Note: Descriptions are shown in the official language in which they were submitted.
CA 02323759 2000-09-08
WO 99!55878 PCTIUS99/07396
NOVEL NUCLEIC ACIDS AND POLYPEPTIDES RELATED TO A - w
FARNESYL-DIRECTED CYSTEINE CARBOXYMETHYL~'RANSFERASE
BACKGROUND OF THE INVENTION
Farnesyl-directed cysteine carboY~emethyltransferases are involved in the
processing of various
proteins, such as proteins involved in signaling pathways, fungal mating
factors. and Ras polvpeptides.
An activity of these methyltransferases is to perform methvlesterification on
prenvlated proteins. See,
e.g., Ashbv et al., Yeast, 9:907-913, 1993; Khosravi-Far et al., Cell Growth
and Di ferentiation, 3:461-
469. 1992.
DESCRIPTION OF THE INVENTION
The present invention concerns carboYynethvltransferases ("MTase"), especially
mammalian
farnesvl-directed cv steine MTases. such as human ST'E 14. MTases catalyze the
transfer of a methyl
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 enzyne
acts and the nature of the
chemical bond which is formed. See. e.g., Kagan and Clarke, Arch. Biochem.
Biophvs. 310: 417-427,
1994.
An aspect of the invention relates to nucleic acids, polvpeptides, and
fragments thereof., coding
for carboY~methyltransferases, especially a manunalian farnesyl-directed
cysteine
carboxvmethvltransferase. such as human STE14 and murine MTase. The invention
further relates to
methods of using such nucleic acids and polvpeptides in therapeutics,
diagnostics, and research. For
example, the nucleic acids and polvpeptides can be utilized in methods to
identify modulators of MTase
activity and to obtain ligands which bind to MTase.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a nucleotide and amino acid coding sequence for a human farnesyl-
directed
cvsteine carboxvmethyltransferase.
Fig. 2 shows a comparison behveen methyltransferase sequences from different
species.
Fig. 3 shows a nucleotide and amino acid coding sequence for a mouse farnesyl-
directed
cvsteine carboxvmethvltransferase
DETAILED DESCRIPTION OF THE INVENTION
Novel nucleic acid and polyeptide sequences have been identified which code
for mammalian
farnesyl-directed cysteine carboxvmethyltransferases. 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-proteins,
nuclear laminins, and fungal mating pheromones. A farnesyl-directed cysteine
CA 02323759 2000-09-08
WO 99155878 PCTIUS99107396
carboxvmethyltransferase has at least one of the following activities: ability
to bind to, or attach to, a~~
methyl donor substrate, e.g., S-adenosyl-L-methionine ("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-famesyl-cysteine;
methylesterification of a newly exposed
alpha-carboxyl group. where the alpha-carboxyl group is a prenylated cysteine.
such as a S-farnesvl-
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 polvpeptide or
poivnucleotide is capable of eliciting an immune response specific for an
MTase of the present
invention.
l0 The above-mentioned activities of an MTase can be measured according to
available assays or
as described in the examples below. See, e.g., Imai et al., Mol. Cell. Bio.,
17:1543-l~~ 1, 1997:
Hrye~na et al., Methods Enzvmol.. 20:261-266. 1995.
Substrate binding is generally considered the first step in enzyme catalysis
because the
substrate, acting as a ligand, must first attach to the enzyme surface to
enable the enzyme to carry out
13 its catalytic reactions. This enzyme surface can be referred to as the
active site of the enzyme. Binding
of the substrate to the enzyne surface can involve multiple interactions with
the enzyme, e.g., chemical
bonding with one or more amino acids and/or functional groups w°hich
comprise the enzyme. A methyl
donor substrate binding activiy as used herein means that a methyl donor
substrate attaches to an
MTase of the present invention. Attachment to the enzyme can be accomplished
by one or more of the
20 interactions which hold its naturally-occurring substrate to it: however, a
polvpeptide can have a methyl
donor substrate binding activity when it holds the substrate with less than
the naturally-occurring
number and qualit~~ of interactions.
Methyl-donor substrate binding and catalyic activity can be dissociated from
each other. Thus.
an MTase pohpeptide in accordance with the invention can possess substrate
binding activity but not a
25 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 in 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
30 substrates which attach to a polvpeptide, or derivative thereof, e.g., by
combining under effective
conditions, a substrate containing a detectable marker, an STE 14 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
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in solid phase. as desired. Solid-phase assays can be performed using high-
throughput procedures, e.g.,
on chips, wafers, etc.
A poh~peptide according to the present invention can also possess a catalyic
activity, e.g.,
transfer of a methyl group to a methyl acceptor substrate. Generally. the
catalysis results in
methvlesterification of a newly exposed alpha-carboxyl group of an amino acid.
especially a prenylated
cysteine. Thus, a catalytic activity in accordance with the present invention
is a carbaxylmethylation
activity. especially of prenylated polvpeptides. This activity is described,
e.g., in Hrycvna et al.,
Methods Enzvmol., 250:251-266, 199; Imai et al., Mol. Cell. Bio.. 17:1543-1~~
1, 1997. This activity
can be measured in vitro or in vivo.
A polyeptide, 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 STE I4 coding sequence
in cells can cause a
transformed phenotype, or it can enhance the phenotype of already transformed
cells. A transfornation-
promoting activity can be enhanced by the presence of defects in other genes
which contribute to
transformation. such as ras, p~3, Rb, cell-cycle regulaton~ genes, etc.
