EP1097226A1 - Usurpin, a mammalian ded-caspase homologue that precludes caspase-8 recruitment and activation by the cd-95 (fas, apo-1) receptor complex - Google Patents

Abstract

The protein Usurpin, an endogenous mammalian regulator of the process of apoptotic cell suicide, is provided. Provided are three Usurpin proteins having an amino acid sequence selected from the group consisting of: SEQ.ID.NO.:4, SEQ.ID.NO.:5, and SEQ.ID.NO.:6 that arise from alternative splicing of the Usurpin gene. The full-length Usurpin polypeptide (Usurpin-α) has features in common with pro-caspase-8 and -10, including tandem 'death effector domains' (DEDs) on the N-terminus and a large subunit/small subunit caspase-like domain, but it lacks key residues that are necessary for caspase proteolytic activity. However, Usurpin heterodimerizes with pro-caspase-8 and precludes pro-caspase-8 recruitment by the FADD/MORT1 adapter protein, thus blocking an important interaction in the CD95 (Fas/APO-1) apoptotic process. Therefore, Usurpin is useful as a modulator of the sensitivity of cells to CD95 (Fas/APO-1)-mediated apoptosis. Accordingly, the present invention also provides methods of modulating apoptosis by introducing Usurpin polypeptides into cells. Also provided are various purified nucleotide sequences encoding Usurpin. Also provided are methods of identifying inhibitors of the interaction between Usurpin and pro-caspase-8. Such inhibitors will be useful in controlling the interaction between Usurpin and pro-caspase-8, thus modulating apoptosis.

Description

TITLE OF THE INVENTION

USURPIN, A MAMMALIAN DED-CASPASE HOMOLOGUE THAT PRECLUDES CASPASE-8 RECRUITMENT AND ACTIVATION BY THE CD-95 (FAS, APO-1) RECEPTOR COMPLEX

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/092,005, filed July 8, 1998, the contents of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not applicable.

REFERENCE TO MICROFICHE APPENDIX Not applicable.

FIELD OF THE INVENTION

The invention relates to the field of molecules involved in the control of apoptosis and uses thereof.

BACKGROUND OF THE INVENTION

Individual cells within multicellular organisms commit suicide in a highly ordered and systematic process which involves a biochemical "cell death" pathway that has been largely conserved throughout evolution. This form of altruistic cell suicide, which is manifest as the apoptotic phenotype, occurs during developmental morphogenesis, in the removal of expended, unnecessary or irreparably damaged cells, and in response to pathogenic infections (Kerr et al., 1972; McConkey et al, 1996; Steller, 1995; Thompson, 1995; Uren and Vaux, 1996).

Apoptotic cell suicide proceeds through a highly regulated series of biochemical events and many of the components of the cell death pathway have been recently identified. At the heart of this pathway lie a family of cysteine proteases, the caspases, which are related to mammalian interleukin-lβ converting enzyme (ICE/caspase-1) and the product of the C. elegans "death gene", CED-3 (Alnemri et al., 1996; Cohen, 1997; Nicholson and Thornberry, 1997). The caspases mediate apoptosis by cleaving a discrete subset of homeostatic, repair, and structural proteins within dying cells. This results in the cessation of normal cellular functions, the dismantling of the genome and structural constituents of the cell, and the packaging of cellular components into apoptotic corpses for engulfment by other cells (Nicholson and Thornberry, 1997; Rosen and Casciola-Rosen, 1997). The central importance of caspases in these processes has been demonstrated with both macromolecular and peptide-based inhibitors, which prevent apoptosis from occurring in vitro and in vivo, and by genetic approaches as well (Bump et al, 1995; Devereaux et al., 1997; Hara et al, 1997; Kuida et al, 1996; Liston et al, 1996; Loddick et al, 1996; Nicholson et al, 1995; Xue and Horvitz, 1995; Zhou et al, 1997).

Ten caspases have so far been identified in human cells. Each is synthesized as a catalytically dormant proenzyme containing an amino-terminal prodomain followed by the large and small subunits of the heterodimeric active enzyme. The subunits are excised from the proenzyme by cleavage at Asp-x junctions. The strict requirement by caspases for Asp in the Pi position of substrates is consistent with a mechanism whereby proenzyme maturation can be either autocatalytic or performed by other caspases. The three dimensional crystal structure of mature caspase-1 and -3 show that the large subunit contains the principal components of the catalytic machinery, including the active site Cys residue which is harbored within the conserved pentapeptide motif, QACxG (SEQ.ID.NO.: 16), and residues that stabilize the oxyanion of the tetrahedral transition state (Rotonda et al, 1996; Walker et al, 1994; Wilson et al, 1994). Both subunits contribute residues which stabilize the Pi Asp of substrates while the small subunit appears to contain most of the determinants that dictate substrate specificity and, in particular, those which form the specificity-determining S4 subsite.

