de PTK et de STK et de prédire leur structure protéique.
PCT/US2001/014431 2000-05-04 2001-05-04 Nouvelles proteases WO2001083782A2 (fr)

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Cited By (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1134286A2 (fr) * 2000-03-14 2001-09-19 Pfizer Products Inc. Adamts polypeptides, acides nucléiques les codant et leurs applications
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WO2002002775A2 (fr) * 2000-06-30 2002-01-10 Basf Aktiengesellschaft Calpaine protease 12
WO2002010406A2 (fr) * 2000-07-28 2002-02-07 Immunex Corporation Polypeptides metalloproteinase-disintegrine et procedes de fabrication et procede d utilisation de ceux-ci
WO2002016564A2 (fr) * 2000-08-22 2002-02-28 Lexicon Genetics Incorporated Nouvelles proteases humaines et polynucleotides codant lesdites proteases
WO2002020805A2 (fr) * 2000-09-11 2002-03-14 Bayer Aktiengesellschaft Regulation de l'enzyme similaire a la carboxypeptidase humaine
WO2002024927A2 (fr) * 2000-09-20 2002-03-28 Bayer Aktiengesellschaft Regulation de la proteine humaine de type precurseur de adam-ts1
WO2002026955A2 (fr) * 2000-09-29 2002-04-04 Lexicon Genetics Incorporated Nouvelles proteases humaines et polynucleotides codant pour ces proteases
WO2002026948A2 (fr) * 2000-09-25 2002-04-04 Millennium Pharmaceuticals, Inc. 55054, une nouvelle metalloprotease humaine et ses applications
WO2002029026A2 (fr) * 2000-10-04 2002-04-11 Lexicon Genetics Incorporated Proteases humaines et polynucleotides codant pour ces proteases
WO2002033087A2 (fr) * 2000-10-17 2002-04-25 Curagen Corporation Proteines et acides nucleiques codant les memes
GB2369363A (en) * 2000-08-17 2002-05-29 Glaxo Group Ltd Matrix metalloproteases
WO2002051995A1 (fr) * 2000-12-22 2002-07-04 Millennium Pharmaceuticals, Inc. Element metalloprotease humain de la famille adam, et utilisations de celui-ci
WO2002051991A2 (fr) * 2000-12-22 2002-07-04 Millennium Pharmaceuticals, Inc. 17903, nouvelle aminopeptidase humaine et utilisations correspondantes
WO2002057461A2 (fr) * 2001-01-18 2002-07-25 Bayer Aktiengesellschaft Regulation de la metalloprotease a zinc de type adam-ts humaine
WO2002088303A2 (fr) * 2001-04-03 2002-11-07 Bristol-Myers Squibb Company Polynucleotide codant pour une nouvelle cysteine protease de la superfamille calpain, can-12 et leurs variantes
WO2003006646A1 (fr) * 2001-07-10 2003-01-23 Bayer Healthcare Ag Regulation de l'aminopeptidase n humaine
WO2003018764A2 (fr) * 2001-08-22 2003-03-06 Lexicon Genetics Incorporated Nouvelles proteases humaines et polynucleotides codant pour celles-ci
WO2003027282A1 (fr) * 2001-09-24 2003-04-03 Daiichi Fine Chemical Co., Ltd. Adamts-15, -16, -17, -18 et -19
WO2003042379A2 (fr) * 2001-11-13 2003-05-22 Universite De Liege Nouveau polynucleotide de desintegrine et metalloproteinase comportant une thrombospondine de type 1 et polypeptide code
WO2003057842A2 (fr) * 2001-12-31 2003-07-17 Wyeth Molecules d'aggrecanase
WO2003064622A2 (fr) * 2002-01-31 2003-08-07 Wyeth Molecules d'aggrecanase
EP1347048A1 (fr) * 2000-12-25 2003-09-24 Yamanouchi Pharmaceutical Co. Ltd. Nouvelle protease
WO2002026949A3 (fr) * 2000-09-29 2003-09-25 Lexicon Genetics Inc Nouvelles proteases humaines et polynucleotides les codant
WO2003089644A1 (fr) * 2002-04-19 2003-10-30 Riken Nouvelles proteines et adn codant pour elles
NL1022371C2 (nl) * 2003-01-13 2003-11-18 Hoffmann La Roche Nieuwe metallo-proteasen met trombospondine domeinen en daarvoor coderende nucle´nezuur samenstellingen.
