Antibodies to protein, FAF1

The present invention identifies a novel, Fas-associated factor 1 termed FAF1 which potentiates Fas-induced cell killing. The invention provides FAF1 nucleic acid and polypeptide compositions as well as methods of using these compositions in the therapeutic treatment of diseases resulting from dysregulation in apoptosis. Also provided are cells carrying and expressing the nucleic acid compositions and methods of using these cells to screen for agonists and antagonists of Fas-mediated apoptosis. Methods of isolating FAF1-interacting proteins are disclosed. Also provided are antibodies that bind FAF1, a hybridoma and a kit comprising the antibodies.

 

What is claimed is:

1. An antibody that specifically binds to an isolated polypeptide comprising a domain of a Fas-associated factor 1 (FAF1) polypeptide, said polypeptide capable of associating with a cytoplasmic domain of Fas, wherein said FAF1 domain is encoded by a nucleic acid sequence that comprises at least 18 nucleotides and hybridizes to the complementary nucleic acid sequence shown in SEQ ID NO:1 or to a degenerate form thereof.

2. The antibody of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:2.

3. A kit comprising an antibody of claim 1.

4. The antibody of claim 1, which is a rabbit antiserum or a monoclonal antibody.

5. A hybridoma capable of producing the monoclonal antibody of claim 4.

BACKGROUND OF THE INVENTION

Apoptosis or programmed cell death is an important physiological process in multicellular organisms, both during development and for homeostasis. Apoptosis allows the elimination of cells that are no longer necessary, that are produced in excess, that have developed improperly or that have sustained genetic damage. Apoptosis occurs in many different tissue systems and must be properly regulated to maximize benefit to the individual; when the mechanism is dysregulated, it may cause significant disease. Both inhibition of cell death and inappropriate cell death may be deleterious. For example, inhibition of cell death may contribute to disease in the immune system to allow persistence of self-reactive B and T cells, thus promoting autoimmune disease (Watanabe-Fukunaga et al., Nature, 356:314-317 (1992)). Most importantly, cancer may result when cells that fail to die undergo further mutations, leading to a transformed state (Korsmeyer, S. J., Blood, 80:879-886 (1992)).

The protein, Fas, mediates apoptosis. A cell surface receptor, Fas plays an important role in the development and function of the immune system. Malfunction of the Fas system causes lymphoproliferative disorders and accelerates autoimmune diseases. Exacerbation of Fas-mediated apoptosis may cause tissue destruction.

SUMMARY OF THE INVENTION

The present invention provides the identification and isolation of a novel Fas-associated factor 1, termed FAF1, and the FAF1-encoding DNA. As a cytoplasmic protein, FAF1 was shown herein to bind to the wild type but not the inactive point mutant of Fas. FAF1 specifically interacts with the cytoplasmic domain of wild type Fas and potentiates Fas-induced cell killing. FAF1 is a signal transducing molecule in the regulation of apoptosis.

The FAF1 nucleic acids and polypeptides find many uses. It would be desirable to be able to control apoptosis by enhancing or decreasing the susceptibility of individual cell types to undergo apoptosis, especially when dysregulation of the process leads to disease. In particular, it would be desirable to provide therapeutic intervention in disease conditions where apoptosis is dysregulated due to Fas or FAF1 malfunction. Blockage or activation of FAF1 function in apoptosis can be used for example, in the treatment of cancer, immune disorders such as autoimmune diseases, infectious diseases, and myocardial infarction and neuronal infarction in cardiovascular diseases. Heretofore, such therapeutic approaches involving FAF1 were not possible. The present discovery of FAF1 fulfills these and other needs.

The present invention provides the nucleotide and amino acid sequence of FAF1 as well as polypeptide and nucleic acid compositions based on FAF1. The uses and methods of use of the FAF1 nucleic acid and polypeptide compositions are disclosed.

One aspect of this invention is to provide an isolated nucleic acid comprising at least 85% sequence identity with the nucleotide sequence of SEQ ID NO:1, an allelic or species variation thereof, or a fragment thereof. A nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 is provided. Also provided is a recombinant DNA molecule comprising the nucleotide sequence of SEQ ID NO:1 or a fragment thereof. In one embodiment, the recombinant DNA molecule encodes a FAF1-GAL4 transactivation domain fusion protein. A cell is provided which contains the recombinant DNA molecule comprising the nucleotide sequence of SEQ ID NO:1 or a fragment thereof.

Another aspect of the invention is to provide an isolated polypeptide comprising at least 85% sequence identity with the sequence of SEQ ID NO:2, an allelic or species variation thereof, or a fragment thereof. The polypeptide can be provided in a kit. The polypeptide may further comprise an influenza virus HA epitope tag. An isolated polypeptide comprising the sequence of SEQ ID NO:2 is also provided. In a specific embodiment, the polypeptide is one capable of associating with the cytoplasmic domain of Fas.

Yet another aspect of the invention is to provide an isolated polypeptide comprising the sequence of SEQ ID NO:2, an allelic or species variation thereof, or a fragment thereof, wherein the isolated polypeptide is a fusion protein. A FAF1-GAL4 activation domain fusion protein is specifically provided. In one aspect, the fusion protein comprises a tag, or a product of a second gene or fragment of that second gene product. A fusion protein is provided wherein the tag is GST, an epitope tag or an enzyme or wherein the second gene is lacz.

A different aspect of the invention is the provision of antibodies that specifically bind a polypeptide comprising the sequence of SEQ ID NO:2, an allelic or species variation thereof, or a fragment thereof. These antibodies can be polyclonal, such as the rabbit antiserum provided herein, or monoclonal. A hybridoma capable of producing a monoclonal antibody to a FAF1 polypeptide is provided. Also provided is a kit comprising any antibody preparation to the above-mentioned polypeptides.

In another aspect of the invention, a method is provided for isolating a FAF1 gene or fragment thereof, comprising screening a DNA library using a FAF1 probe to identify a hybridizing clone and isolating said FAF1 gene or gene fragment from said hybridizing clone. A FAF1 probe suitable for use in this method is one which comprises the nucleotide sequence of SEQ ID NO:1 or a fragment thereof. The method is useful to isolate a human FAF1 gene as well as FAF1 genes from other species.

Another aspect of the invention is to provide a method of modulating or blocking Fas activity comprising providing a Fas-interacting domain polypeptide of FAF1 in a cell expressing Fas protein wherein said Fas-interacting domain polypeptide of FAF1 binds to said Fas protein to block Fas activity. In one embodiment, the Fas-interacting domain polypeptide of FAF1 is provided by introducing an expression vector encoding a Fas-interacting domain polypeptide of FAF1 into the Fas expressing cell. A method of activating FAF1-mediated apoptosis in a cell is disclosed, the method comprising providing a constitutively active FAF1 to the cell.

