WO1999035275A1 - Sequences genetiques de l'homme homologues des genes de levures impliques dans le traitement - Google Patents

Sequences genetiques de l'homme homologues des genes de levures impliques dans le traitement Download PDF

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Publication number
WO1999035275A1
WO1999035275A1 PCT/ES1999/000001 ES9900001W WO9935275A1 WO 1999035275 A1 WO1999035275 A1 WO 1999035275A1 ES 9900001 W ES9900001 W ES 9900001W WO 9935275 A1 WO9935275 A1 WO 9935275A1
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WIPO (PCT)
Prior art keywords
seq
sequences
proteins
sequence
human
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PCT/ES1999/000001
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English (en)
Spanish (es)
Inventor
José María PEREZ FREIJE
Gloria Velasco Cotarelo
Alberto Martin Pendas
Pilar Blay Albors
Milagros Balbin Felechosa
Carlos Lopez Otin
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Universidad De Oviedo
Fuji Yakuhin Kogyo Kabushiki Kaisha
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Application filed by Universidad De Oviedo, Fuji Yakuhin Kogyo Kabushiki Kaisha filed Critical Universidad De Oviedo
Publication of WO1999035275A1 publication Critical patent/WO1999035275A1/fr

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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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention is attached to the field of human oncology. Specifically, the present invention is about the enzymes that process pre-packed proteins and the genes that encode them. More particularly, the present invention addresses the identification of human proteases, and their possible inhibitors, responsible for post-translational modifications suffered by ras and other related proteins to activate and perform their pathological functions.
  • Ras proteins are part of a guanine nucleotide binding protein superfamily that, after being activated by mutation, have the ability to transform eukaryotic cells. Mutated or oncogenic forms of ras genes have been found in a very significant percentage of human tumors, reaching 50% in colon and pancreas carcinomas (Annu. Rev. Biochem., 56, 779, (1987)). These observations indicate that ras genes contribute to the development of various types of human tumors, thus being molecular targets of therapeutic intervention.
  • Ras proteins are synthesized in the cell cytoplasm as precursor molecules that require various post-translational modifications to be inserted into the membrane and perform their biological functions there.
  • the first of these modifications consists in the precursor or farnesylation of a cysteine residue, located in the carboxyl-terminal region, and forming part of a sequence. Cys-AAX, where A is usually an aliphatic amino acid and X is any amino acid. After pre-pilation, the three residues adjacent to the pre-piled cysteine are removed proteolytically and the resulting carboxyl group is methylated.
  • the Rce-1 protein can contribute to the proteolytic processing of both factor-a and yeast ras proteins.
  • the description of these two proteins opens the possibility of searching for analogous proteins in humans through a "homology cloning" strategy.
  • One of the multiple ways of approaching this strategy seeks to search in publicly accessible databases of fragments of sequences of nucleotides of human genes that have similarity to the sequences of the AFC1 and RCE1 genes of Saccharomyces cerevisiae.
  • the hypothetical homologous fragments can be amplified by PCR of total RNA from human tissues in which the expression of said genes is suspected, and used as probes to hybridize cDNA libraries prepared from RNA of the same tissues .
  • An object of the present invention is to identify the human gene that encodes a protein homologous to the Afc-1 protein of Saccharomyces cerevisiae.
  • Another object of the invention is to identify the human gene that encodes a protein homologous to the Rce-1 protein of Saccharomyces cerevisiae.
  • a third object of the invention is to analyze the expression in human tissues of the homologous genes to AFC1 and RCE1 of Saccharomyces cerevisiae.
  • the first object of the invention was the identification of a human gene that could encode a protein homologous to the Afc-1 protein of Saccharomyces cerevi - siae. For this, the amino acid sequence described for this protein was compared with the "Expressed Sequence Tags" (ESTs) division of the GenBank database used. leaving the TBLASTN program (J. Mol. Biol. 215, 403, (1990)). Six overlapping human ESTs were identified, whose access numbers are AA210930, F11310, Z43272, R54272, T35312 and N76181.
  • AFC1 (5 '-ATGAGGAGGTACTCGCTGTACTAGG-3') and AFC2 (5 '-GCTGGAACATGCTGCCCAGGAC-3').
  • AFC2 5 '-GCTGGAACATGCTGCCCAGGAC-3'.
  • the resulting 516 base pair (bp) DNA fragment was purified by agarose gel electrophoresis and extraction with GeneClean. The identity of the amplified fragment with the partial sequence deduced for Face-1 was verified after subcloning it into pUC18 and determining its nucleotide sequence by standard Molecular Biology techniques.
  • nucleotide sequence of this plasmid was determined by the method of chain terminators described by Sanger (PNAS, 74 5463, (1977)). Sequencing revealed the existence of an open reading phase, which encodes a 475 amino acid protein that we call human Face-1. The comparison of this amino acid sequence with all the sequences present in the publicly accessible databases showed that the highest degree of similarity (40%) corresponded to the Afc-1 protein of Saccharomyces cerevisiae. A significant degree of similarity was also detected with the so-called hypothetical protein p59 of Schizosaccharomyces pombe and with an Escherichia coli protein of unknown function and called htpX.
  • Face-1 is the human homologue of the Afc-1 protein of Saccharomyces cerevisiae and therefore its participation in the proteolytic maturation of prenilated proteins is presumable.
  • Both the isolated DNA and the encoded polypeptide, represented in SEQ ID NO: 1, as partial sequences obtained from both, can also be chemically synthesized.
  • the second object of the invention was the identification of a human gene that could encode a protein homologous to the Rce-1 protein of Saccharomyces cerevisiae. For this, the amino acid sequence described for this protein was compared with the "Expressed Sequence Tags" (ESTs) division of the GenBank database using the TBLASTN program.
  • the isolated phage DNA was converted into the corresponding pDR2 plasmids by excision in vivo, following the instructions of the library provider. Analysis of the four plasmids revealed that they all contained cDNA inserts of similar size.
  • the nucleotide sequence of the clone we call 1. Ib was determined by the method of chain terminators described by Sanger (PNAS, 74, 5463, (1977)). Sequencing revealed the existence of an open reading phase, which encodes a 329 amino acid protein that we call human Face-2. The comparison of this amino acid sequence with all the sequences present in the publicly accessible databases showed that the highest degree of similarity (30%) corresponded to the Rce-1 protein of Saccharomyces cerevisiae.
  • the third object of the invention is to analyze the expression in human tissues of the homologous genes to AFC1 and RCE1 of Saccharomyces cerevisiae.
  • two membranes containing polyadenylated RNA from multiple human tissues are they hybridized with the radioactively labeled Face-1 and Face-2 probes.
  • Two micrograms of polyadenylated RNA from the indicated tissues were hybridized with the Face-1 and Face-2 cDNAs.
  • FIG. 1A As can be seen in Figure 1A, after hybridization with the Face-1 probe, a minor RNA of approximately 3.5 kilobases was detected in all the tissues analyzed. Similarly, when the filters hybridized with the Face-2 probe, a messenger RNA was detected. about 1.5 kilobases in all tissues, being especially abundant in testis.
  • the ubiquitous expression of the Face-1 and Face-2 genes is consistent with the wide tissue distribution of pre-packed proteins, which in turn implies the need for the proteases responsible for their maturation to be present in all body tissues.
  • FIG. 1 Northern analysis of human tissue expression of the Face-1 (A), Face-2 (B) genes and an Actin (C) control that is expressed in all tissues.
  • the size of the RNAs used as markers is indicated on the left.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des séquences génétiques de l'homme homologues des gènes de levures impliqués dans le traitement protéolytique de protéines prénylées. L'invention consiste à identifier des fragments homologues de gènes humains AFC1 et RCE1 de Saccharomyces cerevisiae, à assurer leur amplification par réaction en chaîne de la polymérase de l'ARN total, à utiliser les fragments ainsi amplifiés comme sondes pour hybrider l'ADNc et déterminer la séquence des clônes d'ADNc qui s'hybrident avec les sondes. Les séquences identifiées sont SEQ ID NO: 1 et SEQ ID NO: 2. Ces séquences présentent une grande utilisation pour le diagnostic et le traitement des maladies oncogènes.
PCT/ES1999/000001 1998-01-08 1999-01-08 Sequences genetiques de l'homme homologues des genes de levures impliques dans le traitement WO1999035275A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES9800016A ES2149672B1 (es) 1998-01-08 1998-01-08 Secuencia genica humana homologa al gen afc1 de levaduras implicado enel procesamiento proteolitico de proteinas preniladas.
ESP9800016 1998-01-08