A mammalian farnesyi-directed cysteine carboxymethyltransferase (e.g.. human
STE14) is a
mammalian polvpeptide, or fragment thereof, having an amino acid sequence
which is obtainable from
a natural source. It therefore includes naturally-occurring, 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 primary and immortalized cell fines,
biopsied tissues, etc. The
present invention also relates to fragments of a full-length mammalian MTase,
such as human STE 14 or
murine MTase. The fragments are preferably biologically-active. By
biologically-active. it is meant
that the pohpeptide 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
immunogenic activity.
Fragments can be prepared according to any desired method. including, chemical
synthesis, genetic
engineering, cleavage products, etc. See, below.
The present invention also relates to a human farnesyl-directed cvsteine
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 STE 14 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, andlor enhancer
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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
various
methods. For example, hybridization with an oligonucleotide (see below)
selective for a mammalian
farnesyl-directed cysteine carboxvmethvltransferases can be employed to select
such homologs, e.g., as
described in Sambrook et al., Molecular Cloning, 1989, Chapter 11. Such
homologs can have varying
amounts of nucleotide and amino acid sequence identity and similarity to
farnsyl-directed
carboxynethyltransferase. 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 cvsteine carboxwnethvl-
transferase specific
amino acid sequences, e.g., a defined amino 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 STE 14, 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 amino acid sequence can be found routinely, e.g., by searching a
genelprotein
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
carboxymethyltransferases
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 farnesv I-directed cysteine carboxvmethyltransferases protein for
diagnostic or research
purposes.
A polvpeptide of the invention. e.g., having a pohpeptide sequence as shown in
Fig. 1 or Fig. 3.
can by analyzed by available methods to identify structural and/or functional
domains in the
polypeptide. For example, when the polvpeptide coding sequence set forth in
Fig. 1 is analyzed by
hydropathy and hydrophilicity analysis (e.g., Ky~te and Doolittle, J. Mol.
Bio.,157:105, 1982) putative
membrane spanning regions are identified at: L16 to T34: L44 to Y59: I68 to
F85; I156 to L173, and
V225 to W241. A putative catalytic region is V 1 I O to L284. Various other
programs can be used to
analyze its structure and routinely predict functional domains, including,
EMBL Protein Predict: Rost
3o and Sander. Proteins, 19:55-72, 1994.
As mentioned polypeptides of the present invention can comprise a complete
coding sequence
for a mammalian farnesyl-directed cysteine carboYVmethyltransfer-ase, or
fragments thereof. For
example, an N-terminal region of a mammalian famesyl-directed cysteine
carboxymethyltransferase can
modulate its enzymatic activity, e.g., by enhancing its activity or
stabilizing it. Thus, useful fragments
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include, about amino acids I-6~ of the murine 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 polvpeptides
by joining them in reading-fi~ame with the polypeptide of interest. One or
more fi~agments can be used,
e.g., a human or murine STE 14 can comprise two or more N-terminal regions
which possess a
modulatory activity. A fragment of a farnesyl-directed cysteine
carboYVmethyltransferases polvpeptide
can be selected to have a specific biological activity, e.g., a methyl donor
binding activity, a
methvlesterification 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 activiy. The measurement of these activities is described below and in
the examples. These
peptides can also be identified and prepared as described in EP 496 162.
A polvpeptide 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 identitv~ means that the same nucleotide or amino acid which is found
in 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 various 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 in
the sequence over which
the farnesyl-directed cysteine carboxvmethyltransferases polvpeptide is
compared is equal to the percent
sequence similarity. 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 comprise e.g., about 99%,
97%, 9~% , preferably
about greater than 7I% homology, such as 75% or more, with the proviso that
the sequence is not
Xenopus Xmam4, S. pombe mam4, S. cerevisiae STE 14, or mouse MTase. See, Imai
et al., Mol. Cell..
Bio., 17:1 S43-1551, 1997; Sapperstein et al., Mol. Cell. Bio., 14:1438-1449.
The fragment of Fig 3
can also be excluded.
A mammalian farnesyl-directed cysteine carbo~cymethyltransferases polypeptide,
fi~agment, or
substituted polvpeptide can also comprise various modifications, where such
modifications include lipid
modification. methylation, phosphorvlation, glycosylation, covalent
modifications (e.g., of an R group
of an amino acid), amino acid substitution, amino acid deletion, or amino acid
addition. Modifications
to the polvpeptide can be accomplished according to various methods, including
recombinant, synthetic,
chemical, etc.
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WO 99155878 PCT/US99107396
Polypeptides of the present invention (e.g., human STE 14 or mouse MTase,
fragments thereof,
mutations thereof] can be used in various ways, e.g., in assays, as immunogens
for antibodies as
described below, as biologically-active agents (e.g., having one or more of
the activities associated with
STE 14).
A pohpeptide coding for a farnesyl-directed ~~steine
carboxy~rnethvltransferase, a derivative
thereof, or a fragment thereof, can be combined with one or more structural
domains, functional
domains. detectable domains. antigenic domains. andlor a desired polvpeptides
of interest. in an
arrangement which does not occur in nature. i.e., not naturally-occurring.
e.g., as in a human or murine
STE 14 gene. a genomic fragment prepared from the genome of a living organism,
e.g., an animal.
t0 preferably a mammal, such as human. mouse, or cell lines thereof. A
polvpeptide comprising such
features is a chimeric or fusion polypeptide. Such a chimeric polvpeptide can
be prepared according to
various methods. including, chemical. synthetic. quasi-swthetic. and/or
recombinant methods.