US6683165B1 (en) 1999-04-13 2004-01-27 Genome Therapeutics Corporation Human gene relating to respiratory diseases and obesity
US6689599B1 (en) * 2000-10-20 2004-02-10 Genetics Institute, Llc Aggrecanase molecules
WO2002055702A3 (fr) * 2000-10-26 2004-02-12 Curagen Corporation Protéines humaines, polynucléotides les codant et procédés d'utilisation correspondants
EP1401470A2 (fr) * 2001-06-04 2004-03-31 Curagen Corporation Nouveaux anticorps se fixant a des polypeptides antigeniques, acides nucleiques codant les antigenes et modes d'utilisation
WO2004073657A2 (fr) * 2003-02-19 2004-09-02 Protein Design Labs, Inc. Methode de diagnostic du cancer, composition et methodes de criblage destinees a identifier des modulateurs du cancer
US6855532B2 (en) 1999-11-19 2005-02-15 Solvay Pharmaceuticals B.V. Human enzymes of the metalloprotease family
EP1508619A1 (fr) * 2002-06-20 2005-02-23 Yamanouchi Pharmaceutical Co. Ltd. Nouveau promoteur
WO2005031359A2 (fr) * 2003-09-27 2005-04-07 Bayer Healthcare Ag Agents diagnostiques et therapeutiques pour maladies associees a l'arginyl aminopeptidase (rnpep)
WO2005040792A2 (fr) * 2003-10-21 2005-05-06 Bayer Healthcare Ag Diagnostics et therapeutiques pour des maladies associees a l'arginyl aminopeptidase (aminopeptidase b)-de type 1 (rnpepl1)
US6903201B2 (en) 2001-01-05 2005-06-07 Curagen Corporation Proteins and nucleic acids encoding same
US7034134B2 (en) * 2001-04-26 2006-04-25 Bristol-Myers Squibb Company Polynucleotide encoding a novel metalloprotease highly expressed in the testis, MMP-29
US7125701B2 (en) 2001-07-27 2006-10-24 Wyeth Aggrecanase molecules
US7892750B1 (en) * 2004-12-23 2011-02-22 University Of Notre Dame Du Lac Cathepsin E as a marker of colon cancer

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0123928A2 (fr) * 1983-03-31 1984-11-07 Codon Genetic Engineering Laboratories ADN recombinant codant pour un polypeptide présentant une activité de coagulation du lait
WO1998031818A2 (fr) * 1997-01-21 1998-07-23 Human Genome Sciences, Inc. Polypeptides semblables a tace ou a la matrilysime
WO2000053774A2 (fr) * 1999-03-08 2000-09-14 Neurocrine Biosciences, Inc. Metalloproteinases et leurs procedes d'utilisation
WO2001036605A2 (fr) * 1999-11-19 2001-05-25 Millennium Pharmaceuticals, Inc. 2786, nouvelle aminopeptidase humaine
WO2001098468A2 (fr) * 2000-06-16 2001-12-27 Incyte Genomics, Inc. Proteases

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0123928A2 (fr) * 1983-03-31 1984-11-07 Codon Genetic Engineering Laboratories ADN recombinant codant pour un polypeptide présentant une activité de coagulation du lait
WO1998031818A2 (fr) * 1997-01-21 1998-07-23 Human Genome Sciences, Inc. Polypeptides semblables a tace ou a la matrilysime
WO2000053774A2 (fr) * 1999-03-08 2000-09-14 Neurocrine Biosciences, Inc. Metalloproteinases et leurs procedes d'utilisation
WO2001036605A2 (fr) * 1999-11-19 2001-05-25 Millennium Pharmaceuticals, Inc. 2786, nouvelle aminopeptidase humaine
WO2001098468A2 (fr) * 2000-06-16 2001-12-27 Incyte Genomics, Inc. Proteases

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] 11 January 2000 (2000-01-11) "Homo sapiens matrix metalloprotease (ADAMTS1) mRNA" Database accession no. AF207664 XP002210995 *