An important aspect of the invention is a method of screening for an agonist or an antagonist of FAF1, comprising contacting a cell expressing both Fas and FAF1 with a test molecule, activating Fas, and analyzing the cell for any effects on apoptosis, increased apoptosis indicative that the test molecule is an agonist and decreased or loss of apoptosis indicative that the test molecule is an antagonist. The test molecules can be peptides, oligonucleotides, lipids, toxins, hormones, small proteins, drugs and compounds from plant or animal sources and recombinantly produced substances. In a specific embodiment, peptide libraries are screened.

The cells are analyzed for effects on apoptosis such as cell membrane blebbing and/or DNA fragmentation. Fas is activated by binding to Fas ligand or crosslinking with antibodies. Fas and/or FAF1 can be expressed as a fusion protein. In a particular embodiment, the cell expresses CD4/fas and HA epitope tagged FAF1 fusion proteins. An L cell expressing both these fusion proteins is specifically provided in the invention.

The invention further provides a pharmaceutical composition useful in the treatment of a disease resulting from dysregulated apoptosis, comprising a FAF1 polypeptide and a pharmaceutically acceptable carrier. Instead of the FAF1 polypeptide, the pharmaceutical composition can comprise an expression vector capable of expressing the FAF1 polypeptide in an affected cell. The diseases contemplated for such treatment include cancer, autoimmune disease and viral infections. For these diseases, a constitutively active FAF1 polypeptide can be provided in the pharmaceutical composition. In a different set of diseases comprising myocardial infarction, stroke and reperfusion injury, arrest or blockage of apoptotic cell death is targeted. In the latter set of disease conditions, it may be therapeutically effective to express a Fas-interacting domain polypeptide in the affected cell to block the interaction between endogenous Fas and FAF1, thus blocking FAF1-potentiated apoptosis. Also provided are methods of alleviating a patient suffering from the aforementioned diseases by administering to the patient, a therapeutically effective amount of the above pharmaceutical compositions.

Finally, the invention provides several methods of isolating a FAF1-interacting protein. One method requires contacting a cell lysate suspected of containing a FAF1 interacting protein with a FAF1 polypeptide and isolating any protein bound to the FAF1 polypeptide, as a FAF1-interacting protein. A second method comprises labeling the cellular proteins, activating Fas expressed on the cell, immunoprecipitating FAF1 from the cell lysate and identifying a labeled protein that coimmunoprecipitates with FAF1. In a third method, a peptide library is exposed to a FAF1 protein to allow one or more peptides to bind to the FAF1 protein. Any bound peptide will be isolated as a FAF1-interacting protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D shows surface expression of CD4/fas and CD4/fas786A detected by fluorescent activated cytometry scanning (FACS) analysis.

FIG. 2 shows fragmentation of DNA from CD4/fas786A-23 cells (lanes 1-3) and from CD4/fas-16 cells (lanes 4-6) after anti-CD4 crosslinking. Lanes 1 and 4 show DNA from control cells incubated with actinomycin D (0.5 ng/ml) only. Lanes 2 and 5 show DNA from cells crosslinked with L3T4 Rat anti-CD4 (GK1.5) (1 .mu.g/ml) alone. Lanes 3 and 6 show DNA from cells incubated with both L3T4 (1 .mu.g/ml) and anti-Rat IgG (0.5 .mu.g/ml of Rabbit anti-Rat IgG, Zymed).

FIGS. 3A-3B shows the morphology of cells after anti-CD4 crosslinking. Cells were crosslinked as described as in FIG. 2 and photographs were taken ten hours later. Top panel (FIG. 3A) shows CD4/fas-16 cells, bottom panel (FIG. 3A) shows CD4/fas786A-23 cells.

FIGS. 4A-4F shows the nucleotide sequence (SEQ ID NO:1) of the cDNA encoding FAF1.

FIGS. 5A-5D is an amino acid sequence (SEQ ID NO:2) deduced from the nucleotide sequence of the FAF1 cDNA.

FIG. 6A shows expression of FAF1 in Cos cells detected in whole cell lysates by Western blot analysis using anti-HA epitope antibody (12CA5). The arrow indicates HA-tagged FAF1. In FIGS. 3A-6C, the lanes are as follows: Cos cells were transfected with PSM plus PCGN8.1 (lane 1), PSMCD4/fas plus PCGN (lane 2), PSMCD4/fas plus PCGN8.1 (lane 3) or PSMCD4/fas786A plus PCGN8.1 (lane 4).

FIG. 6B shows a Western blot analysis blotting with 12CA5 antibody after immunoprecipitation of CD4.

FIG. 6C shows immunoprecipitated chimeric molecules of CD4/fas or CD4/fas786A detected by Western blot. The blot from FIG. 6B was re-probed with anti-CD4 serum (a gift of Dr. D. R. Littman).

FIGS. 7A-7L show the morphological changes of cells expressing FAF1 after CD4/fas antibody crosslinking. FIGS. 7A-7F show CD4/fas-16 cells. G-L show CD4/fas786A-23 cells. FIGS. 7A-7C and 7G-7I show cells transiently expressing FAF1. D-F and J-L show cells mock transfected with vector alone. CD4/fas-16 and CD4/fas786A-23 cells were co-transfected with PSV-.beta. Gal and PCGN8.1 or PCGN alone (1:5 ratio) by DEAE Dextran method or Lipofectomin (BRL). Forty-eight to seventy-two hours later, transfectants were crosslinked by 1 .mu.g/ml (FIGS. 7C, 7F, 7I, and 7L) or 200 ng/ml (FIGS. 7B, 7E, 7H, and 7K) of L3T4 or a control rat IgG (FIGS. 7A, 7D, 7G, and 7J) as described in FIG. 2. The cells were fixed and photographed one hour later.

FIG. 8 shows the percentage of cells undergoing apoptosis 1 hour after L3T4 treatment. The fixed cells, from the experiment described in FIGS. 4A-4F, were assayed for .beta.-galactosidase expression and blue cells were counted by light microscopy. The percentage of apoptotic cells is the number of cells with cell membrane blebbing among one hundred blue cells counted. The bars indicate means and standard deviations for four independent experiments.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Fas antigen, a member of TNF/NGF (Tumor Necrosis Factor/Nerve Growth Factor) receptor family, is a cell surface protein that mediates apoptosis. Fas induces apoptosis when activated by Fas ligand (FasL) binding or anti-Fas antibody crosslinking (Nagata, S., Seminars in Immunol., 6:3-8 (1994)). Fas plays an important role in the development and function of the immune system (Nagata, supra, Lowin et al., Nature, 370:650-652 (1994)). Malfunction of the Fas system causes lymphoproliferative disorders and accelerates autoimmune diseases, whereas exacerbation of Fas activity causes tissue destruction.