A chimeric nucleic acid coding for a chimeric polvpeptide can contain the
various domains or desired
polvpeptides in a continuous (e.g., with multiple N-terminal domains to
stabiiize or enhance activity) or
i5 interrupted open reading frame, e.g., containing introns, splice sites,
enhancers, etc. The chimeric
nucleic acid can be produced according to various 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, hydro-
20 philic. membrane-spanning. etc., receptor-ligand 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, Nnt:ire Biotechnology, 14:606: Levy et al., 1996. Natr~re
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 amino acid sequence
according to the present
25 invention can be fused in frame to a desired coding sequence and act as a
tag for purification, selection,
or marking purposes. The region of fusion can encode a cleavage site to
facilitate expression, isolation,
purification. etc.
A polvpeptide 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.
30 Modifications to the polvpeptide imparted by such system include,
glycosylation, amino acid
substitution (e.g., by differing codon usage), polypeptide processing such as
digestion, cleavage,
endopeptidase or exopeptidase activity, attachment of chemical moieties,
including lipids and
phosphates, etc.
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A poh~peptide 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. hvdroxyapatite 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 farnesvl-directed cysteine carboxvmeth~~ltransferase nucleic acid.
or fragment
thereof; is a nucleic acid having a nucleotide sequence obtainable from a
natural source. or comprising a
coding sequence coding for a mammalian farnesyl-directed cvsteine
carboxymethvltransferase. See,
above. It therefore includes naturally-occurring, normal, mutant, polymorphic.
degenerate sequences,
etc., alleles. Natural sources include. e.g., living cells obtained from
tissues and whole organisms,
cultured cell lines, including primary: and immortalized cell Lines.
Expression of human STE14 is
relatively ubiquitous, e.g., it is expressed in e.g., heart, brain, placenta.
lung, liver. skeletal muscle,
kidney, pancrease, spleen. thynus, prostate. testis. overt'. small intestine,
colon. and peripheral blood
leucocytes. It is also expressed in various 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 6361. The approximate
size of the
transcripts are about 2 kb, 3.5 kb, and S kb.
A nucleic acid sequence of a human allele of a mammalian farnesyl-directed
cysteine
carboxvmethyltransferase, STE14, is shown in Fig. 1. It contains open-reading
frame of 284 amino
acids at nucleotide positions 90 to 944. It contains ~' 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 amino acid 1 to amino acid 284 (i.e., foil-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 comprise 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
variety 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
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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), ete.
As for the polvpeptides mentioned above, a nucleic acid comprising a
nucleotide sequence
coding for a polvpeptide 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 ~' or 3' end. or dispersed in the coding
sequence. e.g., introns. A
nucleic acid comprising a nucleotide sequence coding W thout interruption for
a
polvpeptide means that the nucleotide sequence contains an amino acid coding
sequence for a farnesyl-
directed cysteine carboxvmethyltransferase. with no non-coding nucleotides
interrupting or intervening
in the coding sequence, e.g., absent intron(s). Such a nucleotide sequence can
also be described 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 described above. The phrase
"expression control
sequence" means a nucleic acid sequence which regulates expression of a
polvpeptide coded for by a
nucleic acid to which it is operably linked. Expression can be regulated at
the level of the mRNA or
polt~peptide. Thus, the expression control sequence includes mRNA-related
elements and protein-
related elements. Such elements include promoters, enhancers (viral or
cellular). ribosome binding
s~uences, transcriptional 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 operably linked
~' to a coding sequence, expression of the coding sequence is driven 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 hybridization. The ability of tvo single-stranded nucleic acid
preparations to hybridize together is
a measure of their nucleotide sequence complementarily, e.g., base-pairing
between nucleotides, such as
A-T, G-C, ete. The invention thus also relates to nucleic acids which
hybridize to a nucleic acid
comprising 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 polvmerase (i.e., an appropriate nucleic
acid synthesizing enzyme).
The present invention includes both strands of nucleic acid, e.g., a sense
strand and an anti-sense strand.
Hybridization conditions can be chosen to select nucleic acids which have a
desired amount of
nucleotide complementarily with the nucleotide sequence set forth in Fig. 1. A
nucleic acid capable of
hybridizing to such sequence, preferably, possesses about 95%, more
preferably, 97%, etc.,
CA 02323759 2000-09-08
WO 99155878 PCT/US99/07396
compiementarity. between the sequences. The present invention particularly
relates to DNA sequences
which hybridize to the nucleotide sequence set forth in Fig. I . 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,
heparin. denatured,
fragmented salmon sperm DNA) at 42 C (or 68 C if the fonnamide is omitted).
Washing and
hybridization can be performed as described in Sambrook et al., Molecular
Cloning, 1989, Chapter 9.
Hybridization can also be based a calculation of melting temperature (Tm) of
the hybrid formed
ber<veen the probe and its target. as described in Sambrook et al. Such
stringent 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 STEI4. 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 in the nucleotide or amino acid sequence set forth in Fig. I or
Fig. 3. Changes or
modifications to the nucleotide andlor 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 comprise
nucleotides which occur in a naturally-occurring MTase gene e.g., naturally-
occurring polvmorphisms,
normal or mutant alleles (nucleotide or amino acid), mutations which are
discovered in a natural
population of mammals. such as humans. monkeys, pigs, mice. rats, or rabbits.