DATABASE EMBL [Online] 18 October 2000 (2000-10-18) KRISTENSEN, T. : "Human aminopeptidase B cDNA cloning" Database accession no. AJ296161 XP002210996 *
KAGEYAMA TAKASHI: "New World monkey pepsinogens A and C, and prochymosins. Purification, characterization of enzymatic properties, cDNA cloning, and molecular evolution." JOURNAL OF BIOCHEMISTRY (TOKYO), vol. 127, no. 5, May 2000 (2000-05), pages 761-770, XP002195979 ISSN: 0021-924X *
RAWLINGS NEIL D ET AL: "MEROPS: The peptidase database." NUCLEIC ACIDS RESEARCH, vol. 27, no. 1, 1 January 1999 (1999-01-01), pages 325-331, XP002195980 ISSN: 0305-1048 cited in the application *

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US6855532B2 (en) 1999-11-19 2005-02-15 Solvay Pharmaceuticals B.V. Human enzymes of the metalloprotease family
EP1134286A2 (fr) * 2000-03-14 2001-09-19 Pfizer Products Inc. Adamts polypeptides, acides nucléiques les codant et leurs applications
EP1134286A3 (fr) * 2000-03-14 2003-05-02 Pfizer Products Inc. Adamts polypeptides, acides nucléiques les codant et leurs applications
WO2001078894A3 (fr) * 2000-04-13 2003-03-20 Schering Corp Nouveau gene humain implique dans les maladies respiratoires, l'obesite et les maladies intestinales inflammatoires
WO2001078894A2 (fr) * 2000-04-13 2001-10-25 Schering Corporation Nouveau gene humain implique dans les maladies respiratoires, l'obesite et les maladies intestinales inflammatoires
WO2002002775A2 (fr) * 2000-06-30 2002-01-10 Basf Aktiengesellschaft Calpaine protease 12
WO2002002775A3 (fr) * 2000-06-30 2002-12-12 Basf Ag Calpaine protease 12
WO2002010406A3 (fr) * 2000-07-28 2003-01-16 Immunex Corp Polypeptides metalloproteinase-disintegrine et procedes de fabrication et procede d utilisation de ceux-ci
WO2002010406A2 (fr) * 2000-07-28 2002-02-07 Immunex Corporation Polypeptides metalloproteinase-disintegrine et procedes de fabrication et procede d utilisation de ceux-ci
GB2369363A (en) * 2000-08-17 2002-05-29 Glaxo Group Ltd Matrix metalloproteases
WO2002016564A3 (fr) * 2000-08-22 2002-07-25 Lexicon Genetics Inc Nouvelles proteases humaines et polynucleotides codant lesdites proteases
WO2002016564A2 (fr) * 2000-08-22 2002-02-28 Lexicon Genetics Incorporated Nouvelles proteases humaines et polynucleotides codant lesdites proteases
WO2002020805A3 (fr) * 2000-09-11 2002-09-19 Bayer Ag Regulation de l'enzyme similaire a la carboxypeptidase humaine
WO2002020805A2 (fr) * 2000-09-11 2002-03-14 Bayer Aktiengesellschaft Regulation de l'enzyme similaire a la carboxypeptidase humaine
WO2002024927A2 (fr) * 2000-09-20 2002-03-28 Bayer Aktiengesellschaft Regulation de la proteine humaine de type precurseur de adam-ts1
WO2002024927A3 (fr) * 2000-09-20 2002-11-07 Bayer Ag Regulation de la proteine humaine de type precurseur de adam-ts1
WO2002026948A2 (fr) * 2000-09-25 2002-04-04 Millennium Pharmaceuticals, Inc. 55054, une nouvelle metalloprotease humaine et ses applications
WO2002026948A3 (fr) * 2000-09-25 2002-12-27 Millennium Pharm Inc 55054, une nouvelle metalloprotease humaine et ses applications
WO2002026949A3 (fr) * 2000-09-29 2003-09-25 Lexicon Genetics Inc Nouvelles proteases humaines et polynucleotides les codant
