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WO1999035275A1 - Human genetic sequences which are homologous to the yeast genes involved in the proteolytic processing of prenylated proteins - Google Patents

Human genetic sequences which are homologous to the yeast genes involved in the proteolytic processing of prenylated proteins Download PDF

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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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seq
sequences
proteins
sequence
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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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  • 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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Abstract

Human genetic sequences which are homologous to the yeast gene involved in the proteolytic processing of prenylated proteins. The invention comprises the identification of homologous fragments of human genes AFC1 and RCE1 of Saccharomyces cerevisiae, amplifying them through PCR of total RNA, using the amplified fragments as probes to hybridize cDNA and determining the sequence of the clones of cDNA which hybridize with the probes. The identified sequences are SEQ ID NO: 1 and SEQ ID NO: 2. The applications of said sequences concern essentially diagnosis and treatment of oncogenic troubles.

Description

Secuencias génicas humanas homologas a los genes de levaduras implicados en el procesamiento proteolítico de proteínas preniladas.Human gene sequences homologous to yeast genes involved in proteolytic processing of pre-piled proteins.
Campo de la invenciónField of the Invention
La invención se adscribe al campo de la oncología humana. En concreto la presente invención versa sobre las enzimas procesadoras de proteínas preniladas y sobre los genes que las codifican. Más particularmente, la presente invención aborda la identificación de las proteasas humanas, y de sus .posibles inhibidores, encargadas de las modificaciones post-traduccionales que sufren las proteínas ras y otras relacionadas para activarse y poder desempeñar sus funciones patológicas .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.
Estado de la técnicaState of the art
Las proteínas ras forman parte de una superfamilia de proteínas enlazantes de nucleótidos de guanina que, tras activarse por mutación, tienen la capacidad de transformar las células eucariotas . Las formas mutadas u oncogénicas de los genes ras se han encontrado en un porcentaje muy significativo de tumores humanos, alcanzando el 50% en los carcinomas de colon y páncreas (Annu. Rev. Biochem. , 56 , 779, (1987)). Estas observaciones indican que los genes ras contribuyen al desarrollo de diversos tipos de tumores humanos, siendo por tanto dianas moleculares de interven- ción terapéutica.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.
Las proteínas ras se sintetizan en el citoplasma celular como moléculas precursoras que requieren diversas modificaciones post-traduccionales para insertarse en la membrana y desempeñar allí sus funciones biológicas. La primera de estas modificaciones consiste en la prenilación o farnesilación de un residuo de cisteína, localizado en la región carboxilo-terminal , y formando parte de una secuen- cia Cys-A-A-X, donde A suele ser un aminoácido alifático y X cualquier aminoácido. Tras la prenilación, los tres residuos adyacentes a la cisteína prenilada son eliminados pro- teolíticamente y el grupo carboxilo resultante es metilado. Hasta el momento se han identificado diversas proteínas que participan en las etapas de prenilación y metilación de las proteínas ras y de otras proteínas que experimentan modificaciones post-traduccionales análogas, y entre las que se pueden citar feromonas fúngicas como el factor-a de lévadura, las subunidades γ de diversas proteínas G triméricas o pequeñas proteínas enlazantes de GTP implicadas en el tráfico vesicular celular (Annu. Rev. Biochem. 6JL, 355, (1992)) . Sin embargo, actualmente no se conoce ninguna proteína humana responsable de la eliminación proteolítica de los residuos adyacentes a la cisteína prenilada, etapa esencial en la maduración de ras y proteínas relacionadas.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. So far, several proteins have been identified that participate in the pre-translation and methylation stages of ras proteins and other proteins that undergo analogous post-translational modifications, and among which fungal pheromones such as the yeast factor-a, γ subunits of various trimeric G proteins or small GTP binding proteins involved in cell vesicular traffic (Annu. Rev. Biochem. 6JL, 355, (1992)). However, there is currently no known human protein responsible for the proteolytic removal of residues adjacent to the pre-piled cysteine, an essential stage in the maturation of ras and related proteins.
