WO2006074914A2 - Helicase d'arn humaine et ses utilisations therapeutiques - Google Patents

Helicase d'arn humaine et ses utilisations therapeutiques Download PDF

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WO2006074914A2
WO2006074914A2 PCT/EP2006/000190 EP2006000190W WO2006074914A2 WO 2006074914 A2 WO2006074914 A2 WO 2006074914A2 EP 2006000190 W EP2006000190 W EP 2006000190W WO 2006074914 A2 WO2006074914 A2 WO 2006074914A2
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nucleic acid
seq
rhau
peptide
amino acid
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PCT/EP2006/000190
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WO2006074914A3 (fr
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Yoshikuni Nagamine
Hoanh Tran
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Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)

Definitions

  • RNA helicases are enzymes that unwind or rearrange duplex and structured RNA molecules and are required at almost every discrete step of major RNA processing events. RNA helicases are also involved in mRNA degradation. RNA helicases of the DExH/D family not only can unwind duplex RNA but are reported to disrupt high-affinity RNA-protein interactions using the energy released by ATP hydrolysis, although the precise mechanism of action remains to be characterized.
  • ARE AU-rich element
  • RNA helicase ⁇ 14 RNA helicase associated AU-rich element
  • RHAU ⁇ 14 RNA helicase associated AU-rich element
  • RHAU can be used as a novel drug target useful in the diagnosis, prevention, and treatment of human diseases, particularly neoplasia.
  • the invention provides methods for identifying modulators of RHAU activity and gene expression and the use of such modulators, e.g., for the treatment of cancer.
  • the invention also provides pharmaceutical compositions comprising said modulators.
  • the instant invention relates to the identification and isolation of RNA helicase polypeptides, allelic variants and other mammalian orthologs thereof.
  • These unique polypeptide sequences, and nucleic acid sequences that encode these peptides can be used as models for the identification and development of therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of therapeutic agents that modulate enzyme activity in cells and tissues that express the enzyme.
  • the present invention provides amino acid sequences of previously uncharacterized human RNA helicases referred to herein as RHAU and RHAU ⁇ 14 , nucleic acid sequences in the form of transcript sequences, and cDNA sequences that encode RHAU peptides and proteins and variants thereof.
  • RHAU and RHAU ⁇ 14 amino acid sequences of previously uncharacterized human RNA helicases referred to herein as RHAU and RHAU ⁇ 14
  • nucleic acid sequences in the form of transcript sequences a sequence of RNA helicases
  • cDNA sequences that encode RHAU peptides and proteins and variants thereof.
  • the peptides that are provided in the present invention may be useful as drug targets.
  • RHAU amino acid sequence of RHAU is provided herein as SEQ ID NO:3 and the amino acid sequence of an allelic variant missing 14 amino acids (amino acids 517-530), RHAU ⁇ 14 , is provided herein as SEQ ID NO:4. These amino acid sequences are referred to interchangeably herein as the enzyme peptides of the present invention, enzyme peptides, or peptides/proteins of the present invention. Unless otherwise apparent from context, the term "RHAU" as used herein is meant to include RHAU ⁇ U . Nucleic acid sequences encoding the RHAU and RHAU ⁇ 14 peptides are provided herein as SEQ ID NO:1 and SEQ ID NO:2, respectively.
  • a peptide is said to be "isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals.
  • the peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components.
  • substantially free of cellular material includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins.
  • culture medium represents less than about 20% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the enzyme peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.
  • the isolated enzyme peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods.
  • a nucleic acid molecule encoding the enzyme peptide may be cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell.
  • the protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
  • proteins that may consist of, consist essentially of, or comprise the amino acid sequences provided in SEQ ID NO:3 and SEQ ID NO:4, for example, proteins encoded by the nucleic acid sequence of SEQ ID NO:1 and SEQ ID NO:2, respectively.
  • a protein "consists of" an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein; a protein "consists essentially of” an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein; and a protein "comprises" an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein.
  • the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences.
  • additional amino acid molecules such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences.
  • Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids.
  • the preferred classes of proteins that are comprised of the enzyme peptides of the present invention are the naturally occurring mature proteins.
  • the enzyme peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins.
  • Such chimeric and fusion proteins comprise an enzyme peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the enzyme peptide.
  • Operatively linked indicates that the enzyme peptide and the heterologous protein are fused in-frame.
  • the heterologous protein can be fused to the N- terminus or C-terminus of the enzyme peptide.
  • the fusion protein does not affect the activity of the enzyme peptide per se.
  • the fusion protein can include enzymatic fusion proteins, for example, beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, Hl-tagged or Ig fusions.
  • Such fusion proteins, particularly poly-His fusions can facilitate the purification of recombinant enzyme peptide.
  • expression and/or secretion of a protein can be increased by using a heterologous signal sequence.
  • a chimeric or fusion protein can be produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence.
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein).
  • An enzyme peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the enzyme peptide.
  • the present invention also provides obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides.
  • variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry.
  • Variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the enzyme peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (J. MoI. Biol. 48: 444-453, 1970) which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux J et al., 1984, Nucleic Acids Res.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of Myers and Miller (CABIOS 4: 11-17, 1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases to, for example, identify other family members or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (J. MoI. Biol. 215: 403-410, 1990).
  • Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the enzyme peptides of the present invention as well as being encoded by the same genetic locus as the enzyme peptide provided herein.
  • Allelic variants of an enzyme peptide can readily be identified as being a human protein having a high degree of, or significant, sequence homology/identity to at least a portion of the enzyme peptide as well as being encoded by the same genetic locus as the enzyme peptide provided herein.
  • two proteins or a region of the proteins
  • a significantly homologous amino acid sequence will be encoded by a nucleic acid sequence that will hybridize to an enzyme peptide encoding nucleic acid molecule under stringent conditions as more fully described below.
  • Paralogs of an enzyme peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the enzyme peptide, as being encoded by a gene from humans, and as having similar activity or function.
  • Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain.
  • Such paralogs will be encoded by a nucleic acid sequence that will hybridize to an enzyme peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.
  • Orthologs of an enzyme peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the enzyme peptide as well as being encoded by a gene from another organism.
  • Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents.
  • Such orthologs will be encoded by a nucleic acid sequence that will hybridize to an enzyme peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.
  • Non-naturally occurring variants of the enzyme peptides of the present invention can readily be generated using recombinant techniques.
  • Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the enzyme peptide.
  • one class of substitutions are conserved amino acid substitution which involve the substitution of a given amino acid in an enzyme peptide by another amino acid of like characteristics. Such substitutions are familiar to one of skill in the art.
  • Variant enzyme peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc.
  • Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions.
  • Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.
  • Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
  • Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis; mutant molecules may be tested for biological activity such as enzyme activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling techniques familiar to one of skill in the art.
  • the present invention further provides fragments of the enzyme peptides, in addition to proteins and peptides that comprise and consist of such fragments.
  • a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from an enzyme peptide.
  • Such fragments can be chosen based on the ability to retain one or more of the biological activities of the enzyme peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen.
  • Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length.
  • Such fragments will typically comprise a domain or motif of the enzyme peptide, e.g., active site, a transmembrane domain or a substrate-binding domain.
  • possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis).
  • Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in enzyme peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art.
  • the enzyme peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature enzyme peptide is fused with another compound, such as a compound to increase the half-life of the enzyme peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature enzyme peptide, such as a leader or secretory sequence or a sequence for purification of the mature enzyme peptide or a pro-protein sequence.
  • a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature enzyme peptide is fused with another compound, such as a compound to increase the half-life of the enzyme peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature enzyme peptide, such as a leader or secretory sequence or a sequence for purification of the mature enzyme peptide or a pro-
  • the proteins of the present invention can be used in various applications, e.g., to raise antibodies or to elicit an immune response; as a reagent (including as a labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state).
  • the protein binds or potentially binds to another protein or ligand (such as, for example, in an enzyme-effector protein interaction or enzyme-ligand interaction)
  • the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products and the methods for performing the uses listed above are well known to those skilled in the art.
  • Enzymes isolated from humans and their human/mammalian orthologs may also serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the enzyme.
  • proteins of the present invention are useful for biological assays related to enzymes that are related to members of the helicase subfamily.
  • assays may involve any of the known enzyme functions or activities or properties useful for diagnosis and treatment of enzyme-related conditions that are specific for the family of enzymes to which the RHAU proteins belong, particularly in cells and tissues that express the enzyme.
  • the proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems.
  • Cell-based systems can be native, i.e., utilizing cells that normally express the enzyme, as a biopsy or expanded in cell culture.
  • cell-based assays involve recombinant host cells expressing the enzyme protein.
  • the polypeptides can be used to identify compounds that modulate enzyme activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the enzyme.
  • the enzymes of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the enzyme. These compounds can be further screened against a functional enzyme to determine the effect of the compound on the enzyme activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the enzyme to a desired degree.
  • the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the enzyme protein and a molecule that normally interacts with the enzyme protein, e.g., a substrate or a component of the signal transduction pathway with which the enzyme protein normally interacts (for example, another enzyme).
  • assays typically include the steps of combining the enzyme protein with a candidate compound under conditions that allow the enzyme protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the enzyme protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover and adenylate cyclase activation, etc.
  • ATPase activity of RHAU was analysed by detection of free pi on this layer chromatography (TLC) after reaction. After incubation of RHAU and [ ⁇ -32 P] ATP, reaction was spotted on TLC, then TLC was developed. Free 32P visualized by phosphor-imager.
  • Candidate compounds may include, for example, peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries; antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab') 2 , Fab expression library fragments, and epitope-binding fragments of antibodies); and small organic and inorganic molecules (e.g., molecules obtained from combinatorial, chemical and natural product libraries).
  • peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids
  • One candidate compound may be a soluble fragment of a receptor that competes for substrate binding.
  • Other candidate compounds include mutant enzymes or appropriate fragments containing mutations that affect enzyme function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.
  • the invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) enzyme activity.
  • the assays typically involve an assay of events in the signal transduction pathway that indicate enzyme activity.
  • the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the enzyme protein dependent signal cascade can be assayed.
  • any of the biological or biochemical functions mediated by the enzyme can be used as an endpoint assay. Specifically, a biological function of a cell or tissues that express the enzyme can be assayed.
  • Binding and/or activating compounds can also be screened by using chimeric enzyme proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions.
  • a substrate-binding region can be used that interacts with a different substrate than that which is recognized by the native enzyme. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in cells other than the specific host cell from which the enzyme is derived.
  • the proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the enzyme (e.g. binding partners and/or ligands). For example, a compound is exposed to an enzyme polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble enzyme polypeptide is also added to the mixture. If the test compound interacts with the soluble enzyme polypeptide, it decreases the amount of complex formed or activity from the enzyme target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the enzyme. Thus, the soluble polypeptide that competes with the target enzyme region is designed to contain peptide sequences corresponding to the region of interest.
  • a fusion protein e.g., a glutathione-S-transferase (GST) fusion protein
  • GST glutathione-S-transferase
  • fusion protein can be created using methods familiar to one of skill in the art which provides a domain that allows the protein to be bound to a matrix.
  • antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of a plate, and the protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the enzyme protein target molecule, or which are reactive with enzyme protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
  • Agents that modulate one of the enzymes of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.
  • Modulators of enzyme protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the enzyme pathway, by treating cells or tissues that express the enzyme.
  • Experimental data as provided in the Examples herein indicate that such modulators of RHAU activity may be useful to treat neoplasia.
  • Methods of treatment may include the steps of administering a modulator of enzyme activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein. Pharmaceutical compositions are discussed in more detail below.
  • the enzyme proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay familiar to one of skill in the art to identify other proteins which bind to or interact with the enzyme and are involved in enzyme activity.
  • Such enzyme-binding proteins are also likely to be involved in the propagation of signals by the enzyme proteins or enzyme targets as, for example, downstream elements of an enzyme-mediated signaling pathway.
  • enzyme-binding proteins may be enzyme inhibitors.
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., an enzyme-modulating agent, an antisense enzyme nucleic acid molecule, siRNA, ribozyme, an enzyme-specific antibody, or an enzyme-binding partner
  • an agent identified as described herein can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • the enzyme proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. The method involves contacting a biological sample with a compound capable of interacting with the enzyme protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.
  • a "biological sample” as used herein, is used in its broadest sense and may comprise tissues, cells and biological fluids isolated from a subject.
  • the peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs.
  • the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification.
  • Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered enzyme activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein.
  • Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.
  • peptides In vitro techniques for detection of peptides include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent.
  • a detection reagent such as an antibody or protein binding agent.
  • the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.
  • the peptides may also be subjects of pharmacogenomic analysis, which takes into consideration that the genotype of an individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound and suggests that the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment be based on the individual's genotype. Accordingly, therapeutically effective dosages could be discerned and modified as necessary to maximize the beneficial effect within a given population containing a particular polymorphism.
  • the peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Accordingly, methods for treatment include the use of the enzyme protein or fragments.
  • the invention also provides antibodies that selectively bind to the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof.
  • an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins.
  • An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross- reactivity.
  • the antibodies of the present invention include, but are not limited to, humanized or chimeric antibodies, single chain antibodies, anti-idiotypic antibodies, polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab') 2 , and Fv fragments. These antibodies may be generated using conventional methods familiar to one of skill in the art.
  • Antibodies are preferably prepared from regions or discrete fragments of the enzyme proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or enzyme/binding partner interaction and include the conserved motifs responsible for the coordinated coupling between substrate binding, NTP binding/hydrolysis and unwinding activity. Methods for detecting antibodies to a given target peptide are also well known to one of skill in the art. Detection of an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • the antibodies can be used to isolate the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation.
  • the antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells.
  • such antibodies are useful to detect the presence of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development.
  • such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression.
  • such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.
  • the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function.
  • a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form
  • the antibody can be prepared against the normal protein.
  • antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.
  • the antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism.
  • the diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.
  • antibodies are useful in pharmacogenomic analysis.
  • antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities.
  • the antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.
  • the antibodies are also useful for tissue typing.
  • tissue typing where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.
  • the antibodies are also useful for inhibiting protein function, for example, blocking the binding of the enzyme peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function.
