EP1071788A1 - Novel nucleic acids and polypeptides related to a farnesyl-directed cysteine carboxymethyltransferase - Google Patents

Abstract

The present invention relates to a farnesyl-directed cysteine carboxymethyltransferase nucleic acid and polypeptide, especially one which is obtainable or derived from a human. The nucleic acid and polypeptide is useful in diagnostics and in assays for identifying agents which modulate signalling pathways, especially pathways involved in the cell cycle, cell proliferation, and cancer.

Description

NOVEL NUCLEIC ACIDS AND POLYPEPTIDES RELATED TO A FARNESYL-DIRECTED CYSTEINE CARBOXYMETHYLTRANSFERASE

BACKGROUND OF THE INVENTION Famesyl-directed cysteine carboxymethyltransferases are involved in the processing of vaπous proteins, such as proteins involved m signaling pathway s. fungal mating factors, and Ras polypeptides An activity of these methyltransferases is to perform methylestenfication on prenylated proteins See. e g , Ashbv et al . Yeast, 9 907-913, 1993, Khosravi-Far et al , Cell Growth and Differentiation. 3 461- 469. 1992 DESCRIPTION OF THE INVENTION

The present invention concerns carboxymethyltransferases ("MTase^"'), especially mammalian farnesN 1-dιrected cy steine MTases. such as human STE 14 MTases catalyze the transfer of a methy 1 group from a methyl donor to a methyl acceptor There are at least seven different categories of MTases. distinguished by the type of methyl acceptor upon which the enzyme acts and the nature of the chemical bond which is formed See. e g . Kagan and Clarke. Arch Biochem Biophys 310 417-427.

1994

An aspect of the invention relates to nucleic acids, polypeptides. and fragments thereof, coding for carboxymethyltransferases. especially a mammalian farnesyl-directed cysteine carboxymethy ltransferase. such as human STEM and muπne MTase The invention further relates to methods of using such nucleic acids and polypeptides in therapeutics, diagnostics, and research For example, the nucleic acids and polypeptides can be utilized in methods to identify modulators of MTase activity and to obtain hgands which bind to MTase

BRIEF DESCRIPTION OF THE DRAWINGS Fig 1 shows a nucleotide and ammo acid coding sequence for a human farnes l-directed cysteine carboxymethyltransferase

Fig 2 shows a comparison between methyltransferase sequences from different species Fig 3 shows a nucleotide and ammo acid coding sequence for a mouse farnesy 1-dιrected cy steine carboxymethyltransferase

DETAILED DESCRIPTION OF THE INVENTION Novel nucleic acid and polypeptide sequences have been identified which code for mammalian farnesyl-directed cysteine carboxymethyltransferases These enzymes are involved in a variety of biological processes, including pathways which are associated with the maturation of signaling molecules, such as ras, rho, rab. rac, gamma-subunits of GTP -binding protein, related G-protems, nuclear lammins. and fungal mating pheromones A farnesyl-directed cysteine carboxymethyltransferase has at least one of the following activities ability to bind to. or attach to, a methyl donor substrate, e g . S-adenosyl-L-methionme ("AdoMet^"'). ability to catalyze the transfer of a methyl group to a methyl acceptor ("methyltransferase activity^"), where the methyl acceptor is preferably a prenylated cysteine. such as a S-farnesyl-cysteine. methylestenfication of a newly exposed alpha-carboxy 1 group, where the alpha-carboxyl group is a prenvlated cysteine. such as a S-farnesyl- cysteine. ability to bind to, or attach to. a prenylated cysteine. promote protein/protein interactions, a transformation-modulating activity activity, or, an immunogenic activity, e g . the polypeptide or polynucleotide is capable of eliciting an immune response specific for an MTase of the present invention The above-mentioned activities of an MTase can be measured according to available assays or as descπbed m the examples below See, e g . Imai et al . Mol Cell Bio , 17 1543-1551. 1997. Hrycyna et al . Methods Enzymol 250 251-266. 1995

Substrate binding is generally considered the first step in enzyme catalysis because the substrate, actmg as a gand. must first attach to the enzyme surface to enable the enzyme to carry out its catalytic reactions This enzyme surface can be referred to as the active site of the enzyme Bindmg of the substrate to the enzyme surface can involve multiple interactions with the enzyme, e g , chemical bonding with one or more ammo acids and/or functional groups which comprise the enzyme A methyl donor substrate binding activity as used herein means that a meth\ 1 donor substrate attaches to an MTase of the present invention Attachment to the enzyme can be accomplished by one or more of the interactions which hold its naturall -occurnng substrate to it. however, a polypeptide can have a methyl donor substrate binding activity when it holds the substrate w ith less than the naturally-occurnng number and quality of interactions

Methy 1-donor substrate binding and catalytic activity can be dissociated from each other Thus, an MTase polypeptide in accordance with the invention can possess substrate binding activity but not a catalytic activity A methyl donor substrate binding activity can optionally be effective to achieve catalysis of the substrate, to competitively or noncompetitively bind to the active site, to irreversibly attach to the enzyme, to result m the loss of catalytic activity (e g . where it is a suicide substrate), etc

Methyl donor substrate binding activity (e g . binding of S-adenosyl-L-methionine) can be measured conventionally For instance, a competition binding assay can be employed to identify substrates which attach to a polypeptide. or denvative thereof, e g . by combining under effective conditions, a substrate containing a detectable marker, an STE14 polypeptide. or fragments thereof, and a compound which is to be tested for substrate binding activity The assay can be accomplished in liquid phase, where bound and free substrate are separated by a membrane, or. it can be accomplished in solid phase, as desired Solid-phase assays can be performed using high-throughput procedures, e g , on chips, wafers, etc

A polypeptide according to the present invention can also possess a catalytic activity, e g . transfer of a methyl group to a methyl acceptor substrate Generally, the catalysis results in methylestenfication of a newly exposed alpha-carboxyl group of an ammo acid, especially a prenylated cysteine Thus, a catalytic activity in accordance with the present invention is a carboxylmethylation activity, especially of prenylated polypeptides This activity is descπbed. e g . in Hrycyna et al . Methods Enzvmol . 250 251-266. 1995. Imai et al , Mol Cell Bio , 17 1543-1551. 1997 This activity can be measured in vitro or in vivo A polypeptide. or a nucleotide coding sequence thereof, can also possess a ^"transformation- modulating activity^"' This can be an activity that modulates a transformed phenotype of cells, e g , induces cell division, induces anchorage independent growth, increases ras activity, etc The effect can be partial or incomplete For example, expression of a STE14 coding sequence in cells can cause a transformed phenotype. or it can enhance the phenotype of already transformed cells A transformation- promoting activity can be enhanced by the presence of defects in other genes which contnbute to transformation, such as ras. p53, Rb. cell-cycle regulatory genes, etc

A mammalian farnesyl-directed cysteine carboxymethyltransferase (e g . human STEM) is a mammalian polypeptide. or fragment thereof, having an amino acid sequence which is obtainable from a natural source It therefore includes naturally -occurπng. normal, mutant, polymorphic, etc . amino acid sequences Natural sources include, e g , living cells, e g , obtained from tissues or whole organisms, cultured cell lines, including pnmary and immortalized cell lines, biopsied tissues, etc The present invention also relates to fragments of a full-length mammalian MTase. such as human STEM or munne MTase The fragments are preferably biologically -active By biologically-active, it is meant that the polypeptide fragment possesses an activity in a living system or with components of a living system Biological-activities include those mentioned, e g . a methyltransferase activity, a methyl donor substrate binding activity, a transformation-modulating activity, and/or an lmmunogenic activity Fragments can be prepared according to any desired method, including, chemical synthesis, genetic engineeπng. cleavage products, etc See, below

The present invention also relates to a human farnesyl-directed cysteine carboxymethyltransferase having an amino acid sequence of amino acids 1 to 284 See, Fig 1 The

284 amino acid polypeptide has a predicted molecular weight of 31 9 kilodaltons

In addition to the human STEM sequence, an MTase from another mammalian species, mouse, has been cloned and identified A sequence of this is identified in Fig 2 and 3 A full-length nucleic acid containing, e g . a complete coding sequence for a mouse MTase. its promoter, and/or enhancer region, etc . can be routinely identified and obtained, e g , by using the above-mentioned fragments as probes for a cDNA or genomic library, by PCR. etc