Una de las estrategias para la identificación de las proteasas humanas activadoras de proteínas preniladas consistiría en la búsqueda de proteínas homologas en otras especies filogenéticamente alejadas, como son las levaduras, pero que también son capaces de desarrollar reacciones análogas de procesamiento proteolítico en secuencias Cys-A- A-X. En este sentido, cabe señalar que recientemente Boyartchuk et al., (Science 275, 1797, (1997)) han descrito la existencia en Saccharomyces cerevisiae de dos proteínas (Afc-1 y Rce-1) que participan en la maduración proteolítica de proteínas preniladas. La proteína Afc-1 se ha descrito como una metaloproteasa que participa en el procesamiento de la feromoma denominada factor-a. Por el con- trario, la proteína Rce-1 puede contribuir al procesamiento proteolítico tanto de factor-a como de proteínas ras de levaduras. La descripción de estas dos proteínas abre la posibilidad de búsqueda de proteínas análogas en humanos a través de una estrategia de "clonación por homología" . Una de las múltiples formas de abordar esta estrategia, persigue en un primer paso la búsqueda en bancos de datos accesibles públicamente, de fragmentos de secuencias de nucleótidos de genes humanos que tengan similitud con las secuencias de los genes AFC1 y RCE1 de Saccharomyces cerevisiae . Tras su identificación, los hipotéticos fragmentos homólogos se pueden amplificar mediante PCR de RNA total de tejidos humanos en los que se sospeche la expresión de dichos genes, y utilizarlos como sondas para hibri- dar genotecas de cDNA preparadas a partir de RNA de los mismos tejidos. Finalmente, la secuenciación y posterior caracterización de los clones humanos aislados mediante técnicas estándar de Biología Molecular, permitiría confirmar el posible papel de las proteínas codificadas por dichos clones en el procesamiento proteolítico de proteínas preniladas. Basándose en esta idea, los autores de la invención, tras los pertinentes estudios experimentales, han llegado a los objetivos antes enumerados que constituyen los diversos aspectos de la presente invención.One of the strategies for the identification of human protease activators of pre-piled proteins would be the search for homologous proteins in other phylogenetically remote species, such as yeasts, but which are also capable of developing analogous proteolytic processing reactions in Cys-A sequences. - AX. In this regard, it should be noted that recently Boyartchuk et al., (Science 275, 1797, (1997)) have described the existence in Saccharomyces cerevisiae of two proteins (Afc-1 and Rce-1) that participate in protein proteolytic maturation piled up. The Afc-1 protein has been described as a metalloprotease that participates in the processing of the pheromome called factor-a. On the contrary, 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, in a first step, 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. Upon identification, 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 . Finally, the sequencing and subsequent characterization of human clones isolated by standard Molecular Biology techniques would confirm the possible role of the proteins encoded by said clones in the proteolytic processing of pre-piled proteins. Based on this idea, the authors of the invention, after the relevant experimental studies, have reached the objectives listed above that constitute the various aspects of the present invention.
Breve descripción de la invenciónBrief Description of the Invention
Un objeto de la presente invención es identificar el gen humano que codifica una proteína homologa a la proteína Afc-1 de Saccharomyces cerevisiae .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.
Otro objeto de la invención es identificar el gen humano que codifica una proteína homologa a la proteína Rce-1 de 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.
Un tercer objeto de la invención es analizar la expresión en tejidos humanos de los genes homólogos a AFC1 y RCE1 de 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.