  • An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity.
  • Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane.
  • kits for using antibodies to detect the presence of a protein in a biological sample can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use.
  • a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array using conventional methodologies.
  • the present invention further provides isolated nucleic acid molecules that encode an enzyme peptide or protein of the present invention.
  • nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the enzyme peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.
  • an "isolated" nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5 1 and 3 1 ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • flanking nucleotide sequences for example up to about 5KB, 4KB, 3KB, 2KB, or 1 KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence.
  • nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.
  • an "isolated" nucleic acid molecule such as a cDNA molecule
  • a cDNA molecule can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
  • recombinant DNA molecules contained in a vector are considered isolated.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • nucleic acid molecules that consist of, consist essentially of or comprise the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 or any nucleic acid molecule that encodes the proteins provided in SEQ ID NO:3 and SEQ ID NO:4.
  • a nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.
  • a nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.
  • a nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule.
  • the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences.
  • Such a nucleic acid molecule can have a few additional nucleotides or can comprise several hundred or more additional nucleotides.
  • the isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be removed from the mature protein by cellular enzymes.
  • the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the enzyme peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5' and 3 1 sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA.
  • the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.
  • Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form of DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof.
  • the nucleic acid, especially DNA can be double-stranded or single-stranded.
  • Single- stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).
  • the invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the enzyme proteins of the present invention that are described above.
  • nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis.
  • non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.
  • the present invention further provides for the addition of non-coding fragments to the nucleic acid molecules provided in SEQ ID NO:1 and SEQ ID NO:2.
  • Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents and methodologies for their manipulation are well known to one of skill in the art.
  • a "fragment" comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could be at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.
  • a probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.
  • Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described above, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence disclosed herein or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown herein or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other.
  • the conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1- 6.3.6.
  • stringent hybridization conditions are hybridization in 6 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 50-65°C. Examples of moderate to low stringency hybridization conditions are also well known in the art.
  • the nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays.
  • the nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptides described in SEQ ID NO:3 and SEQ ID NO:4 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides disclosed herein.
  • the probe can correspond to any sequence along the entire length of the nucleic acid molecules provided herein. Accordingly, it could be derived from 5' non- coding regions, the coding region, and 3' non-coding regions.
  • the nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.
  • the nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.
  • the nucleic acid molecules are also useful for constructing recombinant vectors.
  • Such vectors include expression vectors that express a portion of, or all of, the peptide sequences.
  • Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product.
  • an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.
  • the nucleic acid molecules are also useful for expressing antigenic portions of the proteins.
  • the nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods.
  • nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.
  • the nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in enzyme gene expression.
  • recombinant cells which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired enzyme protein to treat the individual.
  • Gene therapy methodologies are familiar to one of skill in the art.
  • nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.
  • the nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.
  • the nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.
  • the nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. DNA or RNA levels may be determined. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, issue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in enzyme protein expression relative to normal results.
  • In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detecting DNA include Southern hybridizations and in situ hybridization.
  • Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express an enzyme protein, such as by measuring a level of an enzyme-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if an enzyme gene has been mutated.
  • Nucleic acid expression assays are useful for drug screening to identify compounds that modulate enzyme nucleic acid expression.
  • the invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the enzyme gene, particularly biological and pathological processes that are mediated by the enzyme in cells and tissues that express it.
  • the method typically includes assaying the ability of the compound to modulate the expression of the enzyme nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired enzyme nucleic acid expression.
  • the assays can be performed in cell-based and cell-free systems.
  • Cell-based assays include cells naturally expressing the enzyme nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.
  • the assay for enzyme nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the enzyme protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.
  • modulators of enzyme gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined.
  • the level of expression of enzyme mRNA in the presence of the candidate compound is compared to the level of expression of enzyme mRNA in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression.
  • expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression.
  • nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.
  • the invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate enzyme nucleic acid expression in cells and tissues that express the enzyme.
  • Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.
  • a modulator for enzyme nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule modulates the enzyme nucleic acid expression in the cells and tissues that express the protein.
  • the nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the enzyme gene in clinical trials or in a treatment regimen.
  • the gene expression pattern can serve as an indication of the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance.
  • the gene expression pattern can also indicate the physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant.
  • administration of the compound could be commensurately decreased.
  • the nucleic acid molecules are also useful in diagnostic assays for qualitative changes in enzyme nucleic acid expression, and particularly in qualitative . changes that lead to pathology.
  • the nucleic acid molecules can be used to detect mutations in enzyme genes and gene expression products such as mRNA.
  • the nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the enzyme gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the enzyme gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of an enzyme protein.
  • Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis.
  • RNA or cDNA can be used in the same way.
  • detection of the mutation may involve the use of such conventional methods as the use of a probe/primer in a polymerase chain reaction (PCR) such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (the latter of which can be particularly useful for detecting point mutations in the gene).
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences. Alternatively, mutations in an enzyme gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.
  • nucleic acid e.g., genomic, mRNA or both
  • sequence-specific ribozymes can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature according to conventional methods.
  • Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method.
  • sequence differences between a mutant enzyme gene and a wild-type gene can be determined by direct DNA sequencing.
  • a variety of automated sequencing procedures can be utilized when performing the diagnostic assays, including, e.g., sequencing by mass spectrometry as disclosed in PCT International Publication No. WO 94/16101; Cohen et al., 1996, Adv. Chromatogr. 36: 127-162; and Griffin et al., 1993, Appl. Biochem. Biotechnol. 38: 147-159).
  • Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes, electrophoretic mobility of mutant and wild- type nucleic acid is compared and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis, techniques which are all familiar to one of skill in the art. Examples of other conventional methods for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.
  • the nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality.
  • nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the enzyme gene in an individual in order to select an appropriate compound or dosage regimen for treatment.
  • nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.
  • the present invention includes modulators of the nucleic acids encoding the RHAU proteins described herein.
  • modulators include ribozymes, antisense oligonucleotides, triple helix DNA, RNA aptamers, siRNA and/or double stranded RNA directed to an appropriate nucleotide sequence encoding the RHAU proteins disclosed herein.
  • These inhibitory molecules may be created using conventional techniques by one of skill in the art without undue burden or experimentation. For example, modifications (e.g.
  • antisense molecules DNA or RNA
  • the control regions of the genes encoding the polypeptides discussed herein i.e., to promoters, enhancers, and introns, which can act to prevent transcription and production of enzyme protein.
  • oligonucleotides derived from the transcription initiation site e.g., between positions -10 and +10 from the start site may be used.
  • all regions of the gene may be used to design an antisense molecule in order to create those which give strongest hybridization to the mRNA and such suitable antisense oligonucleotides may be produced and identified by standard assay procedures familiar to one of skill in the art.
  • triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • triplex DNA Recent therapeutic advances using triplex DNA have been described in the literature (Gee JE et al., In: Huber BE and Carr Bl, eds, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994). These molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to inhibit gene expression by catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples which may be used include engineered "hammerhead” or "hairpin” motif ribozyme molecules that can be designed to specifically and efficiently catalyze endonucleolytic cleavage of gene sequences, for example, a gene for RHAU.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • Ribozyme methods include exposing a cell to ribozymes or inducing expression in a cell of such small RNA ribozyme molecules (Grassi and Marini, 1996, Annals of Medicine 28: 499-510; Gibson, 1996, Cancer and Metastasis Reviews 15: 287-299). Intracellular expression of hammerhead and hairpin ribozymes targeted to mRNA corresponding to at least one of the genes discussed herein can be utilized to inhibit protein encoded by the gene.