A point mutation in the cytoplasmic domain of Fas (a single base pair change from T to A at base number 786), replacing isoleucine with asparagine, abolishes the apoptotic signal transducing property of Fas (Watanabe-Fukunaga et al., Nature, 356:314-317 (1992)). Mice homozygous for this mutant allele (lpr.sup.cg /lpr.sup.cg mice) develop lymphadenopathy and an autoimmune disease that resembles human systemic lupus erythematosus (Watanabe-Fukunaga, supra). They produce large quantities of IgG and IgM including autoantibodies such as anti-DNA, and rheumatoid factor (Cohen et al., Annu. Rev. Immunol., 9:243 (1991)) and develop nephritis or arthritis. Patients have been described with phenotypes similar to that of lpr mice (Sneller et al., J. Clin. Invest., 90:334 (1992)) and patients with altered Fas were recently reported (Rieux-Laucat et al., Abstracts of the 12th European Immunology Meeting, Barcelona (European Federation of Immunological Societies) (June 1994)).

The present invention identifies a novel, Fas-associated factor 1, termed FAF1. FAF1, which was isolated using the two-hybrid screen in yeast (Durfee et al., Genes Dev., 7:555-569 (1993), specifically interacts with the cytoplasmic domain of wild type Fas but not the lpr.sup.cg mutated Fas protein. This interaction occurs not only in yeast but also in mammalian cells. When expressed in L cells, FAF1 potentiated Fas-induced apoptosis. A search of available DNA and protein sequence data banks did not reveal any significant homology between FAF1 and other known proteins. Therefore, FAF1 is a novel protein that binds to the wild type but not the inactive point mutant of Fas; it potentiates Fas-induced cell killing and is a signal transducing molecule in the regulation of apoptosis.

Blockage or activation of FAF1 function in apoptosis can be used in the treatment of diseases including cancer, immune disorders such as autoimmune diseases, infectious diseases, and myocardial infarction and neuronal infarction in cardiovascular diseases.

Characterization of Apoptosis

The characteristics of cell death by apoptosis include cytoplasmic and nuclear condensation, loss of membrane integrity and extensive fragmentation of chromosomal DNA. The degraded DNA from apoptotic cells forms a characteristic ladder when analyzed by gel electrophoresis (Vaux, D., Proc. Natl. Acad. Sci., 90:786-789 (1993)).

Yeast Two Hybrid Screening

The yeast two-hybrid system (Durfee et al., Genes Dev., 7:555-569 (1993)) can be utilized to detect proteins capable of interacting with a known protein. Briefly, the method is as follows. Plasmids are constructed to encode two hybrid proteins which are coexpressed in Saccharomyces cerevisiae. One hybrid consists of the DNA-binding domain of the yeast transcriptional activator protein GAL4, fused to the known protein; the other hybrid consists of the GAL4 activation domain fused to protein sequences encoded by a library of DNA fragments. Interaction between the known protein and a protein encoded by one of the library plasmids leads to transcriptional activation of a reporter gene containing a binding site for GAL4. A suitable reporter gene is the Saccharomyces cerevisiae HIS3 gene and the E. coli lacZ gene (encoding .beta.-galactosidase (.beta.-gal)). Yeast cells are tested for growth in media lacking histidine and for expression of .beta.-gal activity which can be assayed by detecting blue colonies on a plate containing the substrate 5-bromo-4-chloro-3-indolyl .beta.-D-galactoside.

In the present invention, the hybrid construct encoding the known protein consists of the .lambda. repressor dimerization domain/Fas cytoplasmic domain chimera fused to the DNA-binding domain of GAL4. Using this two-hybrid screening system, cDNA clones encoding a protein that interacted with the Fas fusion molecule were isolated. A probe prepared from the longer cDNA clone was used to screen a murine thymus cDNA library from which two full length cDNA clones encoding FAF1 were isolated.

Definitions

As used herein, a "Fas-associated factor 1" is a protein which has an affinity for Fas and binds or physically interacts with Fas.

A "FAF1 interacting molecule" or "FAF1 associating molecule" is a molecule which has an affinity for FAF1 and binds or physically interacts with FAF1. If the interacting molecule is a protein, it is referred to herein as "FAF1 interacting protein". The interaction can be transient, lasting only a fraction of a second or it can be stable so as to enable the detection of the complex of FAF1--FAF1 interacting molecule. Preferably, this interaction persists for at least ten seconds, ideally several minutes. The term "FAF1 interacting molecule" does not imply any particular molecular size or other structural or compositional feature other than that the molecule or compound in question is capable of binding or otherwise interacting with FAF1. The interacting molecule may be a substrate of FAF1, an enzyme that acts on FAF1, a protein that FAF1 is involved in localizing, an effector molecule of FAF1 and/or Fas or a molecule that alters the conformation of FAF1 upon interaction. Interacting or associating molecules that can be investigated by this invention include but are not restricted to agonists and antagonists of FAF1, cellular proteins encoded by oncogenes or proto-oncogenes, lipids, toxins, hormones, sugars, cofactors, peptides, proteins, enzyme substrates, drugs and compounds from plant or animal sources.

A "Fas-interacting domain polypeptide or peptide of FAF1" is defined as a polypeptide or peptide having a sequence corresponding to a region in the wild-type FAF1 protein, which physically interacts with the cytoplasmic region of Fas. The peptide will typically be in the range of 10-30, preferably 25, amino acids.

An "isolated nucleic acid" is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule. An "isolated polypeptide" or protein carries a similar meaning with the polypeptide or protein being substantially separated from any cellular contaminants and components naturally associated with the protein in vivo.

An "allelic variation" in the context of a nucleic acid or a gene is an alternative form (allele) of a gene that exists in more than one form in the population. At the polypeptide level, "allelic variants" generally differ from one another by only one, or at most, a few amino acid substitutions. A "species variation" of a nucleic acid or a polypeptide is one in which the variation is naturally occurring among different species of an organism.

A "fragment" of a nucleic acid is a stretch of at least about 18 nucleotides, more typically at least about 50 to 200 nucleotides but less than 2 kb. A polypeptide "fragment" or "segment" is a stretch of amino acid residues of at least about 6 contiguous amino acids from a particular sequence, more typically at least about 12 amino acids but can be up to 20 amino acids.

The term "recombinant" or "recombinant DNA molecule" refers to a nucleic acid sequence which is not naturally occurring, or is made by the artificial combination of two otherwise separated segments of sequence. By "recombinantly produced" is meant artificial combination often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the common natural forms. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design. "Recombinant DNA molecules" include cloning and expression vectors.