Members of the caspase family can be divided into three functional subgroups based on their substrate specificities which have been defined by a positional-scanning combinatorial substrate approach (Rano et al, 1997; Thornberry et al, 1997). The principal effectors of apoptosis (group π caspases, which include caspases-2, -3 and -7 as well as C. elegans CED-3) have specificity for

[P4]DExD[Pι ] (SEQ.ID.NO.:36), a motif found at the cleavage site of most proteins known to be cleaved during apoptosis (Nicholson and Thornberry, 1997). On the other hand, the specificity of group III caspases (caspases-6, -8, -9 and -10, as well as CTL-derived granzyme B) is [P4](I,V,L)ExD[Pι] (SEQ.ID.NO. :37) which corresponds to the activation site at the junction between the large and small subunits of other caspase proenzymes, including group II (effector) family members. This and other evidence indicates that group m caspases function as upstream activators of group π caspases in a proteolytic cascade that amplifies the death signal. The mechanism by which at least one of the group in activator caspases is itself activated (caspase-8/MACH, FLICE, Mch5) has been elucidated by studies on the CD95 (Fas/APO-1) receptor system (Boldin et al, 1996; Medema et al, 1997; Muzio et al, 1996).

CD95 (Fas/APO-1) is a member of the growing family of "death receptors" which also include TNF-Rl , TRAIL-R DR-4/APO-2, TRAIL-R2/DR-5 and TRAMP/DR-3/APO-3/wsl /AIR/LARD. In addition to overall structural similarities, these receptors have in common the ability to communicate a pro- apoptotic signal via carboxy-terminal cytoplasmic "death domains" (DD) (Nagata, 1997; Wallach et al, 1997; Yuan, 1997). In the case of the CD95 (Fas/APO-1) system, receptor ligation by the homotrimeric Fas ligand results in receptor oligomerization and subsequent recruitment of multiple FADD/MORTl adapter proteins (Boldin et al, 1995; Chinnaiyan et al, 1995) to the receptor complex. The FADD/MORTl adapters in turn recruit caspase-8 proenzymes which then become activated, presumably by intermolecular autocatalysis following receptor-mediated proenzyme oligomerization. Whereas interactions between CD95 (Fas/APO-1) and the FADD/MORTl adapter are mediated by the "death domains" contained within both molecules, the interaction between FADD/MORTl and the caspase-8 proenzyme is mediated by interactions between homologous "death effector domains" (DEDs) that are contained in the amino-terminus of FADD/MORTl as well as within the prodomain of caspase-8. The prodomain of caspase-8 contains two serial DEDs as does the prodomain of caspase- 10 (Fernandes-Alnemri et al, 1996). Several viral DED-containing proteins have recently been shown to disrupt the formation of functional death-signaling CD95 (Fas/APO-1) receptor complexes by competing for interactions with the DEDs within either the FADD/MORTl adapter or the DED- caspase proenzymes, and this appears to be a mechanism to delay the host suicide response and facilitate productive viral infection (Bertin et al, 1997; Hu et al, 1997a; Thome et al, 1997). SUMMARY OF THE INVENTION

The present invention provides the protein Usurpin, an endogenous mammalian regulator of the process of apoptotic cell suicide. Provided are Usurpin proteins having an amino acid sequence selected from the group consisting of: SEQ.ID.NO.:4, SEQ.ID.NO.:5, and SEQ.ID.NO.:6. The Usurpin gene is ubiquitously expressed in mammalian tissues and Usurpin protein exists as at least three isoforms arising from alternative mRNA splicing. The full-length Usurpin polypeptide (Usurpin-α, SEQ.ID.NO. :4) has features in common with pro-caspase-8 and -10, including tandem "death effector domains" (DEDs) on the N-terminus and a large subunit/small subunit caspase-like domain, but it lacks key residues that are necessary for caspase proteolytic activity. Usurpin heterodimerizes with pro-caspase-8 in vitro and precludes pro-caspase-8 recruitment by the FADD/MORTl adapter protein, thus blocking an important interaction in the CD95 (Fas/APO-1) apoptotic process. Cell death induced by CD95 (Fas/APO-1) ligation is attenuated in cells transfected with Usurpin. Therefore, Usurpin is useful as a modulator of the sensitivity of cells to CD95 (Fas/APO-1 )-mediated apoptosis. Accordingly, the present invention also provides methods of modulating apoptosis by introducing Usurpin polypeptides into cells.