  • Ribozymes can either be delivered directly to cells, in the form of RNA oligonucleotides incorporating ribozyme sequences, or introduced into the cell as an expression vector encoding the desired ribozymal RNA. Ribozymes can be routinely expressed in vivo in sufficient number to be catalytically effective in cleaving mRNA, and thereby modifying mRNA abundance in a cell (Cotten et al., 1989, EMBO J. 8: 3861-3866).
  • a ribozyme coding DNA sequence designed according to conventional, well known rules and synthesized, for example, by standard phosphoramidite chemistry, can be ligated into a restriction enzyme site in the anticodon stem and loop of a gene encoding a tRNA, which can then be transformed into and expressed in a cell of interest by methods routine in the art.
  • an inducible promoter e.g., a glucocorticoid or a tetracycline response element
  • tDNA genes i.e., genes encoding tRNAs
  • ribozymes can be routinely designed' to cleave virtually any mRNA sequence, and a cell can be routinely transformed with DNA coding for such ribozyme sequences such that a controllable and catalytically effective amount of the ribozyme is expressed. Accordingly the abundance of virtually any RNA species in a cell can be modified or perturbed.
  • Ribozyme sequences can be modified in essentially the same manner as described for antisense nucleotides, e.g., the ribozyme sequence can comprise a modified base moiety.
  • RNA aptamers can also be introduced into or expressed in a cell to modify RNA abundance or activity.
  • RNA aptamers are specific RNA ligands for proteins, such as for Tat and Rev RNA (Good et al., 1997, Gene Therapy 4: 45-54) that can specifically inhibit their translation.
  • Gene specific inhibition of gene expression may also be achieved using conventional RNA Interference and double stranded RNA technologies.
  • a description of such technology may be found in, for example, Fire et al., 1998, Nature 391: 806-811 ; Agrawal et al., 2003, Microbiol. MoI. Biol. Rev. 67: 657-685 and WO 99/32619 which are hereby incorporated by reference in their entirety.
  • Antisense molecules, triple helix DNA, RNA aptamers, dsRNA, siRNA and ribozymes of the present invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the genes of the polypeptides discussed herein. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.
  • the present invention includes the nucleic acid modulators discussed above as well as pharmaceutical compositions comprising these modulators.
  • Pharmaceutical compositions comprising such modulators are also contemplated.
  • kits for detecting the presence of a RHAU nucleic acid in a biological sample can comprise reagents such as a labeled nucleic acid or a nucleic acid that may be labeled or agent capable of detecting enzyme nucleic acid in a biological sample; means for determining the amount of enzyme nucleic acid in the sample; and means for comparing the amount of enzyme nucleic acid in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect enzyme protein mRNA or DNA.
  • the present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided herein in SEQ ID NO:1 and SEQ ID NO:2.
  • arrays or “microarrays” refer to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832 (Chee et al.), PCT application WO 95/11995 (Chee et al.), Lockhart DJ et al. (Nat. Biotech. 14: 1675-1680, 1996) and Schena M et al. (Proc. Natl. Acad. Sci. 93: 10614-10619, 1996), all of which are incorporated herein in their entirety by reference.
  • such arrays are produced by the methods described by Brown et al. (U.S. Pat. No. 5,807,522).
  • the microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support.
  • the oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length.
  • the microarray or detection kit may contain oligonucleotides that cover the known 5 1 , or 3 1 , sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence.
  • Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.
  • oligonucleotides In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest is typically examined using a computer algorithm which starts at the 5 1 or at the 3 1 end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The "pairs" will be identical, except for one nucleotide that preferably is located in the center of the sequence.
  • the second oligonucleotide in the pair serves as a control.
  • the number of oligonucleotide pairs may range from two to one million.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
  • an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO 95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference.
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.
  • RNA or DNA from a biological sample is made into hybridization probes.
  • the mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA.
  • the antisense RNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of non-hybridized probes, a scanner is used to determine the levels and patterns of fluorescence.
  • the scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit.
  • the biological samples may be obtained from any bodily fluid, cultured cells, biopsies, or other tissue preparations.
  • a detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.
  • the present invention provides methods to identify the expression of the enzyme proteins/peptides of the present invention.
  • methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample.
  • assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the enzyme gene of the present invention.
  • Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay.
  • One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the nucleic acid sequences disclosed herein. Examples of such assays can be found in Chard T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands, 1986, Bullock G R et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FIa., Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985), Tijssen P, Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands, 1985.
  • test samples of the present invention include cells, protein or membrane extracts of cells.
  • the test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the system utilized. In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.
  • the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprise: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.
  • a compartmentalized kit includes any kit in which reagents are contained in separate containers.
  • Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica.
  • Such containers allow one to efficiently transfer 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 nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe.
  • wash reagents such as phosphate buffered saline, Tris-buffers, etc.
  • the invention also provides vectors containing the nucleic acid molecules described herein.
  • the term "vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules.
  • the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid.
  • the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, or MAC.
  • a vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules.
  • the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.
  • the invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules.
  • the vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).
  • Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell.
  • the nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription.
  • the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector.
  • a transacting factor may be supplied by the host cell.
  • a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.
  • the regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage ⁇ , the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.
  • expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers.
  • regions that modulate transcription include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
  • expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region, a ribosome binding site for translation.
  • Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals.
  • a variety of expression vectors can be used to express a nucleic acid molecule.
  • Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.
  • Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids.
  • prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • the regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand.
  • a variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.
  • the nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology.
  • the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.
  • Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium.
  • Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.
  • the invention provides fusion vectors that allow for the production of the peptides.
  • Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification.
  • a proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety.
  • Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enteroenzyme.
  • Typical fusion expression vectors are well known in the art and include pGEX, pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione S-transferase
  • suitable inducible non-fusion E. coli expression vectors include pTrc and pET 11d.
  • Recombinant protein expression can be maximized in host bacteria according to conventional methods, e.g., by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein.
  • sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli.
  • the nucleic acid molecules can also be expressed by expression vectors that are operative in yeast.
  • yeast e.g., S. cerevisiae
  • vectors for expression in yeast e.g., S. cerevisiae
  • yeast e.g., S. cerevisiae
  • yeast e.g., S. cerevisiae
  • yeast e.g., S. cerevisiae
  • pYepSed e.g., S. cerevisiae
  • pMFa pFa
  • pJRY88 pJRY88
  • pYES2 Invitrogen Corporation, San Diego, Calif.
  • the nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells e.g., Sf9 cells
  • Sf9 cells are familiar to one of skill in the art and include the pAc series and the pVL series.
  • the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors.
  • mammalian expression vectors and methods of their use are also familiar to one of skill in the art and include pCDM8 and pMT2PC.
  • the expression vectors listed herein are provided by way of example only and a person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein.
  • the invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA.
  • an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).
  • the invention also relates to recombinant host cells containing the vectors described herein.
  • Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.
  • the recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
  • Host cells can contain more than one vector.
  • different nucleotide sequences can be introduced on different vectors of the same cell.
  • the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors.
  • the vectors can be introduced independently, co- introduced or joined to the nucleic acid molecule vector.
  • bacteriophage and viral vectors these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction.
  • Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.
  • Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs.
  • the marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.
  • RNA derived from the DNA constructs described herein can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences.
  • cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.
  • appropriate secretion signals are incorporated into the vector.
  • the signal sequence can be endogenous to the peptides or heterologous to these peptides.
  • the protein can be isolated from the host-cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods. It is also understood that depending upon the host cell utilized for recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or may be non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.
  • Recombinant host cells expressing the peptides described herein have a variety of uses.
  • the cells may be used for producing an enzyme protein or peptide that can be further purified to obtain desired amounts of enzyme protein or fragments.
  • Host cells are also useful for conducting cell-based assays involving enzyme protein or enzyme protein fragments, such as those described above as well as other formats known in the art.
  • a recombinant host cell expressing a native enzyme protein is useful for assaying compounds that stimulate or inhibit enzyme protein function.
  • Host cells are also useful for identifying enzyme protein mutants including those with impaired function. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations may be used to assay compounds that have a desired effect on the mutant enzyme protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native enzyme protein.
  • mutant enzyme protein for example, stimulating or inhibiting function
  • a transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. These animals are useful for studying the function of an enzyme protein and identifying and evaluating modulators of enzyme protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians. Any of the enzyme protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, e.g., a mouse and any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the enzyme protein to particular cells.
  • Clones of transgenic animals can also be produced according to the methods described in Wilmut I. et al. (Nature 385: 810-813, 1997) and PCT International Publication Nos. WO 97/07668 and WO 97/0766.
  • Transgenic animals containing recombinant cells that express the peptides described herein are useful to assay in vivo enzyme protein function, including substrate interaction, the effect of specific mutant enzyme proteins on enzyme protein function and substrate interaction, and the effect of chimeric enzyme proteins. It is also possible to use the transgenic animals described herein to assess the effect of null mutations, that is, mutations that substantially or completely eliminate one or more enzyme protein functions.
  • the present invention includes pharmaceutical compositions comprising, e.g., modulators of RHAU enzyme protein activity or nucleic acid expression which may be used to treat a subject with a RHAU related disorder, e.g., neoplasia.
  • the pharmaceutical compositions disclosed herein are to be administered to a patient at therapeutically effective doses to treat or ameliorate such disorders.
  • a therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of said disorder.
  • compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.
  • modulators and/or compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or topical, oral, buccal, parenteral or rectal administration.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • 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 nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, 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 of the 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.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • 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.
  • 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.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example, antisense oligonucleotides, triple helix DNA, ribozymes, RNA aptamer and double stranded RNA designed to inhibit RHAU gene expression, antibodies to RHAU or related regulatory proteins or fragments thereof, useful to treat and/or ameliorate the pathological effects of a RHAU related disorder.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. Pharmaceutical formulations suitable for oral administration of proteins are described, e.g., in U.S.
  • RHAU was isolated in association with the AU-rich element (ARE) of urokinase plasminogen activator mRNA (ARE uPA ) and further characterized as described below.
  • ARE AU-rich element
  • ARE uPA urokinase plasminogen activator mRNA
  • RHAU and RHAU ⁇ 14 E335A mutants are created using QuickChange (Stratagene, La JoIIa, CA) and suitable oligonucleotide primers.
  • QuickChange Stratagene, La JoIIa, CA
  • the protein coding regions of various RHAU and RHAU ⁇ U cDNAs are subcloned between the EcoR1 and Xho1 sites of the baculovirus expression vector pAcGHLT-A (Pharmingen, San Diego, CA).
  • Expression vectors for GST, GST-HuR, and tetracycline-regulated ⁇ -globin or chimeric ⁇ - globin-ARE uPA mRNAs are described in Tran et al., 2003, MoI. Cell. Biol. 23: 7177-7188.
  • RNAs Small interfering RNAs (siRNAs; Xeragon, Germantown, MD) used to target RHAU mRNA are as follows: sense, GGG AAC UGC GAA GAA GGU AUU-3' (SEQ ID NO:5) and antisense, UAC CUU CUU CGC AGU UCC CUU-3 1 (SEQ ID NO:6). These sequences contain 3' UU overhangs and target both RHAU isoforms.
  • Control siRNA sequences sense, GUA CCU GAC UAG UCG CAG AAG (SEQ ID NO:7); antisense, UCU GCG ACU AGU CAG GUA CGG (SEQ ID NO:8). Gene specificity is ensured by searching the oligoribonucleotide sequences using BLAST. Transfection of siRNA duplexes is performed as described in Kisielow et al., 2002, Biochem. J. 363: 1-5.
  • ARE uPA wild-type or mutant sequences are prepared from annealed DNA oligonucleotides containing T7 promoter sequences as described in Tran et al., 2003, MoI. Cell. Biol. 23: 7177-7188.
  • a control RNA sequence derived from the SV40 large T antigen is similarly prepared (only sense strand is shown): TAA TAC GAC TCA CTA TAG GGT GCA ATG TAC TTG CAA AGA ATG GCC TGA GTG TGC AAA GAA AAT GTC TGC T (SEQ ID NO:9).
  • RNA transcripts composed of 140 nucleotides of the rabbit ⁇ -globin 3' UTR without or with the 47 nucleotides ARE uPA are prepared as detailed previously (Fritz et al., 2000, Sci. STKE 2000, PU ).
  • Non-labeled T7 transcripts are covalently linked to adipic acid dihydrazide agarose beads (Sigma) as described in Caputi et al., 1999, EMBO J. 18: 4060- 4067.
  • RNA-bound beads are equilibrated in buffer D (Dignam et al., 1983, Nucleic Acids Res. 11: 1475-1489) and mixed with 1.5 mg of HeLa nuclear extract (Dignam et al., 1983, Nucleic Acids Res. 11 : 1475-1489) at 4 9 C for 2 hrs. Unbound proteins are removed by washing six times with buffer D.
  • Glutathione S-transferase GST
  • GST-HuR proteins are expressed in E. coli BL21(DE3) and purified using glutathione-Sepharose (Amersham, Little Chalfont, UK) according to the supplier's instructions.
  • GST-RHAU proteins are expressed in Sf9 cells according to the supplier's instructions (Pharmingen) and purified as above.
  • HeLa S100 extracts are prepared as described in Fritz et al., 2000, Sci. STKE
  • RNA is resuspended in loading buffer, resolved on a 7 M urea/5% acrylamide gel and analyzed with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA).
  • lmmunoprecipation and GST Pull-Down Assays are performed using HeLa whole-cell extracts (500 ⁇ g), in the absence of presence of RNases A (10 ⁇ g/ml) and T1 (100 U/ml), together with 4 ⁇ g of a mouse anti-HA monoclonal antibody (12CA5) or 10 ⁇ l of anti- hRrp40p, anti-PM-Scl100, and anti-PARN rabbit anti-sera precoupled to a 50% solution of protein A-Sepharose beads (100 ⁇ l; Amersham) for 2 hour at 4° C.