Two nucleic acids share sequence "identity" or "homology" if the two nucleic acids or designated segments thereof, when optimally aligned with appropriate nucleotide insertions or deletions, are identical in at least about 50% of the nucleotides. "Substantial homology" in the nucleic acid context means that the nucleic acids or their complementary strands, when compared, are identical when optimally aligned with appropriate nucleotide insertions or deletions, in at least about 60% of the nucleotides, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95 to 98% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, using a sequence derived from the FAF1 nucleic acids. Selectivity of hybridization exists when hybridization occurs with a certain degree of specificity rather than being random. Typically, selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See, Kanehisa, Nuc. Acids Res., 12:203-213 (1984).

The technique of "polymerase chain reaction," or "PCR," as used herein generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Pat. No. 4,683,195 issued Jul. 28, 1987. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands on the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See, generally, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, comprising the use of a known nucleic acid (DNA or RNA) as a primer.

"Operably Linked" means that a polynucleotide sequence it is placed into a functional relationship with another polynucleotide sequence. For example, a promoter is operably linked to a coding sequence if it acts in cis to modulate the transcription of the linked sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous and in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.

A "primer" or "oligonucleotide" can be a single-stranded polynucleotide that may be chemically synthesized by known methods. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetr. Lett., 22:1859 (1981), or by the triester method, according to Matteucci et al., J. Am. Chem. Soc., 103:3185 (1981), or by other methods such as by using commercial automated oligonucleotide synthesizers such as Applied Bio Systems oligonucleotide synthesizer, according to the specifications provided by the manufacturer.

"Epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

As used herein, "constitutively active FAF1" means that FAF1 is functionally active independent of prior activation of Fas. Cells expressing the constitutively active FAF1 will exhibit induced apoptosis without prior activation of Fas by Fas ligand binding or Fas cross-linking. "Constitutively active FAF1" shall be deemed to include functional derivatives thereof or homologs thereof of the wild-type FAF1 protein. Derivatives can be produced by modifying any region in the wild-type protein such as by deleting negative regulatory regions or by making amino acid substitutions.

Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry described below are those well known and commonly employed in the art. Standard techniques such as described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), are used for recombinant nucleic acid methods, nucleic acid synthesis, cell culture, and transgene incorporation, e.g., electroporation, injection, lipofection. Generally enzymatic reactions, oligonucleotide synthesis, and purification steps are performed according to the specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references which are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.

Specific Embodiments

Nucleic Acid and Polypeptide Compositions

The present invention provides an "isolated nucleic acid" encoding a novel, Fas-associated factor 1, defined herein as FAF1. The nucleotide sequence of the cDNA encoding full length FAF1, SEQ ID NO:1, is shown in FIGS. 4A-4F and the amino acid sequence, SEQ ID NO:2, is shown in FIGS. 5A-5D.

Specifically provided by the invention is a nucleic acid comprising at least 85% sequence identity, preferably 90%, most preferably 95% to 98% sequence identity with the nucleotide sequence of SEQ ID NO:1, an "allelic or species variation" thereof, or a fragment thereof. The nucleic acid compositions of the present invention, whether RNA, CDNA, genomic DNA or a hybrid of the various combinations, can be isolated from natural sources or may be synthesized in vitro.

The invention also provides certain recombinant DNA molecules comprising the nucleotide sequence of SEQ ID NO:1 or a fragment thereof, such as the expression construct PCGN8.1 encoding FAF1 tagged at the N-terminus with the HA epitope, and the FAF1/GAL4 transactivation domain fusion construct.

Compositions of the FAF1 polypeptide and derivatives thereof are also provided. These compositions will be full length natural forms, the natural forms including allelic and species variations of the polypeptide encoded by SEQ ID NO:2, fragments of the natural forms, fusion proteins with those fragments and modified forms of each. Fusion proteins based on the FAF1 sequence include the HA epitope-tagged FAF1 and the FAF1/GAL4 fusion protein used in the yeast two-hybrid system described in the experimental examples. FAF1 polypeptides will generally be synthesized by expression from recombinant DNA molecules. Alternatively, FAF1 fragments and peptides can be synthesized by chemical methods such as described in Merrifield, B., Science, 232:342 (1986); Kent, S. B. H., Ann. Rev. Biochem., 57:957 (1988); Offord, R. E., Semisynthetic Proteins, Wiley Publishing (1980); and Atherton et al., Solid Phase Peptide Synthesis, A Practical Approach, Oxford University Press, Oxford (1989).

In a preferred embodiment, the FAF1 polypeptide or fragment thereof is capable of associating with the cytoplasmic domain of Fas. In addition, pharmaceutical compositions are provided that include the FAF1 polypeptide and its derivatives with a pharmaceutically acceptable carrier.

Uses of FAF1 Nucleic Acids

The FAF1 nucleic acid and fragments thereof have various uses. In one aspect of the invention, the mouse full length FAF1 nucleic acid according to the sequence of SEQ ID NO:1, or fragments thereof, are used to prepare probes to screen DNA libraries to isolate FAF1 genes or gene fragments encoded by other species, particularly human. The methods of screening DNA libraries are generally well known, see, e.g., Sambrook et al. (1989). If less than the full length FAF1 sequence is used, the probes will be oligonucleotides or DNA fragments having at least about 25 nucleotides, more usually at least about 100 nucleotides, and fewer than about 2 knt (kilonucleotides), usually fewer than about 0.5 knt. Preferably, the probe should be free of vector sequences. The probes are typically prepared labeled. Radiolabels such as .sup.32 P are normally used although non-radioactive labels are also suitable.

Conditions for hybridization can be varied. Initially, less stringent conditions can be used. However, if a high background of non-specific hybridization is observed, more stringent conditions will be employed.

The screening of mammalian cDNA or genomic DNA libraries, especially human DNA libraries, can be targeted although eukaryotes such as yeast and insects are also of interest for evolutionary comparisons. The DNA libraries may be constructed in phage, bacteria or yeast. Clones that hybridize to the probe are identified such as by autoradiography if radiolabeled probes are used. DNA is isolated from hybridizing clones and analyzed for the presence of FAF1 gene sequences as verification that the hybridizing clone carries all or part of the FAF1 gene. The DNA sequence carried by the clone is compared with that of SEQ ID NO:1. The FAF1 gene or fragment thereof will then be isolated from the vector by restriction endonucleases. It may be necessary to isolate several overlapping DNA sequences from different hybridizing clones to recombinantly reproduce the full length gene in one contiguous DNA fragment.

Alternatively, with the availability of the mouse FAF1 sequence, FAF1 genes from other species can be isolated by Polymerase Chain Reaction (PCR) by selecting appropriate pairs of primers based on the known sequence and using genomic DNA or cDNA prepared from cells as the template. Primers can be chemically synthesized and will be at least 10 nucleotides in length, more usually 14 nucleotides, preferably 17 nucleotides, but can be as long as 100 bp nucleotides. Pairs of primers corresponding to the 5' and 3' ends of the gene or to the internal regions of the gene can be used. Several rounds of PCR may be required to prepare overlapping clones that can then be linked by recombinant methods to produce the entire gene in one DNA fragment.