Other homologs from mammalian and non-mammalian can be obtained according to vanous methods For example, hybndization with an ohgonucleotide (see below) selective for a mammalian farnes l-directed cysteine carboxymethy ltransferases can be employed to select such homologs, e g , as descnbed in Sambrook et al , Molecular Cloning. 1989. Chapter 1 1 Such homologs can have varying amounts of nucleotide and ammo acid sequence identity and similarity to farnsyl-directed carboxymethyltransferase Non-mammalian organisms include, e g . vertebrates, invertebrates, zebra fish, chicken. Drosophila. C elegans. roundworms. prokaryotes. plants. Arabidopsis. viruses, etc The invention also relates to farnesyl-directed cy steine carboxymethy 1-transferase specific amino acid sequences, e g , a defined ammo acid sequence which is found in the particular human or mouse sequences of Figs 1 and 3 but not in another amino acid sequence, preferably not in Xenopus Xmam4, S pombe mam4. S cerevisiae STEM, or mouse MTase See. Imai et al . Mol Cell Bio . 17 1543-1551. 1997. Sapperstein et al . Mol Cell Bio . 14 1438-1449. 1994 A specific ammo acid sequence can be found routinely, e g , b\ searching a gene/protein database using the BLAST set of computer programs Mammalian specific sequences can be selected from about the first 65 amino acids of the MTase, e g . CAARAPP. etc A human specific amino acid sequence is. for instance. ICGVSYALTV A farnesyl-directed cysteine carboxymethy ltransferases specific amino acid sequence can be useful to produce peptides as antigens to generate an immune response specific for it Antibodies obtained by such immunization can be used as a specific probe for a mammalian farnesyl-directed cysteine carboxymeth ltransferases protein for diagnostic or research purposes

A polypeptide of the invention, e g . having a polypeptide sequence as shown in Fig 1 or Fig 3. can by analy zed by available methods to identify structural and/or functional domains in the polypeptide For example, when the polypeptide coding sequence set forth in Fig 1 is analyzed by hydropathy and hydrophilicity analysis (e g , Kyte and Doo ttle. J Mol Bio .157 105. 1982) putative membrane spanning regions are identified at LI 6 to T34. L44 to Y59. 168 to F85. 1156 to LI 73. and V225 to W241 A putative catalytic region is V 1 10 to L284 Vanous other programs can be used to analyze its structure and routinely predict functional domains, including. EMBL Protein Predict. Rost and Sander. Proteins, 19 55-72, 1994

As mentioned polypeptides of the present invention can compnse a complete coding sequence for a mammalian farnesyl-directed cysteine carboxymethyltransfer-ase. or fragments thereof For example, an N-termmal region of a mammalian farnesyl-directed cysteine carboxymethyltransferase can modulate its enzymatic activity, e g , by enhancing its activity or stabilizing it Thus, useful fragments include, about amino acids 1-65 of the muπne or human sequences in Fig 1 and Fig 2 These fragments can be used to modulate, stabilize, or enhance activity of other MTases. or other polypeptides by joining them in reading-frame with the polypeptide of interest One or more fragments can be used. e g.. a human or munne STEM can comprise two or more N-terminal regions which possess a modulatory activity A fragment of a farnesvl-directed cysteine carboxymethyltransferases polypeptide can be selected to have a specific biological activity, e g . a methyl donor binding activity, a methylesteπfication activity, a methyltransferase activity, a transformation-modulatory activity, an immunogenic activity, etc A useful fragment can be identified routinely by testing such fragments for a desired activity The measurement of these activities is descnbed below and in the examples These peptides can also be identified and prepared as described in EP 496 162

A polypeptide of the present invention can also have 100% or less amino acid sequence identity to the amino acid sequence set forth in Fig 1 or 3 For the purposes of the following discussion Sequence identity means that the same nucleotide or ammo acid which is found m the sequence set forth in Fig 1 or Fig 3 is found at the corresponding position of the compared sequence(s). e g . Fig 2 A polypeptide having less than 100% sequence identify to the amino acid sequence set forth in Fig 1 or 3 can contain vanous substitutions from the naturally-occurring sequence, including homologous amino acid substitutions See below for examples of homologous amino acid substitution The sum of the identical and homologous residues divided by the total number of residues m the sequence over which the farnesyl-directed cysteine carboxymethyltransferases polypeptide is compared is equal to the percent sequence similanty For purposes of calculating sequence identity and similarity, the compared sequences can be aligned and calculated according to any desired method, algorithm, computer program, etc . including, e g . FASTA. BLASTA A polypeptide having less than 100% amino acid sequence identity to the amino acid sequence of Fig 1 can compπse e g , about 99%. 97%. 95% . preferably about greater than 71% homology, such as 75% or more, with the proviso that the sequence is not Xenopus Xmam4. S pombe mam4. S cerevisiae STEM, or mouse MTase See, Imai et al , Mol Cell ,

Bio , 17 1543-1551, 1997, Sapperstein et al . Mol Cell Bio , 14 1438-1449 The fragment of Fig 3 can also be excluded

A mammalian farnesyl-directed cysteine carboxymethyltransferases polypeptide. fragment, or substituted polypeptide can also compose vanous modifications, where such modifications include pid modification, methylation. phosphorylation. glycosylation. covalent modifications (e g . of an R-group of an ammo acid), ammo acid substitution, ammo acid deletion, or ammo acid addition Modifications to the polypeptide can be accomplished according to vanous methods, including recombinant. synthetic, chemical, etc Polypeptides of the present invention (e g , human STEM or mouse MTase, fragments thereof, mutations thereof) can be used in vanous ways, e g . in assays, as immunogens for antibodies as descnbed below, as biologically-active agents (e g . having one or more of the activities associated with STEM) A polypeptide coding for a farnesyl-directed cysteine carboxymethyltransferase. a denvative thereof, or a fragment thereof, can be combined with one or more structural domains, functional domains, detectable domains, antigenic domains, and/or a desired polypeptides of interest, in an arrangement which does not occur in nature. 1 e . not naturally-occurnng. e g . as in a human or muπne STEM gene, a genomic fragment prepared from the genome of a living organism, e g . an animal. preferably a mammal, such as human, mouse, or cell lines thereof A polypeptide comprising such features is a chimenc or fusion polypeptide Such a chimeπc polypeptide can be prepared according to vanous methods, including, chemical, synthetic, quasi-synthetic, and/or recombinant methods A chimeπc nucleic acid coding for a chimenc polypeptide can contain the vanous domains or desired polypeptides in a continuous (e g . with multiple N-terminal domains to stabilize or enhance activity) or interrupted open reading frame, e g , containing mtrons. splice sites, enhancers, etc The chimenc nucleic acid can be produced according to vanous methods See. e g , U S Pat No 5,439,819 A domain or desired polypeptide can possess any desired property, including, a biological function such as catalytic, signalling, growth promoting, cellular targeting (e g . signal sequence, targeting sequence, such as to endosomes. ly sosomes. ER. nuncleus). etc . a structural function such as hydrophobic. hy dro- philic. membrane-spanning, etc . receptor-hgand functions, and/or detectable functions, e g . combined with enzyme, fluorescent polypeptide. green fluorescent protein. (Chalfie et al . 1994. Science. 263 802, Cheng et al . 1996. Nature Biotechnology. 14 606. Levy et al . 1996. Nature Biotechnology. 14 610. etc In addition, a polypeptide. or a part of it. can be used as selectable marker when introduced into a host cell For example, a nucleic acid coding for an ammo acid sequence according to the present invention can be fused in frame to a desired coding sequence and act as a tag for punfication. selection, or marking purposes The region of fusion can encode a cleavage site to facilitate expression, isolation, punfication. etc

A polypeptide according to the present invention can be produced in an expression system, e g , in vivo, in vitro, cell-free, recombinant. cell fusion, etc . according to the present invention Modifications to the polypeptide imparted by such system include, glycosylation. ammo acid substitution (e g , by differing codon usage), polypeptide processing such as digestion, cleavage, endopeptidase or exopeptidase activity, attachment of chemical moieties, including pids and phosphates, etc A polypeptide according to the present invention can be recovered from natural sources, transformed host cells (culture medium or cells) according to the usual methods, including, detergent extraction (e g , CHAPS, octylglucoside), ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography. phosphocellulose chromatography. hydrophobic interaction chromatography. hydroxyapatite chromatography and lectin chromatography Protein refolding steps can be used, as necessary, in completing the configuration of the mature protein Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps

A mammalian farnesyl-directed cysteine carboxymethyltransferase nucleic acid, or fragment thereof, is a nucleic acid having a nucleotide sequence obtainable from a natural source, or compnsmg a coding sequence coding for a mammalian farnesyl-directed cv steine carboxymethyltransferase See. above It therefore includes naturally-occurnng. normal, mutant, polymorphic, degenerate sequences, etc . alleles Natural sources include, e g . living cells obtained from tissues and whole organisms, cultured cell lines, including pnmarv and immortalized cell lines Expression of human STEM is relatively ubiquitous, e g . it is expressed m e g . heart, brain, placenta, lung, liver, skeletal muscle. kidney, pancrease. spleen, thymus. prostate, testis. overy. small intestine, colon, and penpheral blood leucocytes It is also expressed in vanous cancer cells, including, HL-60, Hela cell S3, chronic myelogenous leukemia K-562. lymphoblastic leukemia MOLT-4. Burkitt's lymphoma Raji. colorectal adenocarcinoma SW 480. lung carcinoma A549. and melanoma G361 The approximate size of the transcnpts are about 2 kb. 3 5 kb. and 5 kb A nucleic acid sequence of a human allele of a mammalian farnesyl-directed cy steine carboxymethyltransferase. STEM, is shown in Fig 1 It contains open-reading frame of 284 ammo acids at nucleotide positions 90 to 944 It contains 5' untranslated sequences at 1 to 89 and 3' untranslated sequences at 945 to 2556 A nucleic sequence of the invention can contain the complete coding sequence from ammo acid 1 to amino acid 284 (I e , full-length, having a start codon and a termination codon). degenerate sequences thereof, and fragments thereof A nucleic acid according to the present invention can also compnse a nucleotide sequence which is 100% complementary, e g , an anti-sense, to any nucleotide sequence mentioned above and below

The present invention also relates to mouse nucleotide sequence coding for all or part of a MTase. e g . as shown in Fig 3 As for the human allele. the invention relates to degenerate sequences thereof, and anti-sense fragments thereof

A nucleic acid according to the present invention can be obtained from a vanety of different sources It can be obtained from DNA or RNA. such as polyadenylated mRNA, e g , isolated from tissues, cells, or whole organism The nucleic acid can be obtained directly from DNA or RNA. or from a cDNA library The nucleic acid can be obtained from a cell at a particular stage of development, having a desired genotype, phenotype (e g . an oncogenically transformed cell or a cancerous cell), etc

As for the polypeptides mentioned above, a nucleic acid compnsmg a nucleotide sequence coding for a polypeptide according to the present invention can include only coding sequence, a coding sequence and additional coding sequence (e g . sequences coding for leader, secretory, targeting. enzymatic, fluorescent or other diagnostic peptides), coding sequences and non-coding sequences, e g , untranslated sequences at either a 5' or 3' end. or dispersed in the coding sequence, e g . introns A nucleic acid compnsmg a nucleotide sequence coding without interruption for a polypeptide means that the nucleotide sequence contains an amino acid coding sequence for a farnesyl- directed c\ steine carboxymethyltransferase. with no non-coding nucleotides interrupting or intervening in the coding sequence, e g , absent ιntron(s) Such a nucleotide sequence can also be descnbed as contiguous A genomic DNA coding for a human or mouse MTase. etc . can be obtained routinely

A nucleic acid according to the present invention also can comprise an expression control sequence operably linked to a nucleic acid as descnbed above The phrase "expression control sequence" means a nucleic acid sequence which regulates expression of a polypeptide coded for by a nucleic acid to which it is operably linked Expression can be regulated at the level of the mRNA or polypeptide Thus, the expression control sequence includes mRNA-related elements and protein- related elements Such elements include promoters, enhancers (viral or cellular), nbosome binding sequences, transcnptional terminators, etc An expression control sequence is operably linked to a nucleotide coding sequence when the expression control sequence is positioned in such a manner to effect or achieve expression of the coding sequence For example, when a promoter is operablv linked 5' to a coding sequence, expression of the coding sequence is dnven by the promoter Expression control sequences can be heterologous or endogenous to the normal gene

A nucleic acid in accordance with the present invention can be selected on the basis of nucleic acid hybndization The ability of two single-stranded nucleic acid preparations to hybndize together is a measure of their nucleotide sequence complementanty, e g , base-pamng between nucleotides, such as A-T, G-C. etc The invention thus also relates to nucleic acids which hybndize to a nucleic acid compnsmg a nucleotide sequence as set forth in Fig 1 or Fig 3, preferably Fig 1 A nucleotide sequence hybridizing to the latter sequence will have a complementary nucleic acid strand, or act as a template for one in the presence of a polymerase (I e . an appropnate nucleic acid synthesizing enzyme)

The present invention includes both strands of nucleic acid, e g , a sense strand and an anti-sense strand Hybndization conditions can be chosen to select nucleic acids which have a desired amount of nucleotide complementanty with the nucleotide sequence set forth in Fig 1 A nucleic acid capable of hybndizmg to such sequence, preferably, possesses about 95%. more preferably. 97%. etc , complementanty. between the sequences The present invention particularly relates to DNA sequences which hybndize to the nucleotide sequence set forth in Fig 1. or its complement, under stringent conditions As used here, stringent conditions means, for example 50% formamide. 6X SSC or 6X SSPE. and optionally, a blocking agent (s)s (e g . Denhardt^'s reagent, BLOTTO, hepaπn. denatured. fragmented salmon sperm DNA) at 42 C (or 68 C if the formamide is omitted) Washing and hybndization can be performed as descnbed in Sambrook et al . Molecular Cloning, 1989. Chapter 9 Hybridization can also be based a calculation of melting temperature (Tm) of the hybnd formed between the probe and its target, as descnbed in Sambrook et al Such stnngent conditions can select sequences which have, e g , at least about 95%. preferably 97%. nucleotide complementarity between the nucleic acids, with the proviso that such nucleic acid is not Xenopus Xmam4. S pombe mam4. S cerevisiae STEM, or mouse MTase. See, Imai et al , Mol Cell Bio , 17 1543-1551. 1997. Sapperstein et al . Mol Cell Bio . 14 1438-1449. 1994

According to the present invention, a nucleic acid or polypeptide can comprise one or more differences m the nucleotide or amino acid sequence set forth in Fig 1 or Fig 3 Changes or modifications to the nucleotide and/or amino acid sequence can be accomplished by any method available, including directed or random mutagenesis

A nucleic acid coding for a human or mouse MTase according to the invention can compnse nucleotides which occur in a naturally-occurnng MTase gene e g , naturally-occurnng polymorphisms, normal or mutant alleles (nucleotide or ammo acid), mutations which are discovered in a natural population of mammals, such as humans, monkeys, pigs. mice. rats, or rabbits By the term naturally- occurnng. it is meant that the nucleic acid is obtainable from a natural source, e g , animal tissue and cells, body fluids, tissue culture cells, forensic samples Naturally-occurnng mutations can include deletions (e g . a truncated amino- or carboxy-terminus). substitutions, or additions of nucleotide sequence These genes can be detected and isolated by nucleic acid hybndization according to methods which one skilled in the art would know It is recognized that, m analogy to other oncogenes. naturally- occurnng vanants include deletions, substitutions, and additions which produce pathological conditions in the host cell and organism

A nucleotide sequence coding for a polypeptide of the invention can contain codons found in a naturally-occurnng gene, transcnpt. or cDNA. for example, e g . as set forth in Fig 1. or it can contain degenerate codons coding for the same ammo acid sequences

Modifications to a sequence of the invention, e g , mutations, can also be prepared based on homology searching from gene data banks, e g . Genbank. EMBL Sequence homology searching can be accomplished using vanous methods, including algonthms descnbed in the BLAST family of computer programs, the Smith-Waterman algonthm. etc For example, conserved amino acids can be identified between vanous sequences See, e g , Fig 2 A mutatιon(s) can then be introduced into a sequence by identifying and aligning amino acids conserved between the polypeptides and then modifying an amino acid in a conserved or non-conserved position

A nucleic acid and corresponding polypeptide of the present invention include sequences which differ from the nucleotide sequence of Fig 1 (or less preferably Fig 3) but which are phenotypically silent These sequence modifications include, e g , nucleotide substitution which do not affect the ammo acid sequence (e g , different codons for the same amino acid or degenerate sequences), replacing naturally-occurnng amino acids with homologous amino acids, e g . (based on the size of the side chain and degree of polanzation) small nonpolar cysteine. prohne. alanine. threonme. small polar senne, glycine, aspartate. asparagme, large polar glutamate, glutamme. lysine, arginine. intermediate polarity tyrosine. histidme. tryptophan. large nonpolar phenylalanine. methiomne, leucme. isoleucine, va ne

Homologous acids can also be grouped as follows uncharged polar R groups, glycine. senne. threonme. cysteine. tyrosine. asparagme, glutamme. acidic amino acids (negatively charged), aspartic acid and glutamic acid, basic ammo acids (positively charged), lysine. arginine. histidme Homologous substitutions also include those descnbed by Dayhoff in the Atlas of Protein Sequence and Structure 5

(1978), and by Argos in EMBO J . 8. 779-785 (1989)