  • RNases A 10 ⁇ g/ml
  • T1 100 U/ml
  • GST pull-down assays are performed with 10 ⁇ g of GST or GST-HuR coupled to a 50% solution of glutathione-Sepharose 4B beads (100 ⁇ l; Amersham) and incubated with HeLa extracts as described above. Precipitated proteins are analyzed by SDS-PAGE and Western blotting.
  • affinity-purified rabbit polyclonal antibody raised against a synthetic peptide corresponding to 17 amino acids RHAU, 991-1007 at the C terminus of RHAU (Davids Biotechnologie, Regensburg, Germany) and the monoclonal antibodies against PKR (p68 kinase; Transduction Laboratories) and DRBP76 (NFAR; BD Biosciences, Bedford, MA) for Westerns are used.
  • Assays are performed as described (Kang et al., 2002, PNAS USA 99: 637-642) with modifications. Each 20 ⁇ l reaction contains 50 mM MOPS, pH 6.5, 3 mM MgCI 2 , 2mM DTT, 0.1 mM ATP 1 5 ⁇ Ci of [ ⁇ - 32 P]ATP (3,000 Ci/mmol; Amersham), 200 ng GST, or GST-RHAU, in the absence or presence of the indicated DNA or RNA species (20 ng/ ⁇ l). Reactions are incubated at 37°C for 30 min., spotted on polyethylene-immine (PEI) cellulose (Merck, Whitehouse Station, NJ) and developed in 0.75 M LiCI/1 M formic acid solution for 20 min. Visualization and quantitation of radioactive signals are performed with a phosphorimager.
  • PEI polyethylene-immine
  • RNAs corresponding to the ARE sequence of uPA mRNA a mutated ARE sequence (ARE MUT), or a control SV40 early message sequence (C RNA) are coupled to agarose beads and used to affinity purify factors from HeLa nuclear extracts.
  • Nuclear proteins which bound to the immobilized RNAs are analyzed by SDS-PAGE.
  • Three proteins are enriched in the ARE WT fraction compared to the ARE MUT or control RNA fraction, or beads alone. These proteins are excised from the gel and further characterized by NanoESI tandem mass spectrometry. Comparison of tryptic peptides against a peptide database (Mann and WiIIm, 1994, Anal. Chem.
  • MLEL1 HuR, NFAR1 and MLE-like 1 (MLEL1) which we renamed RHAU (see below).
  • MLEL1 accession number AF217190
  • Drosophila MLE maleless
  • MLEL1 was named because of sequence similarity to Drosophila MLE (maleless) - a gene required for dosage compensation (Kuroda et al., 1991 , Cell 66: 935-947).
  • our homology searches and alignments of predicted amino acid full-length sequences from different organisms reveal that in Drosophila, there is an uncharacterized sequence (accession number AAF53921) more similar to MLEL1 than MLE.
  • RHAU is a DExH/D RNA Helicase that is Expressed in Two isoforms
  • RHAU belongs to the DExH/D family of ATP-dependent RNA helicases and contains an evolutionary-conserved RNA helicase core region of approximately 440 amino acids which is composed of at least six discrete functional domains (data not shown).
  • the protein coding region of RHAU is amplified by RT-PCR using total RNA isolated from the human monocytic leukemia cell line THP- 1. PCR products are cloned into a HA-tagged eukaryotic expression vector (pcDNA3-HA) and sequenced. Two isoforms of RHAU are obtained, which was confirmed by two independent RT-PCR experiments.
  • RHAU The cellular localization of RHAU is examined by expressing HA-RHAU and HA- RHAU ⁇ 14 in HeLa-Tet-Off (TO) cells, followed by immunocytochemistry. Levels of expression of both RHAU isoforms are examined by Western blotting using an anti-HA epitope antibody. Using the same antibody for indirect immunofluorescence, we found that the two RHAU isoforms displayed distinct subcellular localization. RHAU and RHAU ⁇ 14 are preferentially localized to the nucleus and cytoplasm, respectively. However, partitioning of cellular localization between the nucleus and cytoplasm was not exclusive, as the staining of full length HA-RHAU was also obvious in the cytoplasm. Both isoforms are excluded from the nucleoli (data not shown). These results suggest that RHAU contains a putative nuclear localization sequence of at least 14 amino acids.
  • RHAU was identified as an ARE uPA -associated factor
  • This ⁇ -globin- ARE uPA ( ⁇ -glo-ARE) reporter gene under the control of a tetracycline-responsive element, is stably expressed in HeLa-TO cells and the de novo synthesis of ⁇ -glo-ARE mRNA could be selectively terminated by addition of doxycycline, a tetracycline derivative.
  • ⁇ -glo-ARE mRNA has a half-life (t 1/2 ) of approximately 3 hours in vector-transfected cells (data not shown).
  • HA-RHAU modestly reduced the stability of ⁇ -glo-ARE mRNA (ti /2 approx. 2 hours) (data not shown). Notably, a more significant reduction of ⁇ - glo-ARE mRNA stability (3-fold) is observed when the cytoplasmically localized HA-RHAU ⁇ 14 is overexpressed (data not shown).
  • Overexpression of HA-RHAU and HA-RHAU ⁇ U in HeLa-TO cells stably expressing a ⁇ -globin wild-type mRNA that has an extrapolated half-life of more than 15 hours did not affect the stability of this message (data not shown).
  • RNA substrate is neither deadenylated or degraded in the absence of HeLa S100 proteins (data not shown).
  • GST-RHAU does not significantly stimulate the decay of a non-ARE-containing (glo-A60) RNA substrate and deadenylation was only slightly stimulated (data not shown).
  • GST- RHAU ⁇ U produced similar, if not identical, results in the assays described above (data not shown). These results complement in vivo data with respect to ARE uPA -directed mRNA decay and its enhancement by RHAU.
  • the ATPase Activity of RHAU is Required for Enhancement of mRNA Destabilization In Vivo and In Vitro
  • ATPase activity of DExH/D RNA helicases are known to be stimulated by nucleic acids.
  • RHAU ⁇ U (results described below are equally applicable to RHAU, data not shown) has ATPase activity in vitro and if this activity is enhanced in the presence of dsDNA, dsRNA, tRNA or homopolymeric RNAs.
  • [ ⁇ - 32 P] ATP hydrolysis by recombinant GST- RHAU ⁇ 14 is modestly increased (approx. 2 fold) with tRNA, dsDNA, dsRNA and poly (A 1 C), but markedly stimulated (> 4-fold) by poly(U) (data not shown).
  • HA- RHAU ⁇ 14 and RHAU ⁇ 14 -E335A are transiently transfected in HeLa-TO- ⁇ -globin- ARE uPA cells. Similar expression levels are confirmed for both wild-type and mutant versions of HA- RHAU ⁇ 14 (data not shown). Consistent with results already shown, HA- RHAU ⁇ 14 significantly enhanced the destabilization of ⁇ -globin-ARE-mRNA (data not shown).
  • HA-RHAU ⁇ 14 -E335A did not enhance ⁇ -globin-ARE mRNA decay but even had a moderate, but not significant, negative effect on ARE uPA -mediated mRNA turnover (data not shown).