A gene that hybridizes with the probe and is determined to be substantially homologous to the FAF1 gene in nucleotide sequence will be isolated. The homologous gene will be inserted into an appropriate expression vector and introduced into a suitable host for expression to produce the encoded polypeptide. The encoded polypeptide will then be assayed to determine if it associates with the appropriate species of Fas and functions like mouse FAF1, using the same procedure for analyzing the interaction of FAF1 with Fas, described below in the Experimental Examples.

Other FAF1 structurally- or functionally-related family member proteins will associate and interact with receptors similar to Fas and regulate apoptosis in other systems. The same library screening approach can also be used to identify other members of the family that share "substantial homology" with FAF1.

The FAF1 probes can also be used to determine whether RNA encoding FAF1 or a FAF1 homolog is present in a cell. This can be done by the procedure of Northern Blotting. In situ hybridization can also be performed on tissue sections of the organism to determine developmental regulation and compare expression levels in various tissues. The probes can be labeled using any suitable labels or tags, e.g., radiolabel, biotin-avidin. The procedures of preparing probes, Southern blotting, Northern blotting and in situ hybridizations are well known in the art. See, for example, Sambrook et al., 1989.

The invention also provides a method of determining if the FAF1 gene from a cell of interest is mutated. The cells can be from cultured cell lines or from tissue isolated from an animal or human. For example, cells can be prepared from a human tumor biopsy. PCR can be used to amplify all or part of the FAF1 gene using selected primers and the amplified DNA fragment sequenced or analyzed for restriction enzyme cleavage patterns. The nucleotide sequence or restriction analysis is compared to the wild type sequence of FAF1 from the appropriate species. Therefore, the wild type sequence acts as a standard or positive control.

Another aspect of the invention is the mapping of the functional domains of FAF1. Functional domains of FAF1 include regions of FAF1 that: interact with Fas; interact with effector molecules; exhibit an enzymatic activity; or regulate the activity of FAF1 itself. Manipulating these domains of FAF1 can provide a means by which to control Fas and FAF1 mediated apoptosis. For example, polypeptides or peptides corresponding to the Fas-interacting domain of FAF1 can be used to block Fas mediated apoptosis. Such polypeptides will generally be in the range of 6 to 100 amino acids. Circumstances whereby blocking of apoptotic cell death is desirable are described below under therapeutic uses.

There may be negative regulatory regions in FAF1 that control the activation of FAF1 so that premature or unnecessary cell death does not occur. These regulatory regions also serve as useful targets for mutagenesis to affect the Fas apoptotic signaling pathway. For example, a constitutively active FAF1 could be produced by removing a negative regulatory region. Such a modified FAF1 can be selectively expressed in targeted population of cells to affect cell death. This approach can be used to target elimination of cells that exhibit uncontrolled proliferation such as cancerous cells and autoreactive cells.

The method of mapping functional domains can be accomplished by generating a series of FAF1 deletion mutants which can then be compared to the wild-type FAF1 in biological function. The deletion mutants will be produced recombinantly using the full length FAF1 nucleic acid as starting material, and more conveniently, the HA-epitope tagged FAF1 described in the Experimental Examples. Tagging the mutant FAF1 molecules either using the HA epitope or some other tag is desirable to facilitate detection of the mutant and to distinguish the transfected from the endogenous FAF1.

Generally, the mutants will have overlapping deletions. Initially, mutants will be created that have deletions typically in the range of 10-50 amino acids. For subsequent more precise mapping, the deletions in a particular region will be smaller, typically in the range of 5-10 amino acids. Mutants having single amino acid changes will be useful to define key residues that determine structure or that may be involved in physical contact with Fas or a downstream interacting or effector molecule of FAF1. Such mutants can be generated by known techniques such as by site-directed mutagenesis or by PCR.

Each deletion mutant is then transfected into an appropriate host cell and the affects of each deletion on the function of FAF1, assessed. To facilitate analysis of the transfected FAF1, the host cell preferably expresses little or no endogenous FAF1 or expresses a mutated endogenous FAF1 that is non-functional. Preferably, mammalian cell lines amenable to transfection will be used and these include Cos and L cells.

The mutant transfected FAF1 will be analyzed for various characteristics that indicate increased, reduction or complete loss of Fas-mediated function. For example, mutant FAF1 will be analyzed for physical association with Fas by its ability to coimmunoprecipitate with Fas or a Fas fusion polypeptide. The extent to which mutant FAF1 can potentiate Fas-mediated apoptosis will be quantitated. Such assays are described in more detail under Experimental Examples below.

The invention provides FAF1 nucleic acid and fragments thereof to prepare expression constructs for FAF1 and FAF1 polypeptide fragments. The expression vectors will contain the necessary elements for transcription and translation of the DNA fragments into polypeptide if these elements are not already present in the DNA fragments themselves. These necessary elements include a promoter 5' of the DNA insert to be expressed, a transcription and translation initiation site, stop codons, poly-A signal sequence, splice signals. DNA sequences encoding the protein will be operably linked to a promoter appropriate for expression in a particular cell type. Usually a strong promoter will be employed to provide for high level transcription and expression. Examples of strong promoters include human cytomegalovirus promoter. An enhancer may be necessary to function in conjunction with the promoter. The expression construct normally comprises one or more DNA sequences encoding FAF1 under the transcriptional control of a native or other promoter. Usually the promoter will be a eukaryotic promoter for expression in a mammalian cell, where the mammalian cell may or may not result in the expression of FAF1. The selection of an appropriate promoter will depend upon the host, but promoters such as the human cytomegalovirus promoter, viral LTRs such as SFFV LTR, and prokaryotic promoters such as trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known. In some circumstances, an inducible promoter may be preferred. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Guide, Vols. 1-3 (1989), Cold Spring Harbor Press.

Plasmid, viral or YAC vectors are contemplated. Conveniently available expression vectors which include the replication system and transcriptional and translational regulatory sequences together with polylinker restriction sites for insertion of the protein encoding sequence, may be employed. Such expression vectors are commercially available such as from Stratagene, Inc. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al. (1989); see also, Metzger et al., Nature, 334:31-36 (1988).

It may be desirable to produce the FAF1 protein or fragments thereof in a prokaryotic host, in which case a prokaryotic promoter is preferred. Examples of prokaryotic promoters are trp, lac, and lambda. See Sambrook et al. (1989) for other useful prokaryotic promoters. Usually a strong promoter will be employed to provide for high level transcription and expression.

The expression construct will often be contained in a vector capable of stable extrachromosomal maintenance in an appropriate cellular host or may be integrated into the host genome. The expression construct may be bordered by sequences which allow for insertion into a host, such as transposon sequences, lysogenic viral sequences, or the like. Normally, markers are provided with the expression construct which allow for selection of host cells containing the construct. The marker is preferably on the same DNA molecule but can be on a different DNA molecule that is cointroduced into the host cell. In prokaryotic cells, markers such as a resistance to a cytotoxic agent, complementation of an auxotrophic host to prototrophy, production of a detectable product, etc., serve the purpose.