Descripción detallada de la invenciónDetailed description of the invention
El primer objeto de la invención consistió en la identificación de un gen humano que pudiera codificar una proteína homologa a la proteína Afc-1 de Saccharomyces cerevi - siae . Para ello la secuencia de aminoácidos descrita para esta proteína se comparó con la división de "Expressed Sequence Tags" (ESTs) de la base de datos GenBank utili- zando el programa TBLASTN (J. Mol. Biol . 215, 403, (1990)). Se identificaron seis ESTs humanas solapantes, cuyos números de acceso son AA210930, F11310, Z43272, R54272, T35312 y N76181. Del solapamiento de estas ESTs, dedujimos una secuencia parcial de nucleótidos, que codifica un fragmento de una hipotética proteína humana con un grado de similitud de aproximadamente 40% con la proteína Afc-1 de Saccharomyces cerevisiae . Esta proteína humana fue denominada tentativamente Face-1, (Farnesylated-proteins cpnverting enzyme 3.) . Su secuencia de aminoácidos, así como la secuencia nucleotídica que la codifica se muestra como SEQ ID NO : 1.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. From the overlap of these ESTs, we deduced a partial nucleotide sequence, which encodes a fragment of a hypothetical human protein with a degree of similarity of approximately 40% with the Afc-1 protein of Saccharomyces cerevisiae. This human protein was tentatively called Face-1, (Farnesylated-proteins cpnverting enzyme 3.). Its amino acid sequence, as well as the nucleotide sequence that encodes it, is shown as SEQ ID NO: 1.
A partir de esta secuencia diseñamos y sintetizamos dos oligonucleótidos, AFC1 (5 ' -ATGAGGAGGTACTCGCTGTACTAGG- 3') y AFC2 (5' -GCTGGAACATGCTGCCCAGGAC-3 ' ) . Estos oligonucleótidos fueron utilizados para amplificar el fragmento de cDNA correspondiente utilizando como molde DNA total aislado a partir de una genoteca de cDNA humano de ovario, construida en Lambda DR2 (Clontech N° de Catálogo HL1146x) . Para ello se utilizaron 20 pmoles de cada oligonucleótido, aproximadamente 1 microgramo de cDNA, 0,2 mM dNTPs y 1,25 U de Taq DNA polimerasa en un volumen total de 50 microlitros de "ExpandLong buffer 3 " (Boehringer Mannheim) . La amplificación se llevó a cabo en un aparato GeneAmp2400 de Perkin- Elmer, y consistió en ciclo inicial de desnaturalización (1 min, 94°C), 35 ciclos de desnaturalización (15 s, 94°C), hibridación (15 s, 60°C) y extensión (1 min, 72CC), seguidos de un ciclo final de extensión de 10 min a 72 °C. El fragmento de DNA resultante, de 516 pares de bases (pb) se purificó por electroforesis en gel de agarosa y extracción con GeneClean. La identidad del fragmento amplificado con la secuencia parcial deducida para Face-1 fue verificada tras subclonarlo en pUC18 y determinar su secuencia de nucleótidos mediante técnicas estándar de Biología Molecu- lar.From this sequence we design and synthesize two oligonucleotides, AFC1 (5 '-ATGAGGAGGTACTCGCTGTACTAGG-3') and AFC2 (5 '-GCTGGAACATGCTGCCCAGGAC-3'). These oligonucleotides were used to amplify the corresponding cDNA fragment using as a template total DNA isolated from a human ovarian cDNA library, constructed in Lambda DR2 (Clontech Catalog No. HL1146x). For this, 20 pmoles of each oligonucleotide were used, approximately 1 microgram of cDNA, 0.2 mM dNTPs and 1.25 U of Taq DNA polymerase in a total volume of 50 microliters of "ExpandLong buffer 3" (Boehringer Mannheim). The amplification was carried out in a Perkin-Elmer GeneAmp2400 apparatus, and consisted of an initial denaturation cycle (1 min, 94 ° C), 35 denaturation cycles (15 s, 94 ° C), hybridization (15 s, 60 ° C) and extension (1 min, 72 C C), followed by a final extension cycle of 10 min at 72 ° C. 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.