Muteins in accordance with the present invention include amino acid sequences where a residue in the human sequence is replaced by a residue from a corresponding domain in the Xenopus Xmam4. S pombe mam4. S cerevisiae STEM, or mouse MTase at a corresponding position A nucleic acid can compnse a nucleotide sequence coding for a polypeptide having an amino acid sequence as set forth in Fig 1 or Fig 3, except where one or more positions are substituted by homologous amino acids, or a nucleotide sequence coding for a polypeptide having an ammo acid sequence as set forth in Fig 1 (less preferably Fig 3). except having 1. 5, 10. 15. or 20 substitutions, e g . wherein the substitutions are conservative amino acids The invention also relates to polypeptides coded for by such nucleic acids In addition, it may be desired to change the codons in the sequence to optimize the sequence for expression in a desired host

A nucleic acid according to the present invention can compnse. e g . DNA. RNA. synthetic nucleic acid, peptide nucleic acid, modified nucleotides. or mixtures A DNA can be double- or single- stranded Nucleotides comprising a nucleic acid can be joined via vanous known linkages, e g . ester. sulfamate. sulfamide. phosphorothioate, phosphoramidate. methy lphosphonate, carbamate. etc , depending on the desired purpose, e g , resistance to nucleases, such as RNase H, improved in vivo stability, etc See. e g , U S Pat Nos 5.378.825

Vanous modifications can be made to the nucleic acids, such as attaching detectable markers (avidin. biotin. radioactive elements), moieties which improve hybndization. detection, or stability The

10 nucleic acids can also be attached to solid supports, e g . nitrocellulose, magnetic or paramagnetic microspheres (e g . as descnbed in USP 5.411.863. USP 5.543.289. for instance, compnsmg ferromagnetic, supermagnetic, paramagnetic, superparamagnetic. iron oxide and polysacchande). nylon, agarose. diazotized cellulose, latex solid microspheres. polyacrylamides. etc . according to a desired method See. e g . U S Pat Nos 5.470.967. 5.476.925. 5.478.893

Another aspect of the present invention relates to ohgonucleotides and nucleic acid probes Such ohgonucleotides or nucleic acid probes can be used, e g . to detect, quantitate. or isolate a human or mouse MTase, such as STEM, nucleic acid in a test sample Detection can be desirable for a vanety of different purposes, including research, diagnostic, and forensic For diagnostic purposes, it may be desirable to identify the presence or quantity of a such a nucleic acid sequence in a sample, where the sample is obtained from tissue, cells, body fluids, etc In a preferred method, the present invention relates to a method of detecting a nucleic acid composing, contacting a target nucleic acid in a test sample with an o gonucleotide under conditions effective to achieve hvbndization between the target and ohgonucleotide. and detecting hy bπdization An ohgonucleotide in accordance with the invention can also be used in synthetic nucleic acid amplification such as PCR (e g , Saiki et al , 1988. Science,

241 53. U S Pat No 4,683,202, PCR Protocols A Guide to Methods and Applications. Innis et al , eds . Academic Press. New York. 1990) or differential display (See. e g . Liang et al . Nucl Acid Res . 21 3269-3275, 1993, USP 5.599.672. W097/18454) Useful ohgonucleotides include, e g . 5^"-CAGATAGCCATCCGAGCTTGT-3" (282-302 nucleotide position), 5"-CTCCTGAATCACAGCCTGGAGTA-3^" (462-484 nucleotide position).

5'-CCTGGAGTATACAGTAGCTGCT^"-3^" (476-497 nucleotide position)

Such detection can be accomplished in combination with ohgonucleotides for other genes, such as ras. p53, Rb. cell-cycle regulatatory genes, etc For methods and probes, e g , USP 5.591.582

Another aspect of the present invention is a nucleotide sequence which is unique to human STEM or mouse MTase By a unique sequence to farnsvl-directed carboxymethyltransferase. it is meant a defined order of nucleotides which occurs m human or mouse STEM, e g , in the nucleotide sequence of Fig 1, but rarely or infrequently m other nucleic acids, especially not in an animal nucleic acid, preferably mammal, such as human, rat. mouse, etc Both sense and antisense nucleotide sequences are included A unique nucleic acid according to the present invention can be determined routinely A nucleic acid composing such a unique sequence can be used as a hybndization probe to identify the presence of. e g , human or mouse STEM, in a sample compnsmg a mixture of nucleic acids, e g , on a Northern blot Hybndization can be performed under stnngent conditions to select nucleic acids having at least 95% identity (I e . complementanty) to the probe, but less stnngent conditions can also be used A unique farnsvl-directed carboxymethyltransferase nucleotide sequence

11 can also be fused in-frame, at either its 5' or 3' end. to various nucleotide sequences as mentioned throughout the patent, including coding sequences for other parts of STEM, enzymes. GFP. etc. expression control sequences, etc See. e g. Hrycyna et al . Methods Enzvmol . 250 251-266. 1995

Hybndization can be performed under different conditions, depending on the desired selectivity, e g , as descnbed in Sambrook et al , Molecidar Cloning, 1989 For example, to specifically detect human or mouse MTase. an ohgonucleotide can be hybndized to a target nucleic acid under conditions in which the ohgonucleotide only hybndizes to it. e g . where the ohgonucleotide is 100% complementary to the target Different conditions can be used if it is desired to select target nucleic acids which have less than 100% nucleotide complementarity, at least about, e g . 99%. 97%. 95%. 90%. 70%. 67%. with the proviso that the sequence is not Xenopus Xmam4, S pombe mam4. S cerevisiae STEM. See. Imai et al . Mol Cell . Bio . 17 1543-1551, 1997, Sapperstein et al , Mol Cell Bio . 14 1438-1449. 1994 Since a mutation in a human or mouse MTase of the present invention can cause or enhance diseases or pathological conditions, e g . cancer, benign tumors, an ohgonucleotide according to the present invention can be used diagnostically For example, a patient having symptoms of a cancer or other condition associated with the Ras signaling pathway (see below) can be diagnosed with the disease by using an ohgonucleotide according to the present invention, in polymerase chain reaction followed by DNA sequencing to identify whether the sequence is normal, in combination with other ohgonucleotides to oncogenes or genes in the ras signalling pathway, etc . e g . GRB2. H-. K- and N-ras. c-Raf. MAP kinases. p42. p44. Ser/Thr kmases. Elk-1. c-myc. c-Jun, G-protems. Ftase, PPSEP, PPSMT. etc In a preferred method, the present invention relates to a method of diagnosing a cancer composing contacting a sample composing a target nucleic acid with an ohgonucleotide under conditions effective to permit hybodization between the target and ohgonucleotide. detecting hybodization. wherein the ohgonucleotide composes a sequence of a human or mouse MTase. preferably a unique sequence of. and determining the nucleotide sequence of the target nucleic acid to which the ohgonucleotide is hybodized The sequence can be determined according to vaoous methods, including isolating the target nucleic acid, or a cDNA thereof, and determining its sequence according to a desired method

Ohgonucleotides of the present invention can compose any continuous nucleotide sequence of Fig 1 These ohgonucleotides (nucleic acid) according to the present invention can be of any desired size, e g , about 10-200 nucleotides, 12-100, preferably 12-50, 12-25. 14-16. at least about 15, at least about 20, etc The ohgonucleotides can have non-naturally-occurong nucleotides, e g , mosine The ohgonucleotides have 100% identity or complementaoty to a sequence of Fig 1 or Fig 3. or it can have mismatches or nucleotide substitutions, e g , 1, 2, 3, 4, or 5 substitutions In accordance with the present invention, the ohgonucleotide can compnse a kit. where the kit includes a desired buffer (e g ,

12 phosphate, tos. etc ), detection compositions, etc The ohgonucleotide can be labeled or unlabeled. with radioactive or non-radioactive labels as known in the art

Anti-sense nucleic acid can also be prepared from a nucleic acid according to the present, preferably an anti-sense to a coding sequence of Fig 1. less preferably Fig 3 Antisense nucleic acid can be used in vaoous ways, such as to regulate or modulate expression of farnsvl-directed carboxymethyltransferase, e g . inhibit it. to detect its expression, or for in situ hybndization These ohgonucleotides can be used analogously to USP 5.576.208 describing inhibition of ras For the purposes of regulating or modulating expression of farnsy 1-dιrected carboxymethyltransferase. an anti- sense ohgonucleotide can be operably linked to an expression control sequence T e nucleic acid according to the present invention can be labelled according to am desired method The nucleic acid can be labeled using radioactive tracers such as ^3:P, ^S, ^I251, ³H. or ¹⁴C, to mention only the most commonly used tracers The radioactive labelling can be earned out according to any method such as. for example, terminal labeling at the 3' or 5' end using a radiolabeled nucleotide. polynucleotide kinase (with or ithout dephosphorylation with a phosphatase) or a gase (depending on the end to be labelled) A non-radioactive labeling can also be used, combining a nucleic acid of the present invention with residues having lmmunological properties (antigens, haptens). a specific affinity for certain reagents (ligands). properties enabling detectable enzyme reactions to be completed (enzymes or coenzymes. enzyme substrates, or other substances involved in an enzymatic reaction), or charactenstic physical properties, such as fluorescence or the emission or absoφtion of light at a desired avelength, etc