WO2002026955A3 (fr) * 2000-09-29 2002-07-11 Lexicon Genetics Inc Nouvelles proteases humaines et polynucleotides codant pour ces proteases
WO2002026955A2 (fr) * 2000-09-29 2002-04-04 Lexicon Genetics Incorporated Nouvelles proteases humaines et polynucleotides codant pour ces proteases
WO2002029026A2 (fr) * 2000-10-04 2002-04-11 Lexicon Genetics Incorporated Proteases humaines et polynucleotides codant pour ces proteases
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WO2002033087A3 (fr) * 2000-10-17 2003-07-24 Curagen Corp Proteines et acides nucleiques codant les memes
US6689599B1 (en) * 2000-10-20 2004-02-10 Genetics Institute, Llc Aggrecanase molecules
WO2002055702A3 (fr) * 2000-10-26 2004-02-12 Curagen Corporation Protéines humaines, polynucléotides les codant et procédés d'utilisation correspondants
WO2002051995A1 (fr) * 2000-12-22 2002-07-04 Millennium Pharmaceuticals, Inc. Element metalloprotease humain de la famille adam, et utilisations de celui-ci
WO2002051991A3 (fr) * 2000-12-22 2002-09-12 Millennium Pharm Inc 17903, nouvelle aminopeptidase humaine et utilisations correspondantes
WO2002051991A2 (fr) * 2000-12-22 2002-07-04 Millennium Pharmaceuticals, Inc. 17903, nouvelle aminopeptidase humaine et utilisations correspondantes
EP1347048A1 (fr) * 2000-12-25 2003-09-24 Yamanouchi Pharmaceutical Co. Ltd. Nouvelle protease
EP1347048A4 (fr) * 2000-12-25 2004-07-21 Yamanouchi Pharma Co Ltd Nouvelle protease
US6903201B2 (en) 2001-01-05 2005-06-07 Curagen Corporation Proteins and nucleic acids encoding same
WO2002057461A2 (fr) * 2001-01-18 2002-07-25 Bayer Aktiengesellschaft Regulation de la metalloprotease a zinc de type adam-ts humaine
WO2002057461A3 (fr) * 2001-01-18 2002-12-27 Bayer Ag Regulation de la metalloprotease a zinc de type adam-ts humaine
US7186564B2 (en) 2001-04-03 2007-03-06 Bristol-Myers Squibb Company Polynucleotides encoding novel cysteine proteases of the calpain superfamily, CAN-12v1 and CAN-12v2.
WO2002088303A2 (fr) * 2001-04-03 2002-11-07 Bristol-Myers Squibb Company Polynucleotide codant pour une nouvelle cysteine protease de la superfamille calpain, can-12 et leurs variantes
WO2002088303A3 (fr) * 2001-04-03 2005-07-07 Bristol Myers Squibb Co Polynucleotide codant pour une nouvelle cysteine protease de la superfamille calpain, can-12 et leurs variantes
US7034134B2 (en) * 2001-04-26 2006-04-25 Bristol-Myers Squibb Company Polynucleotide encoding a novel metalloprotease highly expressed in the testis, MMP-29
US7285633B2 (en) 2001-04-26 2007-10-23 Bristol-Myers Squibb Company Metalloprotease highly expressed in the testis, MMP-29
EP1401470A4 (fr) * 2001-06-04 2005-05-25 Curagen Corp Nouveaux anticorps se fixant a des polypeptides antigeniques, acides nucleiques codant les antigenes et modes d'utilisation
EP1401470A2 (fr) * 2001-06-04 2004-03-31 Curagen Corporation Nouveaux anticorps se fixant a des polypeptides antigeniques, acides nucleiques codant les antigenes et modes d'utilisation
US7129077B2 (en) 2001-07-10 2006-10-31 Bayer Healthcare Ag Regulation of human aminopeptidase N
WO2003006646A1 (fr) * 2001-07-10 2003-01-23 Bayer Healthcare Ag Regulation de l'aminopeptidase n humaine
US7125701B2 (en) 2001-07-27 2006-10-24 Wyeth Aggrecanase molecules