By the term naturally-
occurring, 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-
occurring 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
hybridization according to methods
which one skilled in the art would knrnv. It is recognized that. in analogy to
other oncogenes, naturallv-
occurring variants include deletions, substitutions, and additions which
produce pathological conditions
in the host cell and organism.
A nucleotide sequence coding for a polvpeptide of the invention can contain
colons found in a
naturally-occurnng gene, transcript, or cDNA, for example, e.g., as set forth
in Fig. 1, or it can contain
degenerate colons coding for the same amino 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 various methods, including algorithms described in the
BLAST family of
computer programs, the Smith Waterman algorithm, etc. For example, conserved
amino acids can be
9
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WO 99/55878 PCT/US99/07396
identified between various sequences. See, e.g., Fig. 2. A mutations) can then
be introduced into a w-
sequence by identifying and aligning amino acids conserved between the
polvpeptides and then
modifying an amino acid in a conserved or non-conserved position.
A nucleic acid and corresponding polvpeptide of the present invention include
sequences which
differ from the nucleotide sequence of Fig.l (or less preferably Fig. 3) but
which are phenotvpically
silent. These sequence modifications include, e.g., nucleotide substitution
which do not affect the amino
acid sequence (e.g., different colons for the same amino acid or degenerate
sequences), replacing
naturally-occurring amino acids with homologous amino acids, e.g., (based on
the size of the side chain
and degree of polarization) small nonpolar: cysteine, proline, alanine,
threonine; small polar: serine,
glycine, aspartate, asparagine; large polar: glutamate, glutamine. lysine,
arginine: intermediate polarity:
yrosine. histidine, trytophan; large nonpolar: phenylalanine, methionine,
leucine, isoleucine, valine.
Homologous acids can also be grouped as follows: uncharged polar R groups,
glycine. serine.
threonine, cysteine, tyrosine, asparagine, glutamine: acidic amino acids
(negatively charged), aspartic
acid and glutamic acid; basic amino acids (positively charged). lysine.
arginine, histidine. Homologous
substitutions also include those described by Dayhoff in the Atlas of Protein
Sequence and Structure ~
(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 STEIN, or mouse MTase.at a corresponding
position.
A nucleic acid can comprise a nucleotide sequence coding for a poly~peptide
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 polvpeptide
having an amino 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 polvpeptides
coded for b~~ such nucleic acids. In addition. it may be desired to change the
colons in the sequence to
optimize the sequence for expression in a desired host.
A nucleic acid according to the present invention can comprise, 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 various
known linkages, e.g., ester,
sulfamate,. . .sulfamide, phosphorothioate, phosphoramidate,
methylphosphonate, 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.
Various modifications can be made to the nucleic acids, such as attaching
detectable markers
(avidin. biotin, radioactive elements), moieties which improve hybridization.
detection, or stability. The
CA 02323759 2000-09-08
WO 99155878 PCTIUS99107396
nucleic acids can also be attached to solid supports. e.g., nitrocellulose,
magnetic or paramagnetic
microspheres (e.g., as described in USP 5.411.863: USP 5,543,289: for
instance. comprising
ferromagnetic, supermagnetic, paramagnetic, superparamagnetic, iron oxide and
polysaccharide). 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 oligonucleotides and
nucleic acid probes.
Such oligonucleotides or nucleic acid probes can be used. e.g., to detect.
quantitate, or isolate a human
or mouse MTase, such as STE14, nucleic acid in a test sample. Detection can be
desirable for a variety
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 comprising. contacting a
target nucleic acid in a test
sample W th an oligonucleotide under conditions effective to achieve
hybridization bet<veen the target
and oligonucleotide; and detecting hybridization. An oligonucleotide in
accordance with the invention
can also be used in swthetic 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 fo Methods and
Applications, Innis et al.,
eds., Academic Press, New York, 1990) or differential display (See, e.g.,
Liang et al., Nacl. Acid. Res. ,
21:3269-3275, 1993; USP 5,599,672: W097/18454). Useful oligonucleotides
include. e.g.,
~'-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 oligonucleotides 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
23 STE 14 or mouse MTase. Bv a unique sequence to farnsvl-directed
carboxvmethyltransferase, it is
meant a defined order of nucleotides which occurs in human or mouse STE 14,
e.g., in the nucleotide
sequence of Fig. 1, but rarely or infrequently in 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 comprising such a unique sequence can be used as a
hybridization probe to
identify the presence of, e.g., human or mouse STE 14, in a sample comprising
a mixture of nucleic
acids, e.g., on a Northern blot. Hybridization can be performed under
stringent conditions to select
nucleic acids having at least 95% identity (i.e., complementarity) to the
probe, but less stringent
conditions can also be used. A unique farnsyl-directed
carboxvmethyltransferase nucleotide sequence
11
CA 02323759 2000-09-08
WO 99/55878 PCT/US99/07396
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 STE 14.
enzynes, GFP, etc,
expression control sequences, etc. See, e.g, Hrv_ cyna et al., Methods
Enzvmol.. 2~0:2~ 1-266, 1995.