The expression construct can be joined to a replication system recognized by the intended host cell. Various replication systems include viral replication systems such as retroviruses, simian virus, bovine papilloma virus, or the like.

While the wild-type sequences of FAF1 will generally be employed, in some situations one or more mutations or minor modifications may be introduced, such as deletions, substitutions or insertions resulting in changes in the amino acid sequence, providing silent mutations or modifying amino acid residues or amino or carboxyl terminal groups. Conservative amino acid substitutions can be introduced. These amino acid changes can be made using techniques such as PCR or site-directed mutagenesis.

There will be circumstances where gene fusions between FAF1 and another protein can be useful. The fusion proteins will be recombinantly produced. The recombinant nucleic acid sequences used to produce fusion proteins of the present invention will often be derived from natural or synthetic sequences. The FAF1 nucleic acid or fragments thereof find use in the construction of vectors encoding fusion proteins.

Fusion proteins encoding FAF1 fused to the GAL4 DNA binding domain can be used in the yeast two hybrid system to isolate any proteins other than Fas that interact with the FAF1 protein, in particular, proteins that act downstream of FAF1. The yeast two-hybrid system is described above and in the experimental examples. This method allows the isolation of the cloned genes for the interacting proteins and eventually the identification of the interacting proteins. Knowledge of the interacting proteins in the Fas signaling pathway will allow the screening of drugs for agonists and antagonists of Fas dependent apoptosis.

The nucleic acid constructs will be useful to introduce into cells, providing an efficient and economical means to produce commercially useful quantities of the protein compositions. Transfected cells producing varying quantities of full length FAF1 will also be useful in evaluating the effect of overexpression of FAF1 on Fas function and apoptosis. Nucleic acid constructs expressing various lengths and mutant forms of FAF1 can be used to determine structure-function relationships.

The means of introduction of the expression construct into a host cell will vary depending upon the particular vector and the target host. Introduction can be achieved by any convenient means, including fusion, conjugation, transfection, transduction, electroporation, injection, or the like. See, e.g., Sambrook, et al. (1989), supra. Transient or stable transfection procedures can be used. The FAF1 nucleic acid compositions are introduced into the appropriate cellular host under conditions which favor expression of the polypeptide and isolation of the resultant expressed polypeptide. This implies using an expression vector compatible with the host cell, the vector containing the necessary elements described above for expression of the polypeptide. The transfected cells are then provided with the optimum nutrient, gas and temperature conditions for optimal protein production. These conditions will depend on the cell type.

The host cells will normally be immortalized cells, i.e., cells that can be continuously passaged in culture. For the most part, these cells will be convenient mammalian cell lines which are able to express a FAF1 protein and, where desirable, process the polypeptide so as to provide an appropriate mature polypeptide and transport the mature protein to the appropriate cell compartment. By processing is intended glycosylation, ubiquitination, disulfide bond formation, general post-translational modification, or the like.

A wide variety of both prokaryotic and eukaryotic hosts will be employed for expression of the proteins and peptides. Useful hosts include bacteria, such as E. coli., yeast, filamentous fungi, insect cells such as Sf9, mammalian cells, typically immortalized, e.g., various mouse cell lines, monkey cell lines, Chinese hamster ovary cell lines, human cell lines, derivatives of them, or the like. Since FAF1 appears to be ubiquitous in expression by Northern analysis, it can be expressed in most mammalian cell type. Cells that are amenable to transfection and in vitro cell culture manipulation are preferred. In a specific embodiment, Cos and L cells are used. In a preferred embodiment, FAF1 and fragments thereof are expressed in tumor cells and cells of lymphoid, thymus or heart smooth muscle and epithelial cells of murine or human origin, depending on the FAF1 species used. In some cases, the cells will be derived from a neoplastic host cell or wild-type cells will be transformed with oncogenes, tumor causing viruses or the like. Cells carrying the FAF1 nucleic acid compositions are covered by this invention.

Uses of FAF1 Transfected Cells

Cells transformed with the nucleic acid compositions can be used to create transgenic mice. Such transgenic mice are useful e.g. to study the effect of overexpression of FAF1 on growth and development of the animal. The procedure for producing transgenic mice is known in the art and are described in detail in Hogan et al., Manipulating the Mouse Embryo, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1986). Mice having homozygous deletion of the FAF1 gene (knockout mice) can also be generated by homologous recombination using gene targeting techniques (see, e.g., Capecchi, Science, 244:1288-1292 (1989). Such knockout mice are of interest to study the affect of FAF1 deficiency on development and cell differentiation.

Another aspect of the invention relates to methods of screening for agonists and antagonists of FAF1 which can affect apoptosis mediated by the Fas pathway. The agonists and antagonists are preferably small molecules defined as being in the range of MW 5-20 kD and can cross the plasma membrane or be taken up by the cell. Test molecules will include oligonucleotides, lipids, toxins, hormones, sugars, cofactors, peptides, small proteins, drugs and compounds from plant or animal sources and recombinantly produced substances. FAF1 transfected cells (FAF1 transfectants) are useful for such screening although cells naturally expressing Fas and FAF1 can also be used.

The screening method involves contacting a cell expressing both Fas and FAF1 with a test molecule, activating Fas, and analyzing the cell for any effects on apoptosis, increased apoptosis indicative that the test molecule is an agonist and decreased or loss of apoptosis indicative that the test molecule is an antagonist. Fas can be activated by providing Fas ligand to the cell culture media or solution of the cell sample, to bind the Fas receptor on the cell surface. Alternatively, Fas can be activated by crosslinking with antibodies to the extracellular domain of Fas. If Fas is expressed as a fusion protein, an antibody specific to the extracellular domain portion of the fusion protein will be used to crosslink Fas. For example, an anti-CD4 antibody can be used to crosslink a CD4/Fas fusion protein. FAF1 can be naturally or recombinantly produced in the wild type form or expressed as a fusion protein. Appropriate positive and negative control cells will be required for comparison.

In one embodiment, L cells expressing a Fas fusion protein, such as the CD4/fas-16 cells described in the Experimental Examples, are further transfected with FAF1 or mock transfected with vector as described. The mock transfected CD4/fas-16 serve as negative control for the functional assays. Transfectants expressing both Fas and FAF1 (test cells) and mock transfectants expressing Fas only (negative control cells), are contacted with a solution sample containing one or more test compounds. A sample of double transfectants will be treated under the same conditions but in the absence of the test molecule. The cells can be exposed to the test compound added to the culture media. The test compounds can initially be screened in pools comprising about 2-10 different compounds or molecule types per sample solution.