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WO1999035275A1 true WO1999035275A1 (fr) 1999-07-15

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WO (1) WO1999035275A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998005786A2 (fr) * 1996-08-07 1998-02-12 The Regents Of The University Of California Enzymes de transformation afc1 et rce1: caax isoprenyles
WO1998054333A2 (fr) * 1997-06-02 1998-12-03 Acacia Biosciences Inc. Enzymes de maturation caax de mammifere
EP0887415A2 (fr) * 1997-06-24 1998-12-30 Smithkline Beecham Corporation RCE1 humain
EP0887416A2 (fr) * 1997-06-24 1998-12-30 Smithkline Beecham Corporation Enzyme AFC1 humaine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998005786A2 (fr) * 1996-08-07 1998-02-12 The Regents Of The University Of California Enzymes de transformation afc1 et rce1: caax isoprenyles
WO1998054333A2 (fr) * 1997-06-02 1998-12-03 Acacia Biosciences Inc. Enzymes de maturation caax de mammifere
EP0887415A2 (fr) * 1997-06-24 1998-12-30 Smithkline Beecham Corporation RCE1 humain
EP0887416A2 (fr) * 1997-06-24 1998-12-30 Smithkline Beecham Corporation Enzyme AFC1 humaine

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ASHBY M.N. ET AL.: "Ras and a - factor converting enzyme", METHODS IN ENZYMOLOGY,, vol. 250, 1995, pages 235 - 251 *
BASSETT D.E. ET AL.: "Genome cross-referencing and XREF db: Implications for the identification and analysis of genes mutated in human disease", NATURE GENETICS,, vol. 15, 1997, pages 339 - 344 *
BOYARTCHUK V.L. ET AL.: "Modulation of Ras and a-Factor funtion by carboxylterminal proteolysis", SCIENCE,, vol. 275, 1997, pages 1796 - 1800 *
FUJIMURA KAMADA K. ET AL.: "A novel membrane-associated metalloprotease, Step 24, is required for the first step of NH2-terminal processing of the yeast a - factor precursor", THE JOURNAL OF CELL BIOLOGY,, vol. 136, no. 2, 1997, pages 271 - 285 *
TAM A. ET AL.: "Dual roles for Step 24 in yeast a - Factor maturation: NH2- terminal proteolysis and COOH-terminal CAAX processing", THE JOURNAL OF CELL BIOLOGY,, vol. 142, no. 3, 1998, pages 635 - 649 *

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Publication number Publication date
ES2149672A1 (es) 2000-11-01
ES2151459B1 (es) 2001-07-01
ES2149672B1 (es) 2001-05-16
ES2151459A1 (es) 2000-12-16

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