Con objeto de obtener una secuencia de cDNA que contuviera la información codificante de la proteína Face-1 completa, el producto de PCR obtenido con los oligonucleó- tidos AFC1 y AFC2 , anteriormente descrito, se marcó radiactivamente y se híbrido con 106 clones de la genoteca de cDNA de ovario antes citada siguiendo procedimientos están- dar. Se obtuvieron 11 clones de fago lambda que hibridaban específicamente con la sonda utilizada. El DNA de los fagos aislados fue convertido en los plásmidos pDR2 correspondientes por excisión in vivo, siguiendo las instrucciones del proveedor de la genoteca. Los plásmidos pDR2 recombi- nantes fueron analizados mediante Southern blot, análisis que nos condujo a seleccionar el clon 1.3a, por ser el que contenía el inserto de mayor longitud. La secuencia de nucleótidos de este plásmido se determinó por el método de terminadores de cadena descrito por Sanger (PNAS, 74 5463, (1977) ) . La secuenciación reveló la existencia de una fase abierta de lectura, que codifica una proteína de 475 aminoácidos a la que denominamos Face-1 humana. La comparación de esta secuencia de aminoácidos con todas las secuencias presentes en los bancos de datos accesibles públicamente demostró que el mayor grado de similitud (40%) correspondía a la proteína Afc-1 de Saccharomyces cerevisiae . Asimismo se detectó un grado significativo de similitud con la denominada proteína hipotética p59 de Schizosaccharomyces pombe y con una proteína de Escherichia coli de función descono- cida y denominada htpX. Un análisis más detallado de la secuencia de aminoácidos de Face-1 reveló la presencia de motivos estructurales que permiten clasificarla como una metaloproteasa de la familia de las gluzincinas (Methods Enzymol . 248 , 183, (1995)). Así, en posición 335 se encuen- tra la secuencia HELGH, que corresponde perfectamente con la secuencia HEXXH presente en las metaloproteasas e implicada en la unión de iones metálicos. Además, en posición 415 se encuentra un residuo de glutámico, conservado en la secuencia de Afc-1, p59 y htpX, y que forma parte asimismo del sitio de unión al metal. A cuatro residuos de distancia de este ácido glutámico se encuentra un residuo de ácido aspártico, esencial para la actividad catalítica de estas proteasas (Eur. J. Biochem. 221, 475, (1994)). La existencia de una única proteína humana con estas propiedades nos lleva a concluir que Face-1 es el homólogo humano de la proteína Afc-1 de Saccharomyces cerevisiae y por tanto es presumible su participación en la maduración proteolítica de proteínas preniladas. Tanto el ADN aislado como el poli- péptido codificado, representados en SEQ ID NO: 1, como secuencias parciales obtenidas de ambos, pueden sintetizarse químicamente también. El segundo objeto de la invención consistió en la identificación de un gen humano que pudiera codificar una proteína homologa a la proteína Rce-1 de Saccharomyces cerevisiae . Para ello la secuencia de aminoácidos descrita para esta proteína se comparó con la división de "Expressed Sequence Tags" (ESTs) de la base de datos GenBank utilizando el programa TBLASTN. Se identificaron seis ESTs humanas solapantes, cuyos números de acceso son: W96412, W96411, AA220236, T97242, T97243 y D20146. Del solapamiento de estas ESTs, dedujimos una secuencia parcial de nucleó- tidos, que codifica un fragmento de una hipotética proteína humana con un grado de similitud de aproximadamente 30% con la proteína Rce-1 de Saccharomyces cerevisiae . Esta proteína humana fue denominada tentativamente Face-2, (Farne- sylated-proteins converting enzyme 2 ) . Su secuencia de ami- noácidos, así como la secuencia nucleotídica que la codifica, se muestran en SEQ ID NO: 2. Análogamente a lo descrito para Face-1, se sintetizaron dos oligonucleótidos, RCE1 (5' -CTCTCACCCCTGTGCGTGCTGCTC-3' ) y RCE2 (5 ' -CGCGCAAACAG CTGGGAAACCC-3 ' ) y se utilizaron para amplificar el fragmento correspondiente de Face-2 utilizando como moldeIn order to obtain a cDNA sequence containing the information encoding the Face-1 protein In full, the PCR product obtained with the oligonucleotides AFC1 and AFC2, described above, was radioactively labeled and hybridized with 10 6 clones of the ovarian cDNA library cited above following standard procedures. 