Hybridization can be performed under different conditions. depending on the
desired selectivity,
e.g., as described in Sambrook et al., Molec~elar Cloning, 1989. For example,
to speci$caliy detect
human or mouse MTase, an oligonucleotide can be hybridized to a target nucleic
acid under conditions
in which the oligonucleotide only hybridizes to it. e.g., where the
oligonucleotide 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 complementarily, 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 STE14, See, Imai et al., Mol. Cell., Bio., 17:1543-151, 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 oligonucleotide
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 oligonucleotide according to the present
invention. in polymerase chain
reaction followed by DNA sequencing to identify whether the sequence is
normal, in combination with
other oligonucleotides to oncogenes or genes in the ras signalling pathway,
etc., e.g., GRB2, H-, K- and
N-ras, c-Raf MAP kinases. p42, p44, SerIThr kinases, EIk-1, c-myc, c-Jun, G-
proteins. Ftase, PPSEP,
PPSMT, etc. In a preferred method, the present invention relates to a method
of diagnosing a cancer
comprising contacting a sample comprising a target nucleic acid with an
oligonucieotide under
conditions effective to permit hybridization bet<veen the target and
oligonucleotide; detecting
hybridization. wherein the oligonucleotide comprises a sequence of a human ar
mouse MTase,
preferably a unique sequence of; and determining the nucleotide sequence of
the target nucleic acid to
which the oligonucleotide is hybridized. The sequence can be determined
according to various methods,
including isolating the target nucleic acid, or a cDNA thereof, and
determining its sequence according to
a desired method.
Oligonucleotides of the present invention can comprise any continuous
nucleotide sequence of
Fig. 1. These oligonucleotides (nucleic acid) according to the present
invention can be of any desired
size, e.g., about 10-200 nucleotides, 12-100, preferably 12-~0, 12-25, 14-16,
at least about 15, at least
about 20, etc. The oligonucleotides can have non-naturally-occurring
nucleotides, e.g., inosine. The
oligonucleotides have 100% identity or complementarily to a sequence of Fig. 1
or Fig. 3, or it can have
mismatches or nucleotide substitutions, e.g., 1, 2, 3, 4, or ~ substitutions.
In accordance with the
present invention, the oligonucleotide can comprise a kit, where the kit
includes a desired buffer (e.g.,
12
CA 02323759 2000-09-08
WO 99/55878 PCT/US99/07396
phosphate, tris, etc.), detection compositions, etc. The oligonucleotide 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 various ways. such as to regulate or modulate expression of
farnsvl-directed
carboxvmethyltransferase, e.g., inhibit it, to detect its expression, or for
in situ hybridization. These
oligonucleotides can be used analogously to USP 5,76.208 describing inhibition
of ras. For the
purposes of regulating or modulating expression of farnsy 1-directed
carboxymethyltransferase, an anti-
sense oligonucieotide can be operably linked to an expression control
sequence.
The nucleic acid according to the present invention can be labelled according
to am° desired
method. The nucleic acid can be iabeled using radioactive tracers such as 3'P,
3'S, '-SI, 3H, or'''C, to
mention only the most commonly used tracers. The radioactive labelling can be
carried out according to
any method such as. for example, terminal labeling at the 3' or ~' end using a
radiolabeled nucleotide.
polvnucleotide kinase (with or without dephosphorylation with a phosphatase)
or a lipase (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 immunological 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
characteristic physical properties. such as fluorescence or the emission or
absorption of light at a
desired wavelength, etc.
A nucleic acid according to the present invention, including oligonucleotides,
anti-sense nucleic
acid, etc., can be used to detect expression of farnsyl-directed
carboxvmethyltransferase in whole
organs, tissues, cells, etc., by various techniques. including Northern blot.
PCR, in sing hybridization,
etc. Such nucleic acids can be particularly useful to detect disturbed
expression, e.g., cell-specific
and/or subcellular aiteratians, of farnsyl-directed carboxymethvltransferase.
The levels of farnsvl-
directed carboxvmethyltransferase can be determined alone or in combination
with other genes products
(e.g., Ras-H, -N, -K4A, -K4B, p~3, Rb, RCE1, etc.).
A nucleic acid according to the present invention can be expressed in a
variety of different
systems, in 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
CA 02323759 2000-09-08
WO 99!55878 PCT/US99/07396
nucleic acid is adjacent to a dhfr gene), cyclohexamide, 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 liposomes,
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 chromosomes) 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 St9
(S. frugipeda) and Drosophila, bacteria. such as E. coli, Streptococcus,
bacillus. yeast. fungal cells,
t0 plants. embryonic stem cells (e.g., mammalian, such as mouse or human),
cancer or tumor cells. Sft7
expression can be accomplished in analogy to Graziani 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
IS 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 drive 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 farnsyl-directed carboxvmethyltransferase, elucidating farnsyl-
directed
20 carboxvmethvltransferase 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 variation, e.g., a mutation, chimera. polymorphism. etc.
A nucleic acid or polvpeptide of the present invention can be used as a size
marker in nucleic
acid or protein electrophoresis, chromatography, etc. Defined restriction
fragments can be determined
25 by scanning the sequence for restriction sites, calculating the size, and
performing the corresponding
restriction digest. The human farnsyl-directed carbo~cymethyltransferase 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.,
30 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 in the
processing of various proteins, e.g., polvpeptides which are lipid-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.
Biophys. Acta 1333, .