  • Parallel effects are observed in cell-free RNA decay assays where GST-RHAU ⁇ 14 , but not GST-RHAU ⁇ 14 -E335A, could enhance ARE-RNA deadenylation and decay (data not shown).
  • E335 in the DEIH motif of RHAU is essential for its ATPase activity and that this activity is required for the function of RHAU in promoting mRNA deadenylation and decay.
  • RHAU Physically Interacts with the Exosome and Poly A Specific Ribonuclease (PARN) and Exhibits RNA-Dependent Interaction with NFAR1 and HuR
  • NFAR1 and HuR are also dependent on RNA.
  • a GST pull-down assay reveals that NFAR1 and RHAU is coprecipitated with GST- HuR from lysates not treated with RNases but this interaction is not detected using RNase-treated lysates (data not shown).
  • PKA protein kinase R
  • PKR is an interferon- and dsRNA-activated kinase known to play a major role in the antiviral response and induction of apoptosis.
  • PKR is known to interact with and phosphorylate DRBP76/NFAR1 in vitro.
  • RHAU is a DExH RNA helicase and is an ARE uPA - associated factor. It was demonstrated that RHAU plays a role in ARE ⁇ PA - mRNA deadenylation and decay and this specificity may be linked to its RNA-dependent interaction with the AU binding proteins (AUBPs) HuR and NFAR1. Additionally, we demonstrated a physical association between RHAU and the human exosome, and the deadenylase PARN (Dehlin et al. 2000, EMBO J. 19: 1079-1086). A causal role for RHAU in ARE uPA -mRNA metabolism is confirmed by overexpression and downregulation experiments.
  • AUBPs AU binding proteins
  • RHAU Overexpression of RHAU enhanced the degradation of uPA and reporter ⁇ -globin-ARE uPA mRNAs, but not GAPDH or a non-ARE uPA -containing ⁇ -globin mRNA, while its downregulation by RNA interference stabilized endogenous uPA mRNA. Furthermore, recombinant RHAU in cell-free RNA decay reactions accelerated the decay of an ARE uPA -containing RNA but not a non-ARE RNA substrate. Importantly, it was demonstrated that the enhancement of ARE uPA -mRNA- destabilization by RHAU is dependent on its ability to hydrolyze ATP.
  • the exosome is functionally coupled to a scavenger decapping activity that follows mRNA deadenylation and decay. It has previously been proposed that this degradation-dependent mRNA decapping activity is complexed to a subset of exosome components specific for 3'-to-5' mRNA decay.
  • RNA helicases which by their functional nature should translocate rapidly along and unwind dsRNA or disrupt RNA-protein interactions, can specifically recognize their RNA substrates.
  • the demonstration that an RNA helicase can directly interact with a specific RNA sequence is lacking, and it is widely believed that substrate specificity is mediated indirectly by protein-protein interactions.
  • Our data supports the idea that the specificity of RHAU toward selective degradation of ARE uPA -mRNA is mediated by its RNA- dependent interaction with the AUBPs HuR and NFAR1. This interaction is demonstrated by ARE-affinity chromatography, NFAR1 coimmunoprecipitation and GST-HuR pull down experiments.
  • RNA helicases are associated with ribonucleases (like the exosome) and specific RNA binding protein complexes to entail target specificity of RNA degradation.
  • others have described a protein complex that contains, among other components, a double-stranded RNA binding protein, the RNase Ill-related Dicer and a DExH RNA helicase that is required to direct RNA interference in Caenorhabditis elegans.
  • the DExH protein Suv3p has been shown to be a component of a yeast mitochondrial 3'-to-5' exoribonuclease activity that is required for degradation of group I intron RNAs.
  • ATP-dependent RNA helicases serve as "molecular motors" coupled to specific RNA binding proteins to drive the mechanics of complex RNA remodeling/decay reactions.
  • DExH/D proteins possess the ability to unwind dsRNA.
  • the primary function of DExH/D RNA helicases may be to disrupt RNA-protein interactions.
  • RHAU RNA-protein interactions
  • HuR and/or NFAR1 mRNA- stabilizing proteins
  • NFAR1 mRNA- stabilizing proteins
  • the exosome may be recruited to the ARE by mRNA- destabilizing AUBPs to begin the rapid exonucleolytic decay of the RNA body.
  • mRNA- destabilizing AUBPs to begin the rapid exonucleolytic decay of the RNA body.
  • RHAU is Important for the Survival of Cancer Cells: RHAU Depletion in Tumor and Non-Tumor Derived Cell Lines
  • HeLa Tet-off cells (Clontech) are maintained in Dulbecco's Modified Eagle's
  • DMEM fetal calf serum
  • ANIMED BioConcept, Allschwil, Switzerland
  • streptomycin 50 units/mL penicillin andlOO ng/ml G-418 (Invitrogen) at 37° C in a humidified 5% CO 2 incubator.
  • PNT-2 and PC3 cells are maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum and the antibiotics described above, except G-418.
  • MCF-10A cells are maintained in DMEM supplemented with 10% (v/v) horse serum and the antibiotics described above, except G-418.
  • the 21-mer oligoribonucleotide sequence of siRNAs used to target RHAU mRNA are as follows: sense, 5'-GGG AAC UGC GAA GAA GGU AUU-3' (SEQ ID NO:5) and antisense, 5'-UAC CUU CUU CGC AGU UCC CUU-3' (SEQ ID NO:6). These sequences contain 3' UU overhangs and target both RHAU isoforms.
  • siRNA sequences targeting NFAR1 are as follows: sense, 5'-AAC UUC UCC CGC CUC UUG UAA-3 1 (SEQ ID NO:10) and antisense, 5'-UUA CAA GAG GCG GGA GAA GUU CGC CUC UUG UAA-3 1 (SEQ ID NO:11).
  • sense 5'-AAC UUC UCC CGC CUC UUG UAA-3 1
  • antisense 5'-UUA CAA GAG GCG GGA GAA GUU CGC CUC UUG UAA-3 1
  • siRNAs were purchased from Xeragon (Huntsville, Alabama).
  • siRNA duplexes A day before transfection of small interfering RNA (siRNA) duplexes, cells are seeded in 35-mm dishes at 1.4 x 105 cell/dish. The next morning, siRNAs are introduced into cells using OligofectAMINE reagent (Life Technologies) according to the manufacturer's intructions, with 10 ⁇ of 20 ⁇ M siRNA and 3 ⁇ l of transfection reagent/dish. Cells are photographed and collected for protein/RNA analysis according to conventional methods two days after transfection.
  • OligofectAMINE reagent Life Technologies
  • PNT-2 and MCF10A are cell lines derived from normal prostate and breast tissue, respectively. The ability of these cells to proliferate and repopulate the culture dish to confluency was not affected 48 hours after transfection with siRNA against RHAU (siRHAU) to downregulate endogenous RHAU protein levels. Similarly, cells transfected with siNFARI showed no effects on cell growth compared with a buffer control. In contrast, PC3 (prostate cancer) cells and HeLa (cervical adenocarcinoma) cells showed signs of apoptotic cell death 48 hours after transfection with siRHAU.