WO2003018764A2 (fr) * 2001-08-22 2003-03-06 Lexicon Genetics Incorporated Nouvelles proteases humaines et polynucleotides codant pour celles-ci
WO2003018764A3 (fr) * 2001-08-22 2003-08-14 Lexicon Genetics Inc Nouvelles proteases humaines et polynucleotides codant pour celles-ci
WO2003027282A1 (fr) * 2001-09-24 2003-04-03 Daiichi Fine Chemical Co., Ltd. Adamts-15, -16, -17, -18 et -19
WO2003042379A3 (fr) * 2001-11-13 2004-02-19 Univ Liege Nouveau polynucleotide de desintegrine et metalloproteinase comportant une thrombospondine de type 1 et polypeptide code
WO2003042379A2 (fr) * 2001-11-13 2003-05-22 Universite De Liege Nouveau polynucleotide de desintegrine et metalloproteinase comportant une thrombospondine de type 1 et polypeptide code
WO2003057842A3 (fr) * 2001-12-31 2004-08-05 Wyeth Corp Molecules d'aggrecanase
WO2003057842A2 (fr) * 2001-12-31 2003-07-17 Wyeth Molecules d'aggrecanase
AU2003207795B2 (en) * 2002-01-31 2009-01-08 Wyeth Aggrecanase molecules
EP1476545A2 (fr) * 2002-01-31 2004-11-17 Wyeth Molecules d'aggrecanase
US7078217B2 (en) 2002-01-31 2006-07-18 Wyeth Aggrecanase molecules
EP1476545A4 (fr) * 2002-01-31 2006-11-29 Wyeth Corp Molecules d'aggrecanase
WO2003064622A3 (fr) * 2002-01-31 2003-11-27 Wyeth Corp Molecules d'aggrecanase
SG158739A1 (en) * 2002-01-31 2010-02-26 Wyeth Corp Aggrecanase molecules
WO2003064622A2 (fr) * 2002-01-31 2003-08-07 Wyeth Molecules d'aggrecanase
WO2003089644A1 (fr) * 2002-04-19 2003-10-30 Riken Nouvelles proteines et adn codant pour elles
EP1508619A1 (fr) * 2002-06-20 2005-02-23 Yamanouchi Pharmaceutical Co. Ltd. Nouveau promoteur
EP1508619A4 (fr) * 2002-06-20 2005-11-02 Astellas Pharma Inc Nouveau promoteur
NL1022371C2 (nl) * 2003-01-13 2003-11-18 Hoffmann La Roche Nieuwe metallo-proteasen met trombospondine domeinen en daarvoor coderende nucle´nezuur samenstellingen.
WO2004073657A3 (fr) * 2003-02-19 2005-04-21 Protein Design Labs Inc Methode de diagnostic du cancer, composition et methodes de criblage destinees a identifier des modulateurs du cancer
WO2004073657A2 (fr) * 2003-02-19 2004-09-02 Protein Design Labs, Inc. Methode de diagnostic du cancer, composition et methodes de criblage destinees a identifier des modulateurs du cancer
WO2005031359A3 (fr) * 2003-09-27 2005-06-23 Bayer Healthcare Ag Agents diagnostiques et therapeutiques pour maladies associees a l'arginyl aminopeptidase (rnpep)
WO2005031359A2 (fr) * 2003-09-27 2005-04-07 Bayer Healthcare Ag Agents diagnostiques et therapeutiques pour maladies associees a l'arginyl aminopeptidase (rnpep)
WO2005040792A3 (fr) * 2003-10-21 2005-07-21 Bayer Healthcare Ag Diagnostics et therapeutiques pour des maladies associees a l'arginyl aminopeptidase (aminopeptidase b)-de type 1 (rnpepl1)
WO2005040792A2 (fr) * 2003-10-21 2005-05-06 Bayer Healthcare Ag Diagnostics et therapeutiques pour des maladies associees a l'arginyl aminopeptidase (aminopeptidase b)-de type 1 (rnpepl1)
US7892750B1 (en) * 2004-12-23 2011-02-22 University Of Notre Dame Du Lac Cathepsin E as a marker of colon cancer

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HK1054571A1 (zh) 2003-12-05
WO2001083782A3 (fr) 2003-01-23
AU2001259473A1 (en) 2001-11-12
EP1294901A2 (fr) 2003-03-26
CA2408105A1 (fr) 2001-11-08

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