14
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WO 99/55878 PCT/US99/07396
F~ I-F71, 1997. Of particular interest, is the role that a mammalian famesyl-
directed cysteine
carbo~cymethyltransferase plays in processing ras polvpeptides since the
latter is involved in
carcinogenesis and transformation. Ras and other polvpetides containing the
above-mentioned motifs
are processed through one or more steps involving, farnsylation,
endoproteolysis, and methylation. See,
e.g., Gelb, Science. 27~. 1751-17~ 1. 1997. Over-expression of ras (wild-type,
mutated, constitutive,
etc., ras), optionally in combination with aberrant expression of other genes,
leads to oncogenic 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 oncogenic effect. In
accordance with the present invention, the ras maturation pathway can be
inhibited by blocking
!0 mammalian farnesyl-directed cysteine carboxvmethvltransferase, such as
STE14, Blocking can be
accomplished in various ways, including by administering: STEl4 antibodies, or
other STE14 ligands,
STE 14 peptides (especially. those that bind to a methyl donor but lack
methyltransferase or
methyltransesterification activity). inhibitors of STE 14 catalytic activity
(e.g., methyl-donor mimics or
competitors or antagonists), inhibitors of STE14 gene expression (e.g., anti-
sense or double-stranded
RNA, such as. Fire et al., Nat:ire, 391:806-811, 1998). Blocking agents can be
identified according to
the methods described herein or those available in the art.
One aspect of the invention relates to identifying compounds which modulate
farnesvl-directed
cysteine carboxvmethyltransferase activity. The activity can be modulated by
increasing, reducing,
antagonizing, promoting, stabilizing, etc. its activity. Thus, one object of
the invention is to facilitate
2U screening for compounds which modulate the incorporation of a methyl group
into a methyl-acceptor
substrate of the methyltransferase enzyme.
Accordingly, the present invention relates to identifying compounds that
modulate a mammalian
farnesyl directed cysteine carboxynethyltransferase, especially a human or
mouse MTase of Fig. 1 or
Fig. 3, comprising: reacting, in the presence of a test compound, a methyl-
donor substrate. a methyl-
acceptor substrate. and a mammalian farnesvl-directed cysteine
carboxymethyltransferase, or a
fragment thereof having methyltransferase activity, under conditions effective
for the mammalian
carboxymethyftransferase, or said fragment, 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.
Any functional methyl-donor substrate is acceptable, including detestably-
labeled S-adenosyl-
methionine, especially S-adenosyl-L-(methyl-''~C]methionine or S-adenosyl-L-
[methyl-'H]methionine.
Likewise, am~ functional methyl-acceptor is satisfactory, including
polvpeptides which comprise a
tenminal fannsylated cysteine, polvpeptides which tenminate in CC, CCXX, or
CXC, and/or are
CA 02323759 2000-09-08
WO 99/55878 PCT/US99/07396
cysteine-prenylated with, for instance, a farnsyl or geranylgeranyl.
Functional methyl-acceptor
substrates include, e.g., Ras-H, -N, -K~4A, -K4B, Lamins A and B, RhoB, RhoE,
others, Rap2, Rheb,
Phosphorylase kinase and , Rhodopsin kinase, Transducin , cGMP
phosphodiesterasae . IFN-
induced guanylate-binding protein l, IP3 ~-phosphatase, PxF, PRL-1/PTPCAAXI
and 2. biotin-Lys-
3 Lys-Ser-Lys-Thr-Lys-(Farnesyl)Cys, etc.
Other substrates include, hepatitis delta virus (Otto et al., J. Biol. Chem.,
271:4369-72, 1996). In
general, any substrate is suitable if it can be acted upon in the
methyltransferase reaction. Thus. a
substrate can comprise 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., comprising, latex, sepharose, silica, agarose, sephadex,
cellulose, polysaccharides, glass,
polymers, etc. A substrate can also be detectable labeled, e.g., with
antibody, avidin, biotin, radioactive
labels, aptamers, fluorescent labels. nucleic acid. etc. The substrates can
also comprise phosphates.
methyl groups, sugars, or lipids.
The test compound is preferably reacted with substrates in a milieu in which
methylation 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 activit<~, 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-terminal cysteine, effective methylation
results in its methylation.
2o After the step of reacting the substrates, test compound, and MTase, under
conditions in which
methylation can be achieved, the next step is to determine the effect of the
test compound on
methylation. Detecting methvlation, can be optimized in the absence of the
test compound to establish a
baseline activity for MTase. Generally. detecting methylation involves
measuring the incorporation of a
labeled methyl group (e.g., 3H or "C) into an acceptor-substrate of the MTase.
In a preferred
embodiment. the methyl-acceptor substrate is biotin-Lys-Lys-Ser-Lys-Thr-Lvs-
(Farnesyl)Cvs and the
methyl-donor substrate is detectably-labeled S-adenosyl methionine (for
instance, S-adenosyl-L-
[methyl-'''C]methionine or S-adenosyl-L-[methyl-'H]methionine) and the
methylation detecting is
accomplished by capturing the methyl-acceptor substrate using an streptavidin-
coated bead: and
measuring the amount of labeled methyl incorporated 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) incorporated 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 incorporate
label from 3H-S-adenosyl
methionine.
16
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WO 99/55878 PCT/US99/0739b
In general assays according to the invention involve capture of the methyl-
substrate acceptor -
and distinguishing whether the substrate has been modified, i.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;
S electrophoretic mobility shifts: chromatography; electrophoresis: etc.