  • RHAU With siNFARI transfection, these cells show impaired proliferation but no significant cell death. Thus, depletion of RHAU appeared to significantly affect the survival of tumor-derived cell lines, suggesting that it is essential for cancer cell biology. If the cell death observed in RHAU- depleted cells is caused by apoptosis, then RHAU may have an anti-apoptotic function or be connected to such cellular mechanisms. Future studies to verify and extend these studies are warranted and may be greatly assisted by targeted gene inactivation of RHAU in mice. As RHAU specific siRNA preferentially suppress the growth of transformed cells it is possible that binding partners of RHAU are different in transformed cells compared to non-transformed cells. Identification of transformed cell-specific partners then may provide additional targets for tumor therapy.
  • antibodies against RHAU can be used in co-precipitation analysis using cell extracts derived from transformed and non-transformed cells. Binding partners can then be identified by SDS-polyacrylamide gel electrophoresis followed by mass spectrometry. For example, antibodies against HA and GST can be used after these cells are transfected with expression vectors for HA-tagged and GST- tagged RHAU.
  • RNAi- mediated RHAU downregulation may be performed in cultured cells of various types using RHAU-specific siRNA according to conventional methods.
  • the prostate and breast cancer cell lines PC3 and MDA-MB-231, respectively died 4 days after RHAU downregulation, while their normal counter parts PNT-2 and MCF-1 OA, respectively, did not.
  • RHAU is essential for the survival of tumor cells but is dispensable for non-transformed cells.
  • studies using a RHAU expression vector with silent mutations at the siRNA-targeting site may be performed and overexpression of RHAU with silent mutations simultaneously with or before RHAU-specific siRNA transfection should alleviate the growth inhibitory effect of siRNA in tumor cells if the inhibition described above is due to RHAU downregulation.
  • These "knockdown-in” studies may be performed according to conventional methods and as previously reported in Kisielow, M et al., 2002, Biochem. J. 363: 1-5.
  • Patterns of global mRNA expression in MDA-MB- 231 or PC3 cells before and after RHAU downregulation may be compared by means of an Affymetrix GeneChip Array using Human Genome U133 Set. mRNAs exhibiting a greater than twofold difference, either positively or negatively, will be selected and reconfirmed individually by Northern blot hybridization. Confirmed mRNAs may then be characterized for regulation of stability by RHAU and for the presence of AREs or common features in the 3' UTRs. The identification of these mRNAs will provide useful insight about the mechanism by which RHAU affects tumor cell growth.
  • Possible non-specific effects of siRNA reactions may be controlled for by preparing HeLa cell lines in which RHAU-specific siRNA can be induced by tetracycline according to methods known in the art which circumvent the lipid- mediated transfection step (van de Wetering, M et al., 2003, EMBO Rep. 4: 609- 615).
  • a HeLa cell line stably expressing a tetracycline-suppressible repressor is available (van de Wetering, M et al., 2003, EMBO Rep. 4: 609-615).
  • the patterns of global mRNA expression in this cell line before and after RHAU- specific siRNA expression may be compared as outlined above.
  • the RHAU gene is composed of 25 exons with the Met start codon residing in the first exon.
  • the first intron includes no open reading frames that could be linked to a downstream coding region without hitting termination codons, it is very unlikely that deletion of the first exon would give rise to an active, truncated RHAU protein. Therefore, a targeting (KO1) vector that replaces the first exon of the murine RHAU gene with a neomycin-resistance gene may be constructed according to methods familiar to one of skill in the art. This vector contains 3 kb and 7 kb of the 5' and 3' flanking regions, respectively, of the first exon of the RHAU gene.
  • the vector may be injected into embryonic stem cells and correctly targeted cells selected by conventional PCR analysis and further processed to derive KO mice according to conventional methods as can be found in, e.g., Joyner, AL, ed., Gene Targeting - A Practical Approach, 2nd ed., Oxford University Press.
  • a targeting vector (KO2) can be constructed in which exon 8 of the RHAU gene is flanked by the LoxP sequences. Exon 8 codes the DExH box, which is essential for RHAU ATPase activity and for stimulating ARE-mRNA decay.
  • the LoxP-RHAU mice may be crossed with mice expressing Cre in brain or other tissues under tissue-specific promoters.
  • RHAU In addition to the RNA helicase core region in the middle of the RHAU, which is highly conserved among many DExH-box RNA helicases, RHAU contains unique amino-terminal and carboxyl-terminal regions that are not shared with other RNA helicases. These regions are conserved as highly as the RNA helicase core regions between human and mice, suggesting that they have important functions. To reveal the specific functions associated with these regions, conventional GST- fusion protein pull-down assays may be used to search for proteins interacting with these regions. Conventional methodologies may be used to fuse the amino terminal or carboxyl terminal region of RHAU with GST in a bacterial expression vector in which expression is under control of IPTG.
  • the hybrid protein may then be coupled to glutathione-Sepharose beads, which may then be used for pulldown assays using nuclear or cytoplasmic extracts from HeLa cells.
  • Specifically bound proteins can be separated by 2-D gel electrophoresis and identified by mass spectrometry.
  • Specific binding proteins identified in this analysis will be tested for their interaction with RHAU both in a cell-free system using purified samples and in the cell using appropriate expression vectors. Once their interaction with RHAU is established, these proteins may be studied with regard to localization in the cell and indispensability for cell growth.

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Abstract

L'invention concerne des séquences d'acides aminés d'une hélicase d'ARN humaine, RHAU. L'invention concerne plus précisément des molécules d'acides nucléiques et des peptides isolés ainsi que des procédés d'identification de modulateurs de molécules d'acides nucléiques et de peptides RHAU.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010034700A1 (fr) * 2008-09-24 2010-04-01 Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Traitement du cancer par modulation d’arn hélicases
WO2010128128A1 (fr) * 2009-05-08 2010-11-11 Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Souris inactivée pour dhx36/rhau utilisée comme modèle expérimental de la dystrophie musculaire

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001057190A2 (fr) * 2000-02-03 2001-08-09 Hyseq, Inc. Acides nucleiques et polypeptides
WO2002030268A2 (fr) * 2000-10-13 2002-04-18 Eos Biotechnology, Inc. Procedes de diagnostic du cancer de la prostate, compositions et procedes de criblage de modulateurs du cancer de la prostate
WO2005001031A2 (fr) * 2003-05-22 2005-01-06 Isis Pharmaceuticals, Inc. Modulation de la voie d'interference arn

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001057190A2 (fr) * 2000-02-03 2001-08-09 Hyseq, Inc. Acides nucleiques et polypeptides
WO2002030268A2 (fr) * 2000-10-13 2002-04-18 Eos Biotechnology, Inc. Procedes de diagnostic du cancer de la prostate, compositions et procedes de criblage de modulateurs du cancer de la prostate
WO2005001031A2 (fr) * 2003-05-22 2005-01-06 Isis Pharmaceuticals, Inc. Modulation de la voie d'interference arn

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Title
TRAN HOANH ET AL: "Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU." MOLECULAR CELL. 16 JAN 2004, vol. 13, no. 1, 16 January 2004 (2004-01-16), pages 101-111, XP002386855 ISSN: 1097-2765 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010034700A1 (fr) * 2008-09-24 2010-04-01 Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Traitement du cancer par modulation d’arn hélicases
WO2010128128A1 (fr) * 2009-05-08 2010-11-11 Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Souris inactivée pour dhx36/rhau utilisée comme modèle expérimental de la dystrophie musculaire

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