A nucleic acid according to the present invention, including ohgonucleotides. anti-sense nucleic acid, etc , can be used to detect expression of farnsvl-directed carboxymethyltransferase in whole organs, tissues, cells, etc , by vanous techniques, including Northern blot. PCR. in situ hybodization. etc Such nucleic acids can be particularly useful to detect disturbed expression, e g . cell-specific and/or subcellular alterations, of farnsy 1-dιrected carboxymethyltransferase The levels of farnsvl- directed carboxymethyltransferase can be determined alone or in combination with other genes products (e g , Ras-H. -N, -K4A, -K4B, p53, Rb, RCE1, etc )

A nucleic acid according to the present invention can be expressed in a vaoety of different systems, m vitro and in vivo, according to the desired purpose For example, a nucleic acid can be inserted into an expression vector, introduced into a desired host, and cultured under conditions effective to achieve expression of a polypeptide coded for the nucleic acid Effective conditions includes any culture conditions which are suitable for achieving production of the polypeptide by the host cell, including effective temperatures. pH, medias. additives to the media in which the host cell is cultured (e g . additives which amplify or induce expression such as butyrate. or methotrexate if the coding

13 nucleic acid is adjacent to a dhfr gene), cvclohexamide, cell densities, culture dishes, etc A nucleic acid can be introduced into the cell by any effective method including, e g . naked DNA. calcium phosphate precipitation, electroporation. injection. DEAE-Dextran mediated transfection. fusion with posomes, associated with agents which enhance its uptake into cells, viral transfection A cell into which a nucleic acid of the present invention has been introduced is a transformed host cell The nucleic acid can be extrachromosomal or integrated into a chromosome(s) of the host cell It can be stable or transient An expression vector is selected for its compatibility with the host cell Host cells include, mammalian cells, e g , COS-7, CHO, HeLa, LTK. NIH 3T3, HEK 293, yeast, insect cells, such as Sf9 (S frugipeda) and Drosophila. bacteoa. such as E co . Streptococcus, bacillus, yeast, fungal cells. plants, embryonic stem cells (e g , mammalian, such as mouse or human), cancer or tumor cells Sf9 expression can be accomplished in analogy to Graziam et al . Oncogene, 7 229-235 When the human gene was expressed in Sf9 cells, its activity was highest in the membrane fraction Expression control sequences are similarly selected for host compatibility and a desired purpose, e g . high copy number, high amounts, induction, amplification, controlled expression Other sequences which can be employed include enhancers such as from SV40. CMV. inducible promoters, cell-type specific elements, or sequences which allow selective or specific cell expression Promoters that can be used to dove its expression, include, e g . the endogenous promoter, SV40 etc

Another gene of interest can be introduced into the same host for purposes of. e g . modulating expression farnsvl-directed carboxymethyltransferase. elucidating farnsvl-directed carboxymethyltransferase function or that of the gene of interest Genes of interest include other oncogenes. genes involved in the cell cycle, such as p53. Rb, etc Such genes can be the normal gene, or a vaoation. e g , a mutation, chimera, polymorphism, etc

A nucleic acid or polypeptide of the present invention can be used as a size marker in nucleic acid or protein electrophoresis. chromatography. etc Defined restoction fragments can be determined by scanning the sequence for restoction sites, calculating the size, and performing the corresponding restnction digest The human farnsvl-directed carboxymethyltransferase cDNA can also be used as a 2 5 kb molecular weight marker on a gel

Another aspect of the present invention relates to the regulation of biological pathways in which an MTase gene is involved, particularly pathological conditions For example cell proliferation (e g , cancer), growth control, apoptosis. differentiation, morphogenesis, mating type, G-protein signalling, cell adhesion, etc For example, a mammalian MTase of the present invention is involved m the processing of vanous proteins, e g , polypeptides which are hpid-modified (for instance, containing a steroid intermediate such as faransyl or geranylgernanyl. or other prenylated species) and/or which contain an N-terminal C. CC, CCXX. CXC, See, e g , Cox and Der, Biochim B ophys Ada 1333,

14 F51 -F71, 1997 Of particular interest, is the role that a mammalian farnesyl-directed cysteine carboxymethy ltransferase plays in processing ras polypeptides since the latter is involved in carcinogenesis and transformation Ras and other polypetides containing the above-mentioned motifs are processed through one or more steps involving, farnsylation. endoproteolysis. and methylation See. e . Gelb. Science. 275. 1751-1751. 1997 Over-expression of ras (wild-type, mutated, constitutive, etc . ras), optionally in combination with aberrant expression of other genes, leads to oncogemc activity One approach to treating ras over-expression is inhibiting the ras maturation pathway so that incompletely processed and/or inactive ras accumulates, eliminating or reducing its oncogemc effect In accordance w ith the present invention, the ras maturation pathway can be inhibited by blocking mammalian farnesyl-directed cysteine carboxymethyltransferase, such as STEM, Blocking can be accomplished in various ways, including by administeπng STEM antibodies, or other STEM ligands. STEM peptides (especially, those that bind to a methyl donor but lack methyltransferase or methyltransestenfication activity), inhibitors of STEM catalytic activity (e g . methyl-donor mimics or competitors or antagonists), inhibitors of STEM gene expression (e g , anti-sense or double-stranded RNA. such as. Fire et al . Nature. 391 806-811, 1998) Blocking agents can be identified according to the methods descnbed herein or those available m the art

One aspect of the invention relates to identifying compounds which modulate farnesyl-directed cy steine carboxymethyltransferase activity The activity can be modulated by increasing, reducing, antagonizing, promoting, stabilizing, etc its activity Thus, one object of the invention is to facilitate screening for compounds which modulate the incorporation of a methyl group into a meth l-acceptor substrate of the methyltransferase enzyme

Accordingly the present invention relates to identifying compounds that modulate a mammalian farnesyl directed cysteine carboxymethyltransferase. especially a human or mouse MTase of Fig 1 or Fig 3. composing reacting, in the presence of a test compound, a methyl-donor substrate, a methyl - acceptor substrate, and a mammalian farnesyl-directed cysteine carboxymethyltransferase, or a fragment thereof having methyltransferase activity, under conditions effective for the mammalian carboxymethy ltransferase, or said fragment, to methylate said methyl-acceptor substrate, detecting the methylation of said methyl-acceptor substrate, and identifying whether the test compound modulates meth ltransferase activity by compaong the amount of methylation in the presence and absence of the test compound

Any functional methyl-donor substrate is acceptable, including detectably-labeled S-adenosyl- methionine. especially S-adenosyl-L-[methyl-¹⁴C]methιonιne or S-adenosyl-L-[methyl-³H]methιonιne Likewise, any functional methyl-acceptor is satisfactory, including polypeptides which compose a terminal farnsylated cysteine. polypeptides which terminate in CC, CCXX. or CXC. and/or are

15 cysteine-prenylated with, for instance, a farnsyl or geranylgeranyl Functional methyl-acceptor substrates include, e g , Ras-H, -N, -K4A. -K4B. Lamms A and B. RhoB. RhoE. others. Rap2. Rheb. Phosphorylase kinase and . Rhodopsm kinase. Transducin . cGMP phosphodiesterasae . IFN- mduced guanylate-bindmg protein 1. IP₃ 5-phosphatase. PxF. PRL-1/PTPCAAXl and 2. biotin-Lys- Lys-Ser-Lys-Thr-Lys-(Farnesyl)Cys. etc

Other substrates include, hepatitis delta virus (Otto et al , J Biol Chem , 271 4569-72, 1996) In general, any substrate is suitable if it can be acted upon in the methyltransferase reaction Thus, a substrate can compnse other atoms, such as additional amino acid residues linked by peptide or other bonds, and can be modified in any desirable way For example, a substrate can be affixed to a solid suport. e g . compnsmg, latex, sepharose. silica, agarose. sephadex. cellulose, polysacchaπdes. glass. polymers, etc A substrate can also be detectably labeled, e g . with antibody, avidin. biotin. radioactive labels, aptamers. fluorescent labels, nucleic acid, etc The substrates can also compnse phosphates, methyl groups, sugars, or pids