Cells treated in the absence or presence of the test molecule will be tested for eg. for disruption of Fas-FAF1 interaction or for any effect on apoptosis with or without stimulation of Fas. In one embodiment, the cells are crosslinked with anti-CD4 antibody (e.g., L3T4) and then assayed for any effects on FAF1 activity essentially as described in the Experimental Examples under the subsection Association of FAF1 with Fas. The cells are analyzed for morphological changes such as cell membrane blebbing and other characteristics of apoptosis. Cellular DNA can be analyzed for fragmentation by gel electrophoresis. Based on these criteria, the percentage of apoptotic cells in the test cells as compared to similar cells treated in the absence of the test molecule or the mock transfected cells can be determined. The test molecule is considered an agonist if the test cells contacted with the test molecule show increased frequency of apoptosis over the negative control. In contrast, loss of or reduction in apoptosis in the test cells is indicative that the test molecule is an antagonist.

In a specific embodiment, peptide libraries expressing peptides of about 8-15 amino acids, preferably 10-12 amino acids in length (10-12 kD), are screened for agonistic or antagonistic effects on FAF1 activity. The generation and screening of synthetic peptide combinatorial libraries is described, e.g., in Houghten et al., Nature, 354:84-86 (1991). Libraries of peptide ligand can also be screened using the bacteriophage surface display technology as described by Djojonegoro et al., Biotechnology, 12:169-172 (1994).

Isolation of FAF1 Polypeptide

The FAF1 polypeptide can be isolated from a normally expressing cell or a transfected cell by immunoprecipitation or affinity chromatography of cell lysates using FAF1-specific antibody. The antibody can be in solution or affixed on a solid substrate. It may be more efficient to isolate the protein from transfected cells that may produce larger quantities of the protein due to particular characteristics of the expression construct, such as a strong promoter. Instead of or in addition to immunological methods, the peptide will generally be isolated by techniques employing FPLC, HPLC, electrophoresis, gradient centrifugation and other methods routinely used in protein purification to provide a substantially pure product, i.e., particularly free of cellular contaminants. For protein purification methods, see, e.g., Jacoby, Methods in Enzymology, Vol. 104 (1984), Academic Press, New York; Scopes, Protein Purification: Principles and Practice, (2nd Ed.) (1987) Springer-Verlag, New York; Deutscher (ed.), Guide to Protein Purification, Methods in Enzymology, Vol. 182 (1990).

Uses of FAF1 Polypeptide Compositions

The FAF1 polypeptide compositions find several uses.

The polypeptide compositions of FAF1 are useful for raising antibodies, both polyclonal and monoclonal. Such antibodies are powerful tools that can be employed in various assays and diagnostic situations particularly where immunoprecipitation, immunoblotting and affinity purification procedures are necessary. Thus, one aspect of the invention is to provide antibodies that specifically binds to the FAF1 polypeptide comprising the sequence of SEQ ID NO:2, an allelic or species variation thereof, or a fragment thereof. Antibodies capable of specifically blocking the binding of FAF1 to Fas are also desirable reagents. The invention provides a rabbit antiserum produced using the entire FAF1 as immunogen. This rabbit anti-FAF1 antiserum is useful in both immunoprecipitating FAF1 and detecting FAF1 on Western blots. The invention also provides hybridoma lines that produce monoclonal antibodies to the FAF1 polypeptide.

These antibodies find use in isolating the FAF1 protein and any structurally related proteins expressing an epitope recognized by the anti-FAF1 antibody. Isolation of FAF1 and structurally related proteins can be accomplished by simply immunoprecipitating the proteins from lysates of normally expressing or transfected cells. Alternatively, affinity purification of, e.g., cell lysates can be performed using the FAF1 antibody fixed on a solid matrix such as a column of beads or a filter paper.

FAF1 antibodies are also useful to study the interaction of FAF1 with Fas in vivo, in normal and growth dysregulated or cancerous cells. The same protocol described in the experimental examples for studying the interaction of FAF1 with Fas in intact cells can be followed.

Thirdly, the FAF1 specific antibodies find use in isolating any FAF1 associating protein that co-immunoprecipitates with FAF1. FAF1 associating proteins are useful to study the downstream effectors of Fas and the regulation of Fas and FAF1 function.

In a different aspect, FAF1 specific antibodies can serve as diagnostic reagents to detect deficiencies in FAF1 such as expression levels in cells from patients suffering from certain autoimmune diseases or tumor cells from cancer patients.

Polyclonal and/or monoclonal antibodies with specificity to FAF1 can be prepared by in vitro or in vivo techniques following standard procedures as described in, e.g., Harlow, et al., Antibodies: A Laboratory Manual (1988), Cold Spring Harbor Press, New York. Antibodies are produced by immunizing an appropriate vertebrate host, e.g., rabbit or rodents, with the entire FAF1 protein or peptides derived thereof, or in conjunction with an adjuvant. Usually two or more immunizations will be involved, and the blood or spleen will be harvested a few days after the last injection.

For immunization, preferably peptides corresponding to regions of the protein comprising hydrophilic residues or residues exposed to the aqueous phase are selected. Immunogens comprising peptides corresponding to the region in FAF1 that interacts with Fas are desirable. Synthetic peptide fragments may be prepared in a peptide synthesizer and coupled to a carrier molecule (e.g., keyhole limpet hemocyanin) and the conjugate injected into rabbits at selected times over several months.

For production of polyclonal antibodies, an appropriate host animal is selected, typically a rabbit or a mouse. The substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal and other parameters well known to immunologists. Typical sites for injection are in the footpads, intramuscularly, intraperitoneally, or intradermally. Of course, another species will sometimes be substituted for a mouse or rabbit, including goats, sheep, cows, guinea pigs, and rats.

The rabbit or mouse sera is tested for immunoreactivity to the FAF1 protein or peptide immunogen by an immunoassay, typically with preimmune sera as one of the negative controls. The immunoassay can be a radioimmunoassay, an enzyme-linked assay (ELISA), a fluorescent assay, or any of many other choices, most of which are functionally equivalent but may exhibit advantages under specific conditions.

The polyclonal antibodies can be provided commercially in the form of antisera or in purified form. From the polyclonal antisera, the immunoglobulins may be precipitated, isolated and purified, such as by affinity purification. Preferably, the purified form is substantially free of non-specific antibodies and cellular contaminants.

Monoclonal antibodies with affinities of 10.sup.8 M.sup.-1 preferably 10.sup.9 to 10.sup.10, or stronger will typically be made by standard procedures as described, e.g., in Harlow et al., Antibodies: A Laboratory Manual, CSH Laboratory (1988); or Goding, Monoclonal Antibodies: Principles and Practice (2d ed) (1986), Academic Press, New York. Normally, mice are used to produce monoclonal antibodies although rats, guinea pigs and other animals can also be used. After the appropriate period of time from the immunization schedule, the spleens of such animals are excised and individual spleen cells fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter the cells are clonally separated and the supernatants of each clone are tested for their production of an appropriate antibody specific for the desired region of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors. See, Huse et al., "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phase Lambda," Science, 246:1275-1281 (1989).