The MTase component can be added to the reaction mixture in a variety forms,
e.g.,
substantially purified, as a component of cell membranes (such as, endoplasmic
reticulum), or as a
soluble extract. In each case, the MTase polvpeptide can be obtained from a
natural source. a
recombinant source (e.g., a human STE14 expressed in an insect cell line or
bacteria as a fusion or non-
fusion protein. for instance as described in Hrycvna et al., Methods Enrymol..
2S0:2S 1-266, 1995), or
it can be produced synthetically (produced chemically or enzvmatically, 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
iS heterologous component of the cell; by heterologous, it is meant that the
MTase is not only expressed in
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 (bacterial, yeast, mammalian, ete.). A human famesyl-directed
eysteine
carboxvmethvitransferase, or a fragment thereof, is a preferred coding
sequence. See, e.g., Fig. 1. A
useful fragment of the human sequence comprises a methvitransferase 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 W ih human STE14 are lysed and the resulting lysate is used
directly in the assay. i.e., a
crude lysate. The crude lysate comprising the recombinant human STE14 can
optionally be refined or
enriched for human STE14. For instance, e.g, a membrane fraction can be
isolated. etc., as described in
Hrycvna et al., Methods Enzvmol.. 2S0:2S 1-266, 1995 .
A purpose 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 carboxvmethyltransferase 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 variety 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
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WO 99/55878 PCT/US99107396
identifying whether the substrate has been methylated. For example, in cell
lines that express ras, a -~~
whole cell assay can comprise: administering 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 methvltransferase substrate. overexpress it, etc. An in vivo
method of assaying for farnesyl-
directed cysteine carboxvmethyltransferase further involves modifying test
compounds to gain entry into
the cell. e.g., derivatizing compounds, encapsulating compounds in liposome,
and other means to
enhance delivery to the cell, e.g., to stimulate phagocvtosis. Agents can also
be administered to such
cells and tested for their ability to inhibit transformation, e.g., by
monitoring cell morphology, etc. See,
to e.g., USP x.688.655. Assays can also be carried out as described in USP
5,710,171; x,703,241;
~,~85,3~9: ~,~57,729; ~,~32,359; x,470.832; x,420, 24~; x;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,
Lamins A and B. RhoB, RhoE, Rap2, Rheb, Phosphorylase kinase and , Rhodopsin
kinase,
IS Transducin . cGMP phosphodiesterasae , IFN-induced guanylate-binding
protein 1, IP3 5-phosphatase,
PxF, PRL-IIPTPCAAX1 and 2.
Compounds identified in this or other manners can be useful to modulate
farnesyl-directed
cysteine .carbox~~rnethyltransferase activity in a cell, a tissue, a whole
organism, in sit:~. in vitro (test
tube. a solid support. etc.), in vivo, or in any desired environment. In
general, a compound having such
20 an in vitro activity will be useful in vivo to modulate a biological
pathway associated «7th farnesyl-
directed cvsteine carboxvraethyitransferase, 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
25 example, the invention relates to a method of treating cancer comprising
administering, 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 carboxvmethyltransferase 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,
30 can be synthetic, naturally-occurring, or a combination. A regulator
compound can comprise amino
acids, nucleotides, hydrocarbons, lipids, polysaccharides, etc. A regulator
compound is preferably a
regulator of farnesyl-directed cysteine carboxvmethyltransferase, 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 ribozyme, an aptamer, a synthetic compound, or a
naturally-occurring
18
CA 02323759 2000-09-08
WO 99/55878 PCT/US99/07396
compound. To treat the disease, the compound, or mixture, can be formulated
into pharmaceutical w
composition comprising 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 carboxvmethyltransferase. Antibodies. e.g., polyclonal, monoclonal.
recombinant, chimeric,
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,
l0 goats. or rabbit subcutaneously and/or intraperitoneaily. 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, IgG 1, etc. Antibodies can also be generated by
administering naked DNA
See, e.g., USP 5,703,055; 5,589,466; 5,580,859.
An antibody specific for farnesyl-directed cysteine carboxymethyltransferase
means that the
15 antibody recognizes a defined sequence of amino acids within or including a
farnesy!-directed cysteine
carbo~c~znethyltransferase, e.g., the human and murine 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 in Fig. l than to
a different epitope(s). e.g., as detected and/or measured by an immunoblot
assay. Thus. an antibody
which is specific for an epitope of human STE 14 is useful to detect the
presence of the epitope in a
20 sample, e.g., a sample oftissue containing human STE14 gene product,
distinguishing it from samples
in which the epitope is absent. Such antibodies are useful as described in
Santa Cruz Biotechnology,
Inc., Research Product Catalog, can be formulated accordingly, e.g., 100
pg/ml.
In additian, ligands which bind to a farnesyl-directed cvsteine
carboxymethyltransferase
polypeptide according to the present invention, or a derivative thereof,. can
also be prepared, e.g., using
25 synthetic peptide libraries or aptamers (e.g., Pitrung et al., U.S. Pat.
No. 5,143,854: Geyser et aL,
1987, J. Immunol. Methods, 102:259-274, Scott et al., 1990, Science, 249:386:
Blaclcwell et al., 1990,
Science, 250:1104; Tuerk et al., 1990, Science, 249: 505.