11 clones of lambda phage were obtained that hybridized specifically with the probe used. The isolated phage DNA was converted into the corresponding pDR2 plasmids by excision in vivo, following the instructions of the library provider. The recombinant pDR2 plasmids were analyzed by Southern blot, analysis that led us to select clone 1.3a, as it contained the longest insert. The 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. A more detailed analysis of the amino acid sequence of Face-1 revealed the presence of structural motifs that allow it to be classified as a metalloprotease of the gluzincin family (Methods Enzymol. 248, 183, (1995)). Thus, in position 335 is the HELGH sequence, which corresponds perfectly with the HEXXH sequence present in the metalloproteases and involved in the binding of metal ions. In addition, in position 415 there is a glutamic residue, conserved in the sequence of Afc-1, p59 and htpX, and which is also part of the metal binding site. Four residues away from this glutamic acid is an aspartic acid residue, essential for the catalytic activity of these proteases (Eur. J. Biochem. 221, 475, (1994)). The existence of a single human protein with these properties leads us to conclude that 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. Six overlapping human ESTs were identified, whose access numbers are: W96412, W96411, AA220236, T97242, T97243 and D20146. From the overlap of these ESTs, we deduced a partial nucleotide sequence, which encodes a fragment of a hypothetical human protein with a degree of similarity of approximately 30% with the Rce-1 protein of Saccharomyces cerevisiae. This human protein was tentatively called Face-2, (Far-sylated-proteins converting enzyme 2). Its amino acid sequence, as well as the nucleotide sequence that encodes it, are shown in SEQ ID NO: 2. Similarly to that described for Face-1, two oligonucleotides, RCE1 (5 '-CTCTCACCCCTGTGCGTGCTGCTC-3') and RCE2 (5 '-CGCGCAAACAG CTGGGAAACCC-3') and were used to amplify the corresponding Face-2 fragment using as a template
DNA total de una genoteca de cDNA de cáncer de mama humano construida en lambda gtll (Clontech N° de Catálogo HL1059b) . El fragmento de DNA obtenido, de 582 pb, se sub- clonó en pUC18 y se secuenció mediante técnicas estándar en Biología Molecular, lo que permitió confirmar su identidad con el fragmento esperado del gen Face-2. Con el fin de obtener una secuencia de cDNA que contuviera la información codificante de la proteína Face-2 completa, el producto de PCR obtenido con los oligonucleó- tidos RCE1 y RCE2 , anteriormente descrito, se marcó radiac- tivamente y se híbrido con 106 clones de la genoteca de cDNA de ovario antes citada siguiendo procedimientos estándar. Se obtuvieron 4 clones de fago lambda que hibridaban específicamente con la sonda utilizada. El DNA de los fagos aislados fue convertido en los plásmidos pDR2 correspon- dientes por excisión in vivo, siguiendo las instrucciones del proveedor de la genoteca. El análisis de los cuatro plásmidos reveló que todos ellos contenían insertos de cDNA de similar tamaño. La secuencia de nucleótidos del clon que denominamos 1. Ib se determinó por el método de terminadores de cadena descrito por Sanger (PNAS, 74, 5463, (1977)). La secuenciación reveló la existencia de una fase abierta de lectura, que codifica una proteína de 329 aminoácidos a la que denominamos Face-2 humana. La comparación de esta secuencia de aminoácidos con todas las secuencias presentes en los bancos de datos accesibles públicamente demostró que el mayor grado de similitud (30 %) correspondía a la proteína Rce-1 de Saccharomyces cerevisiae . Aunque la proteína Rce-1 de Saccharomyces cerevisiae había sido descrita como ligeramente similar a las peptidasas señal tipo Ilb (Science 275, 1797, (1997)), el análisis de Face-2 no confirmó esta hipótesis. Antes al contrario, un análisis más detallado de la secuencia de aminoácidos de Face-2 reveló la presencia de motivos estructurales que permiten clasificarla como una metaloproteasa del denominado Grupo IV, caracterizado por poseer sitios de unión al ligando distintos de la secuencia HEXXH (Methods Enzymol . 