Also provided are various purified nucleotide sequences encoding Usurpin.

Also provided are methods of identifying inhibitors of the interaction between Usurpin and pro-caspase-8. Such inhibitors will be useful in controlling the interaction between Usuipin and pro-caspase-8, thus modulating apoptosis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 A-C shows the primary sequence homology of Usurpin with DED-caspases and the lack of critical catalytic and substrate binding determinants in Usurpin.

Figure 1 A. The Usurpin-α isoform was aligned with the caspase-8 (MACH, FLICE, Mch5) and caspase-10 (Mch4) proenzyme sequences using the "ClustalW Pileup" algorithm of the Genetic Computer Group (version 9) software package using default alignment parameters. Amino acids that are identical in at least two of the three sequences are shaded. The first (DED-A) and second (DED-B) of the tandem "death effector domains" are indicated by large boxes, and the regions of the polypeptides corresponding to the large and small subunits of mature caspase enzymes are indicated on the right. The upward-pointing solid arrow indicates the processing sites between the prodomains and large subunits of caspases-8 and 10, and the downwards-pointing solid arrow indicates the cleavage junction between the large and small subunits. The smaller boxes, labeled a through e, contain critical residues that are important for proteolytic catalysis (boxes b and c) or formation of the Si subsite (boxes a and e) which are changed in Usuipin. Box d indicates the group HI caspase/granzyme B consensus sites contained in all three polypeptides. The rightwards-pointing open arrows indicate points of departure in the primary amino acid sequences of the two alternative Usurpin isoforms, Usurpin-β and Usurpin-γ, which are composed of the Usurpin-α sequence, to the left of the arrows, contiguous with the amino acid sequences indicated at the bottom of the panel. The cDNA sequences and deduced amino acid sequences for Usurpin isoforms have been deposited in GenBank under accession numbers AF015450 (Usurpin-α), AF015451 (Usurpin-β) and AF015452 (Usurpin-γ). The 480 amino acid-long sequence shown as "Usurpin" is Usurpin-α and is SEQ.ID.NO. :4; the 479 amino acid-long sequence shown as caspase-8 is SEQ.ID.NO. :7; the 479 amino acid-long sequence shown as caspase-10 is SEQ.ID.NO.:8; the sequence of Usurpin-β consists of the first 435 amino acids of Usurpin-α plus the 27 amino acids shown. This 462 amino acid-long sequence is SEQ.ID.NO.:5; the sequence of Usurpin-γ consists of the first 264 amino acids of Usurpin-α plus the 28 amino acids shown. This 292 amino acid-long sequence is SEQ.ID.NO. :6.

Figure IB. Representative human and viral "Death Effector Domains" (DEDs) were aligned with the DEDs contained within Usurpin. Amino acids that are identical in at least a third of the sequences are shaded. Leucine periodicity, a feature common to other protei protein interaction domains, is evident in all of the DEDs. The sequence shown as "Usurpin DED-A" is a subset of SEQ.ID.NO. :4; the sequence shown as "Usurpin DED-B" is a subset of SEQ.ID.NO. :4; the sequence shown as "caspase-8 DED-A" is a subset of SEQ.ID.NO. :7; the sequence shown as "caspase-8 DED-B" is a subset of SEQ.ID.NO.:7; the sequence shown as "caspase- 10 DED-A" is a subset of SEQ.ID.NO.:8; the sequence shown as "caspase-10 DED- B" is a subset of SEQ.ID.NO.:8; the sequence shown as "FADD DED" is SEQ.TD.NO..-9; the sequence shown as "MC159 DED-A" is SEQ.ID.NO.: 10; the sequence shown as "MCI 59 DED-B" is SEQ.ID.NO.:! 1; the sequence shown as "E8 DED" is SEQ.ID.NO.: 12; the sequence shown as "KS orfK13 DED-A" is SEQ.ID.NO.: 13; the sequence shown as "KS orfK13 DED-b" is SEQ.ID.NO.: 14.