The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by conjugating, either covalently or non-covalently, a substance which provides for a detectable signal.

A wide variety of labels and conjugation techniques are-known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly, U.S. Pat. No. 4,816,567.

The FAF1 antibodies of the invention can also be provided in a kit, with or without a FAF1 polypeptide, for use in any of the various applications described above. The contents of the kit will vary depending on the intended application of the antibody and may include other reagents and instructions for the use of the antibody preparation and the reagents. At least one aliquot of the antibody will be provided. Different FAF1 antibodies with specificities for different regions of the protein can be provided. Different antibodies may be desirable for verification of an assay result. The aliquots can be contained in any suitable container such as a vial or a tube. The polyclonal antibody can be in the form of antisera or affinity purified. Monoclonal antibodies can be provided in the form of ascites, culture media or a buffer such as phosphate buffered saline solution. The antibody preparation can be provided in solution or in lyophilized form, and may even be immobilized on a substrate such as a column matrix. The antibody preparation may also contain in it preservatives such as sodium azide or protease inhibitors such as EDTA. A carrier protein such as BSA or ovalbumin, usually between 0.5-5%, may be included for stability. The solution form of the antibody, especially the purified form, may contain up to 50% glycerol if the kit is to be stored frozen at -20.degree. C. to -70.degree. C. If the antibody is provided in lyophilized form, the kit can include a reconstitution buffer to reconstitute the antibody.

If the antibody is to be used in Western blot analysis, reagents for use in the Western blot procedure can be included in the kit. A secondary, labeled antibody capable of binding to the FAF1 antibody allowing detection of the bound FAF1 antibody, can be included. The labeled antibody may be conjugated to an enzyme such as alkaline phosphatase or horse radish peroxidase.

A FAF1 polypeptide included in the antibody kit will be useful as a control such as for molecular weight indicator on the blot and antibody detection. FAF1 polypeptides by themselves can also be provided in separate kits or vials for the various uses described. It is possible that FAF1 may have lipase activity. If FAF1 is enzymatically active, it may be provided in a kit for use as an enzyme.

The invention also provides methods of isolating a FAF1 associating protein. As used herein, the terms "FAF1 associating protein" and "FAF1 interacting protein" have the same meaning. A first method comprises contacting a cell lysate suspected of containing a FAF1 associating protein with a FAF1 polypeptide and isolating any protein bound to said FAF1 polypeptide as a FAF1 associating protein. The FAF1 polypeptide can be isolated from cells that normally produce FAF1 at high levels or from FAF1 transfected cells that overexpress FAF1. For this purpose, FAF1 encompasses full length FAF1, fragments of FAF1 or FAF1 fusion proteins. A convenient source of FAF1 are the cells transfected with HA epitope-tagged FAF1 of the present invention.

In one embodiment of the first method, a FAF1 polypeptide is immobilized on a solid matrix. The solid matrix can comprise various materials such as is commonly used in column chromatography, including sepharose, sephadex, agarose, polystyrene and latex beads. The solid matrix can also be filter paper or membrane, such as nitrocellulose and polyvinylidene fluoride (PVDF) membrane. The FAF1 polypeptide can be coupled to the solid matrix directly or indirectly. Direct methods include covalent coupling to sepharose beads using cyanogen bromide. Indirect coupling can take advantage of an FAF1 antibody or some other moiety suitable for linking the two components such as biotin-avidin binding pairs. FAF1 or FAF1 fusion proteins may be more conveniently immobilized if the fusion protein can bind a ligand provided on the matrix.

Lysates are prepared from FAF1 expressing cells. Cell lysates will be contacted with the immobilized FAF1 polypeptide such as by running the lysates over a FAF1 affinity column to allow any FAF1 interacting protein to bind the immobilized FAF1 polypeptide under optimum conditions. Preferably the binding reaction is carried out between 4.degree. C. and normal physiological temperature. Buffer conditions can be modified to favor capture of this binding. For example, the Ph and salt conditions can be varied. The matrix is then washed with buffer to remove any unbound or nonspecifically bound cellular components. Any protein that bound to the FAF1 polypeptide will be isolated by elution off the solid matrix such as by using a salt gradient or using soluble FAF1 polypeptide and fragments thereof to compete for binding.

In a second method, FAF1 expressing cells, preferably transfectants that overexpress FAF1, are biosynthetically labeled in order to label the proteins. For this method, the cells to be transfected with FAF1 or FAF1 fragments are preferably cells that naturally express FAF1 so as to favor the likelihood of the cells also expressing a FAF1 associating protein. It is likely that the Fas pathway has to first be activated to induce a FAF1 interacting protein to associate with FAF1. Therefore, a sample of cells treated to activate Fas will be tested in parallel. As mentioned earlier, Fas can be activated by binding to Fas ligand (FasL) or by anti-Fas antibody cross-linking. If CD4/fas fusion protein expressing transfectants (such as CD4/fas-16 described below) are used, Fas can be cross-linked using anti-CD4 antibodies (e.g., L3T4). The cells are lysed under different detergent conditions and FAF1 is immunoprecipitated from the labeled cell lysate using anti-FAF1 antibody or antiserum. Labeled proteins that co-immunoprecipitate with FAF1 are identified by SDS-PAGE followed by autoradiography.

A third method for isolating FAF1 associating proteins involves screening a phage or bacterial peptide library for peptides capable of binding to FAF1 and isolating any bound peptide by affinity purification. Fodor et al. in U.S. Pat. No. 5,143,854 describe methods of preparing arrays of peptides on a solid matrix, screening of the peptides and automated detection of peptides bound to ligand. The FAF1 polypeptide can be provided in substantially pure form, immobilized directly or indirectly onto a solid matrix. FAF1 can also be provided as an immunocomplex containing FAF1 bound to an antibody that specifically recognizes a tag on FAF1. A HA epitope tag and an anti-HA epitope antibody combination is a suitable example. The tag antibody can be covalently bound to a solid matrix such as protein A-sepharose beads or directly conjugated to plain sepharose beads. Phage or bacterial peptide libraries are exposed to the FAF1 protein to allow one or more peptides to bind to the protein. Peptides which bind FAF1 are considered associated proteins and can be isolated by affinity purification.

Yet another approach to identifying FAF1 associating proteins is by using the yeast two-hybrid system and FAF1 fusion proteins as described earlier. Peptide expression libraries such as by phage display methodology, can be screened for ligand binding to the FAF1 polypeptide or fragments thereof or fusion proteins thereof.

FAF1 or FA