248 , 183,Total DNA of a human breast cancer cDNA library built in lambda gtll (Clontech Catalog No. HL1059b). The obtained DNA fragment, 582 bp, was subcloned into pUC18 and sequenced by standard techniques in Molecular Biology, which allowed confirming its identity with the expected fragment of the Face-2 gene. In order to obtain a cDNA sequence containing the information encoding the complete Face-2 protein, the PCR product obtained with the oligonucleotides RCE1 and RCE2, described above, was radiolabelled and hybridized with 10 6 clones of the ovarian cDNA library cited above following standard procedures. Four clones of lambda phage were obtained that hybridized specifically with the probe used. 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. Although the Rce-1 protein of Saccharomyces cerevisiae had been described as slightly similar to the signal peptidases type Ilb (Science 275, 1797, (1997)), Face-2 analysis did not confirm this hypothesis. On the contrary, a more detailed analysis of the amino acid sequence of Face-2 revealed the presence of structural motifs that allow it to be classified as a metalloprotease of the so-called Group IV, characterized by having ligand binding sites other than the HEXXH sequence (Methods Enzymol . 248, 183,
(1995) ) . La comparación de la secuencia de Face-2 con las proteínas incluidas hasta el momento en este grupo nos lleva a identificar los residuos de histidina en posición 211 y ácido glutámico en posición 214 como residuos implicados en la unión de los iones metálicos. Ambos residuos están conservados en la secuencia de Rce-1 y en la de di- versas carboxipeptidasas . Un residuo adicional de histidina en posición 261, también conservado en estas proteínas, puede participar en la unión del metal . La existencia de una única proteína humana con estas propiedades nos lleva a concluir que Face-2 es el homólogo humano de la proteína Rce-1 de Saccharomyces cerevisiae, tiene motivos estructurales característicos de proteasas y más específicamente de carboxipeptidasas y por tanto es presumible su participación en la maduración proteolítica de proteínas preniladas. Tanto el ADN aislado como el polipéptido codificado, representados en SEQ ID NO: 2, como secuencias parciales obtenidas de ambos, pueden sintetizarse químicamente también.(nineteen ninety five) ) . The comparison of the Face-2 sequence with the proteins included so far in this group leads us to identify histidine residues in position 211 and glutamic acid in position 214 as residues involved in the binding of metal ions. Both residues are conserved in the sequence of Rce-1 and in the sequence of carboxypeptidases verses. An additional histidine residue at position 261, also conserved in these proteins, can participate in metal binding. The existence of a single human protein with these properties leads us to conclude that Face-2 is the human homologue of the Rce-1 protein of Saccharomyces cerevisiae, has structural motifs characteristic of proteases and more specifically of carboxypeptidases and therefore its participation is presumably in proteolytic maturation of pre-packed proteins. Both the isolated DNA and the encoded polypeptide, represented in SEQ ID NO: 2, as partial sequences obtained from both, can also be chemically synthesized.
El tercer objeto de la invención es analizar la expresión en tejidos humanos de los genes homólogos a AFC1 y RCEl de Saccharomyces cerevisiae . Con este fin, dos membranas conteniendo RNA poliadenilado procedente de múltiples tejidos humanos (leucocitos, colon, intestino delgado, ovario, testículo, próstata, timo, bazo, páncreas, riñon, músculo esquelético, hígado, pulmón, placenta, cerebro y corazón) se hibridaron con las sondas de Face-1 y Face-2 marcadas radiactivamente. Dos microgramos de RNA poliadenilado de los tejidos indicados se hibridaron con los cDNAs de Face-1 y Face-2. Tras una prehibridación a 42 °C durante tres horas en formamida al 40%, 5x PBS/EDTA (lx=NaCl 150 mM, NaH2P04 10 mM, EDTA lmM, pH 7,4), lOx Solución de Denhardt (lx= Albúmina de suero bovino, 0,02%, polivinilpirrolidona, 0,02%, ficoll, 0,02 %) , SDS 2% y DNA de esperma de salmón 100 mg/ml , se añadieron las sondas y se hibridaron durante 