Figure lC. The second Usurpin DED (DED-B) contains a region with statistically significant homology to the amino terminus of C. elegans CED-4 (27% identity, 40% similarity; this has been noted in other DEDs as well (Bauer et al, 1997)). The sequence shown as "Usurpin" is a subset of SEQ.ID.NO. :4; the sequence shown as "CED-4" is SEQ.ID.NO.: 15.

Figure 2A-B shows the molecular organization of Usurpin.

Figure 2A. Bars indicate the key structural components of DED- containing polypeptides and Usurpin (N-terminus left; C-terminus right). The FADD/MORTl adapter protein contains an N-terminal "Death Effector Domain" (DED) and a C-terminal "Death Domain" (DD). Caspases -8 and -10 proenzymes are formed as larger precursor proteins with an N-terminal prodomain, containing tandem DEDs, plus the large and small subunits which form the active heterodimeric mature enzyme. Separation of these domains during enzyme activation occurs at Asp-X junctions. The large subunit contains the catalytic cysteine residue within a conserved QACxG motif (SEQ.ID.NO.: 16). The small subunit contains substrate specificity determinants, including the RxxxxGSW motif (SEQ.ID.NO.: 17) which contributes residues to both the Si and S4 subsites. The absence of these motifs (as well as others (see text)) in the three Usurpin variants is represented by strikethrough characters. The calculated molecular mass of each Usurpin variant is indicated at the far right. Unshaded regions at the right of the Usurpin-β and Usurpin-γ bars indicate areas of the polypeptides that are not identical to Usurpin-α (see Fig. IA).

Figure 2B. Depicts the arrangement of exons that generate the three variants.

Figure 3A-B shows the tissue distribution of human Usurpin poly(A)⁺ RNA and protein.

Figure 3A. Northern blot analysis. Human fetal multiple-tissue Northern blots were probed for the presence of Usurpin poly(A)+ RNA using a [32p] cDNA probe corresponding to bases 793-1947 of SEQ.ID.NO.: 1 (which encodes

DED-B and both caspase-like subunits) of Usurpin-α. RNA standards are indicated on the right of the resulting autoradiogram, and the predominant Usurpin bands are indicated by the * symbol. Northern blots from adult tissues were analyzed in an identical manner (right side of panel A) and the relative intensity of the bands was estimated, with ++++ indicating the maximum intensity observed. No symbol indicates that Usurpin bands could not be observed. Similar estimates were made for caspase-8 and -10 based on published Northern blots from the same commercial source. Figure 3B. Western blot analysis. Fifty μg of total homogenate protein from the indicated human organs were resolved on 10% SDS-polyacrylamide gels, transferred to nitrocellulose by electroblotting and then probed for the presence of Usuipin immunoreactivity using a rabbit polyclonal antibody raised against recombinant human ΔDED-A Usurpin. The dominant band (indicated by a) corresponds in mass to the Usurpin-α isoform (55.3 kDa) whereas a minor band of lower mass (indicated by b) migrates just below the predicted mass of the Usurpin-γ isoform (33.3 kDa).

Figure 4A-B shows that Usuipin heterodimerization with pro-Caspase- 8 prevents pro-Caspase-8 association with FADD/MORTl. Figure 4A. Binding of [ 5S] DED-containing polypeptides to immobilized Usurpin. Full length Usurpin (i.e., Usurpin-α ) (lanes 3) and a prodomainless construct (Δpro-Usurpin; lanes 4) were C-terminal tagged with the FLAG epitope (DYKDDDDK) (SEQ.ID.NO.:18), expressed in bacteria, and then immobilized on anti-FLAG agarose beads (the "bait"). The beads were combined with reticulocyte lysates containing the indicated [35s] proteins, which were generated by coupled in vitro transcription/translation, in a buffer containing 20 mM Tris/HCl (pH 7.5), 100 mM NaCl, 2 mM EDTA, 0.1% (w/v) Nonidet P-40, 2 mM dithiothreitol, 0.05% (w/v) BSA plus 5% (v/v) glycerol and incubated for 2 hr. at 10°C. The beads were washed 5 times as described in Example 8 and then the harvested [ 5S] proteins were eluted with SDS-containing sample buffer, resolved on SDS-polyacrylamide gels, and visualized by fluorography. Lanes 2 are controls treated in an identical manner except that no Usurpin was present on the beads.