Antibodies and other ligands which bind fa,rnesyl-directed cysteine
carboxvmethyltransferase
can be used in various ways, including as therapeutic, diagnostic, and
commercial research tools, e.g, to
30 quantitate .the levels of farnesyl-directed cysteine
carboxvmethyltransferase polvpeptide in animals.
tissues, cells, etc., .to identify the cellular localization andlor
distribution of it. to purify it, or a
polypeptide comprising 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
CA 02323759 2000-09-08
WO 99/55878 PCT/US99/07396
An antibody according to the present invention can be used to detect farnesyl-
directed cysteine
carboxvmethvltransferase polvpeptide or fragments thereof in various samples,
including tissue, cells.
body fluid, blood, urine, cerebrospinal fluid. A method of the present
invention comprises. 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. Bv 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 derivatives thereof. The antibodies or
derivatives thereof can also be
used to inhibit expression of farnsyl-directed carbo~cymethyltransferase or a
fragment thereof. The
levels of farnesyl-directed cysteine carboxvmethyltransferase polvpeptide can
be detenmined alone or in
l0 combination with other gene products. in particular, the amount (e.g., its
expression levei) of farnesyl-
directed cvsteine carboxvmethyltransferase polvpeptide can be compared (e.g.,
as a ratio) to the
amounts of other polvpeptides in the same or different sample, e.g., ras,
FTase, endoprotease, etc. A
ligand for farnesyl-directed cysteine carboxvmethvltransferase 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
carboxynethyltransferase can be used in
diagnostic andlor 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 farnsyl-directed
carboxynethyltransferase
polvpeptide. prepared according to a desired method, e.g., as disclosed in
U.S. Pat. No. 5,434.030. A
labelled pohpeptide can be used, e.g., in binding assays, such as to identify
substances that bind or
attach to farnsyl-directed carboxvmethvltransferase, to track the movement of
farnsyl-directed
carboxvmethyltransferase in a cell, in an in vitro, in vivo, or in sine
system, etc.
A nucleic acid, polvpeptide, antibody, farnsyl-directed
carboxymethyltransferase ligand etc.,
according to the present invention can be isolated. The term "isolated" means
that the material is in a
form in which it is not found in its original environment, e.g., more
concentraxed, more purified,
separated from component, etc. An isolated nucleic acid includes, e.g., a
nucleic acid having the
sequence of farnsyl-directed carboxymethyltransferase separated from the
chromosomal DNA found in
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 aiso be substantially purified. By substantially
purified, it is meant that
nucleic acid or polvpeptide is separated and is essentially free from other
nucleic acids or polvpeptides,
i.e., the nucleic acid or polypeptide is the primary and active constituent.
CA 02323759 2000-09-08
WO 99/55878 PCT/US99/07396
The present invention also relates to a transgenic animal; e.g., a non-human-
mammas, such~as a
mouse, comprising a farnsyl-directed carboxvmethyltransferase nucleic acid.
Transgenic animals can
be prepared according to known methods, including, e.g., by pronuclear
injection of recombinant genes
into pronuclei of I-cell embryos, incorporating 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 Sci., 77:7380-7384 ( 1980): Palmiter et ai., Cell,
41:343-345 (1985): Palmiter et
al., An». Rev. Genet., 20:465-499 ( 1986): Askew et al., Mol. Cell. Bto.,
13:4115-4124. 1993; Games et
al. Nature, 373:523-527. 1995; Valancius and Smithies, Mol. Cell. Bio.,
11:1402-1408, 1991; Stacey
l0 et al., Mol. Cell. Bio., 14:1009-1016, 1994; Hasty et al., Natr~re, 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
Morse Embryo: A Laboratorv Manreal, Cold Spring Harbor Laboratory. Cold Spring
Harbor. New
York), pig (Hammer et al., Natr~re, 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 in Biotech. 5:20-24; and DePamphilis et al., 1988, BioTechniques,
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,
2o 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. polvpeptides, antibodies. ete. of the present
invention can be
prepared and use 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, polvpeptides, antibodies, etc.,
reference is made to
standard te~ctbooks 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: Names 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; Cr~rrerrt
Protocols in Hreman 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
CA 02323759 2000-09-08
WO 99155878 PCT/US99l07396
EXAMPLE _ w
We have devised our own version of this assay which utilizes a biotinylated,
prenylated peptide
substrate (Biotin-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 STE14-
expressing bacterial or insect cell
membranes utilize the.co-substrate 3H-S-adenosyl methionine to methylate the
(Farnesyl)Cys-carboxyl
group. The resulting label incorporated into the substrate peptide is
quantified using streptavidin-coated
SPA beads. The methylase is cloned into the pRSET (Invitrogen) and pFastBac
(Gibco BRL) bacterial
and insect cell expression vectors, respectively.
A standard assay is performed in 96-well sample plates (Wallac Part NO. 1450-
401) with a
l0 total assay volume of 100 I which generally contains: 50 1 compound, 25 1
membranes and 25 13H-
SAM/substrate added in that order. Final concentration of HEPES pH 7.4 is i 00
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, i.e.
3H-SAM (--85Ci.mmol; lmCi/ml; 12 M):
0.125 I 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 rains.
The reaction is stopped by adding 150 I Stop Mix which contains SPA beads (250
g) in 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
description, 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 incorporated by reference.
From the foregoing description, one skilled in the art can easily ascertain
the essential
characteristics of this invention, and without departing from the spirit and
scope thereof, can make
various changes and modifications of the invention to adapt it to various
usages and conditions.
22