The test compound is preferably reacted with substrates in a milieu in which meth lation of the acceptor-substrate is accomplished Such a milieu can be referred to as effective conditions These conditions can be determined in the absence of the test compound to establish a baseline activity, e g , as in a control The effective reaction conditions can be routinely selected, e g , using salts, buffers, reducing and/or oxidizing agents. pH's. etc For instance, when utilizing a methyl-acceptor substrate acceptor comprising a farnsylated C-termmal cysteine. effective methylation results in its methylation After the step of reacting the substrates, test compound, and MTase, under conditions m which methylation can be achieved, the next step is to determine the effect of the test compound on methylation Detecting methylation. can be optimized in the absence of the test compound to establish a baseline activity for MTase Generally, detecting methylation involves measunng the mcoφoration of a labeled methyl group (e g . ³H or ¹⁴C) into an acceptor-substrate of the MTase In a preferred embodiment, the methyl-acceptor substrate is bιotιn-Lys-Lys-Ser-Lys-Thr-Lys-(Farnesyl)Cy s and the methyl-donor substrate is detectably-labeled S-adenosyl methiomne (for instance. S-adenosyl-L- [methyl-'⁴C]methιonme or S-adenosyl-L-[methyl-³H]methιonιne) and the methylation detecting is accomplished by captunng the methyl-acceptor substrate using an streptavidin-coated bead, and measunng the amount of labeled methyl incoφorated into said methyl-acceptor substrate However, the capture can be accomplished using any available means, depending upon the anchor (such as biotin in the example) incoφorated into the methyl-acceptor substrate, e g , SPA beads coated with protein A can be bound to anti-Ras antibody which can be used to capture full-length protein from cells If this cellular RAS is unmethylated, recombinant MTase can be used to incoφorate label from ³H-S-adenosyl methionine

16 In general assays according to the invention involve capture of the methyl-substrate acceptor and distinguishing whether the substrate has been modified. 1 e . by the addition of a methyl group This can be accomplished by any effective means, including antibodies (for instance, an antibody that recognizes the methylated substrate but not the unmethylated. or vice-versa), mass spectroscopy. electrophoretic mobility shifts, chromatography. electrophoresis, etc

The MTase component can be added to the reaction mixture in a vanety forms, e g , substantially puofied. as a component of cell membranes (such as. endoplasmic reticulum). or as a soluble extract In each case, the MTase polypeptide can be obtained from a natural source, a recombinant source (e g . a human STEM expressed in an insect cell line or bacteoa as a fusion or non- fusion protein, for instance as descobed in Hrycyna et al , Methods Enzvmol . 250 251-266. 1995). or it can be produced synthetically (produced chemically or enzymatically. e g , cleavage of a full-length MTase

Preferably, the MTase is expressed in a cell line transformed with an MTase coding sequence (e g . a cDNA. a gene, a genomic fragment, etc ) In the latter case, the MTase is present as a heterologous component of the cell, by heterologous. it is meant that the MTase is not only expressed m a cell line of a different species, but it is also coded for by a coding sequence that has been introduced into the cell, e g , by transfection. transformation, etc Preferably, the MTase is expressed at high levels in the cell (bacteoal, yeast, mammalian, etc ) A human farnesyl-directed cysteine carboxymethyltransferase. or a fragment thereof, is a preferred coding sequence See, e g , Fig 1 A useful fragment of the human sequence composes a methvltransferase activity and methyl-donor substrate binding activity

In a preferred aspect of the invention, the MTase is provided as a cell lysate. e g . cells transformed with human STEM are lysed and the resulting lysate is used directly in the assay, l e . a crude lysate The crude lysate composing the recombinant human STEM can optionally be refined or ennched for human STEM For instance, e g. a membrane fraction can be isolated, etc , as descnbed in

Hrycyna et al . Methods Enzvmol . 250 251-266, 1995

A puφose of the assay is select and identify compounds which modulate MTase activity Thus, methylation is typically performed in the presence and absence of the test compound Whether a compound modulates RCE can be determined routinely, e g , by determining whether more or less methylation has occurred in the presence of the test compound

The assay can also be conducted in whole cells For instance, a modulator of farnesyl-directed cysteine carboxymethyltransferase activity can be added to a desired host cell line and then its effect on the cell line can be observed As mentioned, the MTases methylate a vaoety of different proteins in cells See. above Thus, the effect of an inhibitor on a whole cell (or extract, etc ) can be measured by

17 identifying whether the substrate has been methylated For example, in cell lines that express ras. a whole cell assay can compose administeong a test compound to a cell, determining or detecting whether the test compound modulates the processing of ras. e g . by using an antibody or electrophoretic shift to detect whether a ras intermediate has accumulated in the cell Cell lines can be engineered to express the methyltransferase substrate, overexpress it, etc An in vivo method of assaying for farnesyl- directed cysteine carboxymethyltransferase further involves modifying test compounds to gain entry into the cell, e g , deovatizmg compounds, encapsulating compounds in hposome. and other means to enhance delivery to the cell, e g , to stimulate phagocytosis Agents can also be administered to such cells and tested for their ability to inhibit transformation, e g . by monitoring cell moφhology. etc See. e g . USP 5.688.655 Assays can also be earned out as descnbed in USP 5,710.171. 5,703.241,

5.585,359. 5.557.729. 5,532.359. 5,470.832, 5,420. 245. 5, 185,248

Cell lines useful for the in vitro (e g , as a source of membranes) and in vivo assays can express one or more heterologous genes, including FTase. RCE1, Rb. rac. p53, Ras-H. -N, -K4A, -K4B. Lamms A and B. RhoB. RhoE. Rap2. Rheb. Phosphorylase kinase and . Rhodopsin kinase. Transducin . cGMP phosphodiesterasae . IFN-induced guanvlate-binding protein 1. IP₃ 5 -phosphatase.

PxF. PRL-1/PTPCAAXl and 2

Compounds identified in this or other manners can be useful to modulate farnesy 1-dιrected cysteine carboxymethyltransferase activity in a cell, a tissue, a whole organism, in situ, in vitro (test tube, a solid support, etc ). in vivo, or in any desired environment In general, a compound having such an in vitro activity will be useful in vivo to modulate a biological pathway associated with farnesyl- directed cysteine carboxymethyltransferase. e g , to treat a pathological condition associated with the biological and cellular activities mentioned above The present invention thus also relates to the treatment and prevention of diseases and pathological conditions associated with ras. G-protein. etc - mediated signal transduction. e g , cancer, diseases associated with abnormal cell proliferation For example, the invention relates to a method of treating cancer compnsmg admimstenng. to a subject in need of treatment, an amount of a compound effective to treat the disease, where the compound is a regulator of farnesyl-directed cysteine carboxymethyltransferase gene or polypeptide expression Treating the disease can mean, delaying its onset, delaying the progression of the disease, improving or delaying clinical and pathological signs of disease A regulator compound, or mixture of compounds, can be synthetic, naturally-occurong. or a combination A regulator compound can compose ammo acids, nucleotides, hydrocarbons, hpids, polysacchaodes. etc A regulator compound is preferably a regulator of farnesyl-directed cysteine carboxymethyltransferase. e g . inhibiting or increasing its mRNA. protein expression, or processing Expression can be regulated using different agents, e g . an anti-sense nucleic acid, a obozyme, an aptamer. a synthetic compound, or a naturally-occurong

18 compound To treat the disease, the compound, or mixture, can be formulated into pharmaceutical composition composing a pharmaceutically acceptable carrier and other excipients as apparent to the skilled worker See, e g , Remington's Pharmaceutical Sciences. Eighteenth Edition. Mack Publishing Company. 1990 Such composition can additionally contain effective amounts of other compounds. especially for treatment of cancer

The present invention also relates to antibodies which specifically recognize farnesyl-directed cysteine carboxymethyltransferase. Antibodies, e g , polyclonal, monoclonal, recombinant. chimenc, can be prepared according to any desired method For example, for the production of monoclonal antibodies, a polypeptide according to Fig 1 (a specific fragment thereof) can be administered to mice, goats, or rabbit subcutaneously and/or mtrapeotoneally. with or without adjuvant, in an amount effective to elicit an immune response The antibodies can also be single chain or FAb The antibodies can be IgG. subtypes. IgG2a. IgGl. etc Antibodies can also be generated bv administeong naked DNA See. e g , USP 5.703.055, 5,589.466. 5,580,859

An antibody specific for farnesyl-directed cysteme carboxymethyltransferase means that the antibodv recognizes a defined sequence of ammo acids within or including a farnesyl-directed cysteine carboxymethy ltransferase. e g , the human and muone sequences of Fig 1 and Fig 3 Thus, a specific antibody will bind with higher affinity to an amino acid sequence, I e . an epitope. found m Fig 1 than to a different epιtope(s). e g . as detected and/or measured by an immunoblot assay Thus, an antibody which is specific for an epitope of human STEM is useful to detect the presence of the epitope in a sample, e g . a sample of tissue containing human STEM gene product, distinguishing it from samples in which the epitope is absent Such antibodies are useful as descobed in Santa Cruz Biotechnology, Inc . Research Product Catalog, can be formulated accordingly, e g . 100 μg/ml

In addition, ligands which bind to a farnesyl-directed cvsteine carboxymethvltransferase polypeptide according to the present invention, or a deovative thereof, can also be prepared, e g , using synthetic peptide braoes or aptamers (e g . Pitrung et al . U S Pat No 5.143.854. Geysen et al .