Figure 4B. FADD/MORTl recruitment of [35s]pro-caspase-8 in the presence of Usurpin. FADD/MORTl was C-terminal tagged with the StrepTag streptavidin recognition decapeptide (SAWRHPQFGG) (SEQ.ID.NO.: 19), expressed in bacteria, and then bound to streptavidin-agarose beads. Recombinant Usurpin-α (lanes 3), or versions lacking the first DED (ΔDED-A Usurpin; lanes 4) or the entire prodomain (Δpro-Usurpin; lanes 5), were also generated by expression in bacteria. [35s]pro-Caspase-8 (MACH, FLICE, Mch5) was made by coupled in vitro transcription/translation in rabbit reticulocyte lysates. In the competition format (upper panel), [35s]ρro-caspase-8 and the indicated recombinant Usurpins (25 nM for Usurpin-α (lanes 3), 70 nM for ΔDED-A Usurpin (lanes 4), 150 nM for Δpro-Usurpin (lanes 5)) were pre-mixed for 15 min at 4°C (in buffer containing 50 mM Tris/HCl (pH 8.0), 2 mM EDTA) prior to the addition of immobilized-FADD/MORTl beads. After further incubation for 2 hr at 4°C, the beads were washed 3 times, and then the harvested [ 5S]pro-caspase-8 was eluted with 1 mM d-biotin, resolved on SDS- polyacrylamide gels, and visualized by fluorography. In the displacement format, [ 5s]pro-caspase-8 was pre-bound (for 15 min at 4°C) to the immobilized- FADD/MORTl beads prior to the addition of the indicated Usurpin constructs. After further incubation for 2 hr at 4°C, the amount of [35s]pro-caspase-8 that remained bound to the FADD/MORTl beads was assessed as described for the competition format. Lanes 1 are controls with streptavidin-agarose beads that did not contain FADD/MORTl and lanes 2 are controls showing the binding of [35s]pro-caspase-8 to FADD/MORTl in the absence of Usurpin. The graph shows the ability of varying concentrations of full-length Usurpin to prevent [35s]pro-Caspase-8 (MACH, FLICE, Mch5) binding to immobilized FADD/MORTl using the competition format described above (i.e., equivalent to lane 3 of the upper panel of Figure 4B.

Figure 5A-B shows resistance of Usurpin-α to proteolytic cleavage during apoptosis.

Figure 5 A. Human Jurkat cells in logarithmic growth were transferred to serum-free medium, cultured overnight, and then either treated (+) or not treated (-) with 1 μg/ml of anti-CD95 (Fas/APO-1) monoclonal antibody CH11 for an additional 2 hr. The cells (or apoptotic corpses) were harvested by centrifugation, lysed in buffer containing 0.1 % (w/v) CHAPS, and the resulting cytosol fraction (50 μg protein) was resolved on 10-20% SDS-polyacrylamide gradient gels and then electroblotted to nitrocellulose membranes for Western blot/ECL immunodetection. Polyclonal antibodies raised in rabbits against the recombinant large subunit of human caspase-8 (left panel), the recombinant large subunit of human caspase-3 (center panel) or recombinant human Usurpin-α (lacking the first DED; ΔDED-A Usuipin) (right panel) were used as primary antibodies as indicated. For the caspase-8 immunoblot, p55 indicates the 55 kDa proenzyme and pl7 indicates the position that the mature large subunit migrates (not detected in these blots). For the caspase-3 immunoblot, p32 indicates the migration of the 32 kDa proenzyme and p20, pi 7 are the partially mature and fully mature large subunit, respectively. For the Usuφin immunoblot, a indicates the migration of the Usuφin-α isoform whereas b and b ' denote the intact and cleaved forms of what appears to be the Usuφin-γ isoform. The endogenous levels of pro-caspases-8 and -3 as well as Usuφin-α in untreated, healthy Jurkat cells were estimated by quantitative Western immunoblot analysis. Known quantities of recombinant protein standards (caspase-8, caspase-3, or Usuφin) were resolved on SDS-polyacrylamide gels with whole cell lysates derived from known numbers of cultured Jurkat cells. The immunoblot signal from the standards and the cell lysates was quantified by laser densitometry and used to calculate the amount of the three polypeptides present in the cells. The concentrations were calculated with the assumption that a) the average Jurkat cell volume was 0.5 nl, and b) that the proteins were evenly distributed throughout the cells.

Figure 5B. Usuφin cleavage by CTL-derived granzyme B. Cytosol fractions (50 μg protein) were isolated from untreated, non-apoptotic Jurkat cells then incubated with (+) or without (-) purified human granzyme B (67 ng in 25 μl) for 1 hr at 37°C. The samples were resolved on SDS-polyacrylamide gels and immunoblotted as described for panel A using anti-Usuφin antibody.