20 horas en las mismas condiciones. Los filtros se lavaron con lx SSC (NaCl 150 mM, citrato sódico 15 mM, pH 7,0) conteniendo SDS al 0,1 % durante 2 horas a 50 °C, y finalmente se expusieron a autorradiografíaThe third object of the invention is to analyze the expression in human tissues of the homologous genes to AFC1 and RCE1 of Saccharomyces cerevisiae. To this end, two membranes containing polyadenylated RNA from multiple human tissues (leukocytes, colon, small intestine, ovary, testis, prostate, thymus, spleen, pancreas, kidney, skeletal muscle, liver, lung, placenta, brain and heart) 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. After a prehybridization at 42 ° C for three hours in 40% formamide, 5x PBS / EDTA (lx = 150 mM NaCl, 10 mM NaH 2 P0 4 , lmM EDTA, pH 7.4), lOx Denhardt's solution (lx = Bovine serum albumin, 0.02%, polyvinylpyrrolidone, 0.02%, ficoll, 0.02%), 2% SDS and 100 mg / ml salmon sperm DNA, the probes were added and hybridized for 20 hours in The same conditions. The filters were washed with 1 x SSC (150 mM NaCl, 15 mM sodium citrate, pH 7.0) containing 0.1% SDS for 2 hours at 50 ° C, and finally exposed to autoradiography
(Fig. 1) . Como puede observarse en la figura 1A, tras hibridación con la sonda de Face-1, se detectó un RNA men- sajero de aproximadamente 3,5 kilobases en todos los tejidos analizados. De manera análoga, cuando los filtros se hibridaron con la sonda de Face-2, se detectó un RNA mensa- j ero de aproximadamente 1,5 kilobases en todos los tejidos, siendo especialmente abundante en testículo. La expresión ubicua de los genes Face-1 y Face-2 es consistente con la amplia distribución tisular de proteínas preniladas, lo cual implica a su vez la necesidad de que las proteasas responsables de su maduración estén presentes en todos los tejidos del organismo.(Fig. 1). 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.
DESCRIPCIÓN DE LAS FIGURASDESCRIPTION OF THE FIGURES
Figura 1. Análisis Northern de la expresión en tejidos humanos de los genes Face-1 (A) , Face-2 (B) y un control de Actina (C) que se expresa en todos los tejidos. A la izquierda se indica el tamaño de los RNAs utilizados como marcadores. (1) Leucocitos, (2) colon, (3) intestino, (4) ovario, (5) testículo, (6) próstata, (7) timo, (8) bazo, (9) páncreas, (10) riñon, (11) músculo, (12) hígado, (13) pulmón, (14) placenta, (15) cerebro y (16) corazón. Figure 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. (1) Leukocytes, (2) colon, (3) intestine, (4) ovary, (5) testis, (6) prostate, (7) thymus, (8) spleen, (9) pancreas, (10) kidney, (11) muscle, (12) liver, (13) lung, (14) placenta, (15) brain and (16) heart.

Claims

REIVINDICACIONES
1. Procedimiento de identificación de secuencias géni- cas humanas homologas a los genes de levaduras implicados en el procesamiento proteolítico de proteínas preniladas caracterizado porque comprende las siguientes etapas: a) Comparar la secuencia de nucleótidos de los genes AFC1 y RCEl de Saccharomyces cerevisiae con las secuencias parciales de nucleótidos presentes en las bases de datos de genes expresados. b) Identificar fragmentos homólogos y amplificarlos mediante PCR de RNA total de tejidos humanos en los que se puedan expresar dichas secuencias génicas. c) Utilizar los fragmentos amplificados como sondas para hibridar genotecas de cDNA preparadas a partir de RNA de tej idos humanos . d) Aislar los clones de cDNA que hibriden con las sondas y determinar su secuencia completa de nucleótidos. 1. Procedure for the identification of human gene sequences homologous to the yeast genes involved in proteolytic processing of prenilated proteins characterized in that it comprises the following steps: a) Compare the nucleotide sequence of the Saccharomyces cerevisiae AFC1 and RCE1 genes with the Partial nucleotide sequences present in the expressed gene databases. b) Identify homologous fragments and amplify them by PCR of total RNA of human tissues in which said gene sequences can be expressed. c) Use the amplified fragments as probes to hybridize cDNA libraries prepared from RNA from human tissues. d) Isolate the cDNA clones that hybridize with the probes and determine their complete nucleotide sequence.