1987, J Immunol Methods. 102 259-274. Scott et al . 1990. Science. 249 386. Blackwell et al . 1990, Science, 250 1104, Tuerk et al , 1990, Science, 249 505

Antibodies and other ligands which bind farnesyl-directed cysteme carboxymethyltransferase can be used in vaoous ways, including as therapeutic, diagnostic, and commercial research tools, e g, to quantitate the levels of farnesyl-directed cysteme carboxymethyltransferase polypeptide in animals, tissues, cells, etc , to identify the cellular localization and/or distribution of it. to punfy it, or a polypeptide composing a part of it. to modulate the function of it, etc Antibodies to it. or a derivative thereof, can be used in Western blots, ELIZA, immunoprecipitation, RIA, etc The present invention relates to such assays, compositions and kits for performing them, etc

19 An antibody according to the present invention can be used to detect farnesyl-directed cysteine carboxymethyltransferase polypeptide or fragments thereof in vaoous samples, including tissue, cells, body fluid, blood, uone, cerebrospmal fluid A method of the present invention composes, e g , (a) contacting a ligand which binds to a peptide of Fig 1 under conditions effective, as known in the art. to achieve binding, and (b) detecting specific binding between the ligand and peptide By specific binding, it is meant that the ligand attaches to a defined sequence of amino acids, e g , within or including the amino acid sequence of Fig 1 or deovatives thereof The antibodies or deovatives thereof can also be used to inhibit expression of farnsvl-directed carboxymethyltransferase or a fragment thereof The levels of farnesyl-directed cysteine carboxymethyltransferase polypeptide can be determined alone or in combination with other gene products In particular, the amount (e g , its expression level) of farnesyl- directed cy steine carboxymethyltransferase polypeptide can be compared (e g , as a ratio) to the amounts of other polypeptides in the same or different sample, e g . ras. FTase. endoprotease. etc A ligand for farnesyl-directed cysteine carboxymethyltransferase can be used in combination with other antibodies, e g . antibodies that recognize oncological markers of cancer, including, ras. etc In general. reagents which are specific for farnesyl-directed cysteine carboxymethyltransferase can be used in diagnostic and/or forensic studies according to any desired method, e g , as U S Pat Nos 5,397,712, 5,434,050. 5.429.947

The present invention also relates to a labeled farnsvl-directed carboxymethyltransferase polypeptide. prepared according to a desired method, e g . as disclosed in U S. Pat No 5,434.050 A labelled polypeptide can be used, e g , in binding assays, such as to identify substances that bind or attach to farnsvl-directed carboxymethyltransferase, to track the movement of farnsvl-directed carboxymethyltransferase in a cell, in an in vitro, m vivo, or in situ system, etc

A nucleic acid, polypeptide. antibody, farnsvl-directed carboxymethyltransferase ligand etc . according to the present invention can be isolated The term "isolated" means that the mateoal is in a form in which it is not found in its onginal environment, e g , more concentrated, more punfied, separated from component, etc An isolated nucleic acid includes, e g , a nucleic acid having the sequence of farnsvl-directed carboxymethyltransferase separated from the chromosomal DNA found m a living animal. This nucleic acid can be part of a vector or inserted into a chromosome (by specific gene-targeting or by random integration at a position other than its normal position) and still be isolated in that it is not in a form which it is found in its natural environment A nucleic acid or polypeptide of the present invention can also be substantially punfied By substantially punfied, it is meant that nucleic acid or polypeptide is separated and is essentially free from other nucleic acids or polypeptides. l e , the nucleic acid or polypeptide is the pomary and active constituent

20 The present invention also relates to a transgenic animal, e.g., a non-human-mammal, such as a mouse, composing a farnsvl-directed carboxymethyltransferase nucleic acid. Transgenic animals can be prepared according to known methods, including, e.g.. by pronuclear injection of recombinant genes into pronuclei of 1 -cell embryos, incoφorating an artificial yeast chromosome into embryonic stem cells, gene targeting methods, embryonic stem cell methodology. See, e.g.. U.S. Patent Nos. 4.736,866;

4,873,191; 4.873,316; 5,082.779; 5,304,489: 5,174.986; 5,175,384; 5,175,385; 5.221.778: Gordon et al.. Proc. Natl. Acad. Set.. 77:7380-7384 (1980): Palmiter et al.. Cell. 41:343-345 (1985): Palmiter et al., Ann. Rev. Genet.. 20:465-499 (1986): Askew et al.. Mol. Cell. Bio., 13.4115-4124. 1993: Games et al. Nature. 373:523-527. 1995; Valancius and Smithies. Mol. Cell. Bio.. 11: 1402-1408. 1991; Stacey et al.. M>/. Cell. Bio., 14: 1009-1016. 1994; Hasty et al.. Nature, 350:243-246. 1995; Rubinstein et al.,

Nucl. Acid Res.. 21:2613-2617, 1993. A nucleic acid according to the present invention can be introduced into any non-human mammal, including a mouse (Hogan et al.. 1986. in Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor. New York), pig (Hammer et al., Nature. 315:343-345, 1985), sheep (Hammer et al.. Nature. 315:343-345, 1985). cattle, rat. or primate. See also, e.g., Church, 1987, Trends in Biotech. 5: 13-19; Clark et al.,

1987. Trends m Biotech. 5:20-24: and DePamphilis et al., 1988, BioTech ques, 6:662-680. In addition, e.g., custom transgenic rat and mouse production is commercially available. These transgenic animals are useful as a cancer model, e.g., to test drugs, as food for a snake, as genetic markers to detect strain origin, etc. Such transgenic animals can further comprise other transgenes genes, e.g., Rb, p53. RCE1. FTase. rho. rab. rac. gamma-subunits of GTP -binding protein, and any of the above- mentioned genes throughout this disclosure.

Generally, the nucleic acids, polypeptides. antibodies, etc. of the present invention can be prepared and used as described in. U.S. Pat. Nos. 5.501.969. 5.506.133. 5.441.870; WO 90/00607; WO 91/15582; For other aspects of the nucleic acids, polypeptides. antibodies, etc.. reference is made to standard textbooks of molecular biology, protein science, and immunology. See, e.g., Davis et al. (1986), Basic Methods in Molecular Biology, Elsevir Sciences Publishing. Inc.. New York; Hames et al. (1985), Nucleic Acid Hybridization. IL Press. Molecular Cloning. Sambrook et al.; Current Protocols in Molecular Biology. Edited by F.M. Ausubel et al.. John Wiley & Sons, Inc; Current Protocols in Human Genetics. Edited by Nicholas C. Dracopoli et al., John Wiley & Sons, Inc.;

Current Protocols in Protein Science: Edited by John E. Coligan et al., John Wiley & Sons, Inc.; Current Protocols in Immunology; Edited by John E. Coligan et al.. John Wiley & Sons. Inc.