2. Procedimiento de identificación de acuerdo con la reivindicación 1, caracterizado porque las secuencias génicas identificadas codifican proteasas procesadoras de proteínas ras y otras relacionadas.2. Identification method according to claim 1, characterized in that the identified gene sequences encode raster and other related protein processing proteases.
3. Procedimiento de identificación de acuerdo con cualquiera de las reivindicaciones anteriores caracterizado porque la secuencia génica identificada y su secuencia de aminoácidos deducida son SEQ ID NO: 1.3. Identification method according to any of the preceding claims characterized in that the identified gene sequence and its deduced amino acid sequence are SEQ ID NO: 1.
4. Procedimiento de identificación de acuerdo con las reivindicaciones 1 o 2, caracterizado porque la secuencia génica identificada y su secuencia de aminoácidos deducida son SEQ ID NO :2.4. Identification procedure according to claims 1 or 2, characterized in that the identified gene sequence and its deduced amino acid sequence are SEQ ID NO: 2.
5. Secuencia génica SEQ ID NO: 1 y sus mutaciones, derivados o secuencias parciales, que codifiquen para una actividad enzimática proteolítica de proteínas preniladas. 5. Gene sequence SEQ ID NO: 1 and its mutations, derivatives or partial sequences, which code for proteolytic enzymatic activity of pre-piled proteins.
6. Secuencia génica SEQ ID NO: 2 y sus mutaciones, derivados o secuencias parciales, que codifiquen una actividad enzimática proteolítica de proteínas preniladas. 6. Gene sequence SEQ ID NO: 2 and its mutations, derivatives or partial sequences, which encode a proteolytic enzymatic activity of pre-piled proteins.
7. Utilización de las secuencias SEQ ID NO: 1 y/o SEQ ID NO: 2 en el diseño de inhibidores de la actividad enzimática proteolítica de proteínas preniladas.7. Use of the sequences SEQ ID NO: 1 and / or SEQ ID NO: 2 in the design of inhibitors of proteolytic enzymatic activity of pre-piled proteins.
8. Utilización de las secuencias SEQ ID NO: 1 y/o SEQ ID NO: 2 en la producción de proteínas recombinantes o sintéticas .8. Use of the sequences SEQ ID NO: 1 and / or SEQ ID NO: 2 in the production of recombinant or synthetic proteins.
9. Utilización de las secuencias SEQ ID NO : 1 y/o SEQ ID NO: 2 en la producción de anticuerpos.9. Use of the sequences SEQ ID NO: 1 and / or SEQ ID NO: 2 in the production of antibodies.
10. Utilización de las secuencias SEQ ID NO: 1 y/o SEQ ID NO: 2 en la producción de sistemas de detección de proteínas proteolíticas de proteínas preniladas y/o de los genes que codifican para las mismas.10. Use of the sequences SEQ ID NO: 1 and / or SEQ ID NO: 2 in the production of proteolytic protein detection systems of pre-piled proteins and / or the genes encoding them.
11. Utilización de las secuencias SEQ ID NO: 1 y/o SEQ ID NO: 2 en la producción de composiciones activas en el tratamiento de procesos patológicos mediados por proteínas ras u otras proteínas preniladas, y/o por genes que codifican para las mismas.11. Use of the sequences SEQ ID NO: 1 and / or SEQ ID NO: 2 in the production of active compositions in the treatment of pathological processes mediated by ras proteins or other pre-encapsulated proteins, and / or by genes encoding them .
12. Secuencia de aminoácidos completa o partes de la misma, reflejadas en SEQ ID NO: 1. 12. Complete amino acid sequence or parts thereof, reflected in SEQ ID NO: 1.
13. Secuencia de aminoácidos completa o partes de la misma, reflejadas en SEQ ID NO : 2. 13. Complete amino acid sequence or parts thereof, reflected in SEQ ID NO: 2.
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EP0887416A2 (en) * 1997-06-24 1998-12-30 Smithkline Beecham Corporation Human AFC1
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