21 EXAMPLE

We have devised our own version of this assay which utilizes a biotmylated. prenylated peptide substrate (Bιotιn-Lys-Lys-Ser-Lys-Thr-Lys-(Farnesyl)Cys, based on the C-terminal sequence of K-Ras- 4B. but lacking the final three residues) In short, the human STEM-expressmg bactenal or insect cell membranes utilize the co-substrate ³H-S-adenosyl methionine to methylate the (Farnesyl)Cys-carboxyl group The resulting label incoφorated into the substrate peptide is quantified using streptavidm-coated SPA beads The methylase is cloned into the pRSET (Invitrogen) and pFastBac (Gibco BRL) bactenal and insect cell expression vectors, respectively

A standard assay is performed in 96-well sample plates (Wallac Part NO 1450-401) with a total assay volume of 100 1 which generally contains 50 1 compound. 25 1 membranes and 25 1 ³H-

SAM substrate added in that order Final concentration of HEPES pH 7 4 is 100 mM

A volume of 25 1 of membranes in 100 mM HEPES pH 7 4 is added to each well, followed by 25 1 diluted substrate (methylase substrates is stored at -20°C in 100% DMSO but is diluted in 10% DMSO to the required working concentration immediately before use) To this is added the label, l e Η-SAM (~85Cι mmol, lmCi/ml, 12 M)

0 125 1 per well made up to 25 1 with 100 mM HEPES pH 7 4

The plate is then sealed and incubated at room temperature for 60 mins

The reaction is stopped by adding 150 1 Stop Mix which contains SPA beads (250 g) m PBS pH 7 1 + 5 mM EDTA + 0 1% Tween-20 The plate is sealed again the beads are left to settle overnight before reading on a scintillation counter

Without further elaboration, it is believed that one skilled in the art can, using the preceding descnption. utilize the present invention to its fullest extent The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever The entire disclosure of all applications, patents and publications, cited above and in the figures are hereby incoφorated by reference

From the foregoing descnption, one skilled in the art can easily ascertain the essential characteπstics of this invention, and without departing from the spint and scope thereof, can make vanous changes and modifications of the invention to adapt it to vaoous usages and conditions

22

Claims

What is claimed:

1 An isolated mammalian farnesyl-directed cysteine carboxymethyltransferase polypeptide or a biologically-active fragment thereof.

2. An isolated mammalian farnesyl-directed cysteine carboxymethyltransferase, or a biologically- active fragment thereof, of claim 1, wherein said polypeptide has a methyl-donor substrate binding activity or a methyltransferase activity.

3. An isolated mammalian farnesyl-directed cysteine carboxymethyltransferase, or a biologically- active fragment thereof, of claim 1, wherein said polypeptide is capable of transferring a methyl group to a methyl-acceptor substrate comprising a terminal S-farnesyl-cysteine.

4. An isolated mammalian farnsvl-directed carboxymethyltransferase. or a biologically-active fragment thereof, of claim 1 which is human.

5. An isolated mammalian farnsvl-directed carboxymethyltransferase of claim 1. comprising amino acid 1 to amino acid 284 as set forth in Fig. 1.

6. An isolated mammalian farnsvl-directed carboxymethyltransferase of claim 1, comprising amino acid 1 to amino acid 153 as set forth in Fig. 3.

7. An isolated mammalian farnsvl-directed carboxymethyltransferase of claim 6. which consist of the amino acid sequence set forth in Figure 3.

8. An isolated mammalian farnsyl-directed carboxymethyltransferase, or a biologically-active fragment thereof, of claim 1, which is substantially purified.

9. An isolated nucleic acid comprising a nucleotide sequence coding for a mammalian farnsyl- directed carboxymethyltransferase polypeptide or a biologically-active polypeptide fragment thereof.

10. An isolated nucleic acid of claim 9, wherein said coded for polypeptide has a methyl-donor substrate binding activity or a methyltransferase activity.

23 11 An isolated nucleic acid of claim 9. wherein said polypeptide is capable of transfemng a methyl group to a methyl-acceptor substrate compnsmg a terminal S-farnesyl-cysteine

12 An isolated nucleic acid of claim 9 which is human

13 An isolated nucleic acid of claim 9, wherein the nucleotide sequence codes for amino acid 1 to ammo acid 284 as set forth in Fig 1

14 An isolated nucleic acid of claim 9. wherein the nucleotide sequence codes for ammo acid 1 to amino acid 153 as set forth in Fig 3

15 An isolated nucleic acid of claim 9. which consist of the nucleic acid set forth m Fig 3

16 An isolated nucleic acid of claim 9. consisting essentially of any continuous sequence of 12-100 base pairs, or a complement thereto, selected from the nucleotide sequences set forth in Fig I

17 An isolated nucleic acid of claim 16. further composing a detectable label

18 An isolated nucleic acid of claim 9. wherein the nucleotide sequence is operably linked to an expression control sequence

19 An isolated nucleic acid of claim 9. wherein the nucleic acid composes a naturally-occurong nucleotide sequence

20 An isolated nucleic acid of claim 9. wherein the nucleic acid codes for said polypeptide without interruption

21 An isolated nucleic acid of claim 9, wherein the nucleic acid is DNA or RNA

22 An isolated nucleic acid of claim 9. wherein the nucleic acid further composes a detectable label

24 23 An isolated nucleic acid of claim 9. wherein one or more amino acid positions are substituted or deleted, or both, and the polypeptide coded for by the nucleic acid has methyl-donor substrate binding activity or a methyltransferase activity

24 An isolated nucleic acid of claim 23. wherein one or more substituted ammo acid positions are substituted by homologous amino acids

25 An isolated nucleic acid of claim 9, having a naturally-obtainable nucleotide sequence which hvbndizes under stnngent conditions to the nucleotide sequence set forth in Fig 1 , or a complement thereto, with the proviso that said sequence is not Xenopus Xmam4, S pombe mam4, S cerevisiae

STEM, or fragments thereof

26 An isolated nucleic acid of claim 25 compnsmg at least 95% nucleotide sequence identity to the nucleotide sequence set forth in Fig 1

27 An isolated nucleic acid of claim 25, wherein said nucleotide sequence codes for a polypeptide having a methyl-donor substrate binding activity or a methyltransferase activity

28 A method of expressing in transformed host cells, a mammalian famsyl-directed carboxymethy ltransferase polypeptide coded for by a nucleic acid, compnsmg cultunng transformed host cells containing a nucleic acid according to claim 15 under conditions effective to express the polypeptide

29 A method of claim 28. wherein said host cells are Sf9 or HEK293

30 A method of claim 28, further composing isolating the membrane fraction of said host cells composing said polypeptide

31 A method of claim 28. further composing modulating expression of the polypeptide

32 A method of claim 28, wherein said polypeptide contains amino acid 1 to 284 as set forth in Fig 1

33 A transformed host cell contaming a nucleic acid of claim 9

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34. A transformed host cell containing a nucleic acid of claim 13.

35. A vector comprising a nucleic acid of claim 9.

36. A vector comprising a nucleic acid of claim 13.

37. A transgenic non-human mammal comprising a nucleic acid of claim 13.

38. A method of identifying compounds that modulate a mammalian farnesyl directed cysteine carboxymethyltransferase comprising: reacting, in the presence of a test compound, a methyl-donor substrate, a methyl-acceptor substrate, and a mammalian farnesyl-directed cysteine carboxymethyltransferase, or a fragment thereof having methyltransferase activity, under conditions effective for the mammalian carboxymethyltransferase, or said fragment thereof, to methylate said methyl-acceptor substrate; detecting the methylation of said methyl-acceptor substrate; and identifying whether the test compound modulates methyltransferase activity by comparing the amount of methylation in the presence and absence of the test compound.

39. A method of claim 38, wherein the mammalian farnesyl-directed cysteine carboxymethyltransferase, or a fragment thereof, is human.

40. A method of claim 38. wherein the methyl-acceptor substrate comprises a prenylated C-terminal cysteine.

41. A method of claim 38, wherein the methyl -acceptor substrate is biotin-Lys-Lys-Ser-Lys-Thr- Lys-(Farnesyl)Cys and the methyl-donor substrate is detectably-labeled S-adenosyl methionine.

42. A method of claim 38, wherein the methyl-acceptor substrate is biotin-Lys-Lys-Ser-Lys-Thr- Lys-(Famesyl)Cys and the methyl-donor substrate is S-adenosyl methionine comprising a detectably labeled methyl group, and the methylation detecting is accomplished by: capturing the methyl-acceptor substrate and measuring the amount of labeled methyl incoφorated into said methyl-acceptor substrate.

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43. A method of claim 38, wherein the methyl-acceptor substrate is biotin-Lys-Lys-Ser-Lys-Thr- Lys-(Farnesyl)Cys and the methyl-donor substrate is S-adenosyl methionine comprising a detectably- labeled methyl group, and the methylation detecting is accomplished by: capturing the methyl-acceptor substrate using an streptavidin-coated bead; and measuring the amount of labeled methyl incoφorated into said methyl-acceptor substrate.

44. A method of claim 38, wherein the carboxymethyltransferase is substantially purified.

45. A method of claim 38. wherein the carboxymethyltransferase is present as a heterologous component of cell membranes.

46. A method of claim 38. wherein the carboxymethyltransferase is present as a fusion protein.

47. A method of claim 38. wherein the carboxymethyltransferase comprises amino acid 1 to 284 as set forth in Fig. 1.

48. A method of claim 38. wherein said method is performed with intact cells.

49. An isolated antibody which is specific for a mammalian farnsyl-directed carboxymethyltransferase.

50. An isolated antibody of claim 48. which binds to an amino acid sequence selected from Fig. 1.

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