Figure 6A-D shows the effect of Usuφin on apoptosis.

Figure 6A. Jurkat cells were co-transfected by electroporation with pcDNA3 :GFP plus the control vector pcDNA3 (open symbols) or with pcDNA3 :GFP plus pcDNA3 containing Usuφin-α (solid symbols). Twelve hours after transfection, the cells were re-isolated and subjected to treatment with anti CD95 (Fas/APO-1) monoclonal antibody CHI 1 (0.5 μg/ml) for the indicated times. Cell death was determined by surface PS (phosphatidylserine) exposure as measured by the binding of biotinylated Annexin V and visualization with streptavidin PE by FACS analysis. Data are expressed as the percentage of the transfected cells (GFP positive; approx. 45%) that were also Annexin V positive.

Figure 6B. Jurkat cells were co-transfected by electroporation with pcDNA3:GFP plus the control vector pcDNA3 (control) or with pcDNA3:GFP plus pcDNA3 containing Usuφin-α (Usuφin). Twelve hours after transfection, the cells were re-isolated and subjected to treatment with anti CD95 (Fas/APO-1) monoclonal antibody CHI 1 (1.0 μg/ml) for 15 hr (columns 1 and 2) or with recombinant TNFa (125 ng/ml) for 48 hr (columns 3 and 4). Cell death was measured by the degree of DNA fragmentation which was assessed by propidium iodide staining of trypsin- permeabilized cells followed by FACS analysis.

Figure 6C. Jurkat cells were co-transfected by electroporation with pcDNA3:GFP plus the indicated expression constructs in pcDNA3. Twelve hours after transfection, the cells were re-isolated and the transfection efficiency (GFP positive) was determined by FACS analysis. The cell cultures were then treated with anti-CD95 (Fas/APO-1) monoclonal antibody CHI 1 (0.5 μg/ml) for 72 hr, after which the number of viable transfected cells (GFP positive, propidium iodide negative) versus the number of viable non- transfected cells in the same culture dish (GFP negative, propidium iodide negative) was determined by Facs analysis. The percentage of the transfected cells that remained viable versus the non-transfected cells which remained viable is shown.

Figure 6D. Stable HeLa cell lines containing either vector alone (open symbols) or vector containing Usuφin-α lacking the first DED (ΔDED-A Usuφin; solid symbols) were generated following calcium phosphate transfection and continuous selection for four weeks with 0.4 mg/ml G418. The cells were then treated with the indicated concentrations of anti-CD95 (Fas/APO-1) monoclonal antibody CHI 1 for 16 hr and survival was measured by the retention of activity that could convert thiazolyl blue tetrazolium Br to the corresponding formazan. Data are presented as a percentage of controls cells that did not receive the antibody.

Figure 7 depicts a model for Usuφin-mediated attenuation of cell death following CD95 (Fas/APO-1) receptor ligation. The trimeric Fas ligand binds to its receptor, resulting in oligomerization at the plasma membrane and recruitment of the FADD/MORTl adapter protein. In the absence of Usuφin (left panel, Figure 7A), FADD/MORTl in turn recruits the caspase-8 proenzyme to form a "death- inducing signaling complex" (DISC). The accumulation of multiple pro-caspase-8 polypeptides at a common site enables enzyme autoactivation, presumably through proenzyme interdigitation and intermolecular proteolysis. The resulting mature caspase-8 becomes liberated from the receptor complex owing to removal of the prodomain which otherwise links it to FADD/MORTl via DED:DED interactions. Mature caspase-8 then proteo lyrically activates effector caspases, such as caspase-3, which subsequently cleave key cellular proteins that collectively manifest the apoptotic phenotype. In the presence of Usuφin (right panel, Figure 7B), pro- caspase-8 is unavailable for recruitment by FADD/MORTl due to heterodimerization with the catalytically non- functional Usuφin polypeptide. Pro-caspase-8 is precluded from homodimerization and concomitant intermolecular proteolysis and therefore cannot launch an apoptotic response.

Figure 8A-B shows the presence of Usuφin in cardiac myocytes except for regions undergoing apoptotic cell death following ischemia/reperfusion injury.

Figure 8 A shows localization of Usuφin in rat heart.

Figure 8B shows localization of Usuφin relative to apoptotic myocyte nuclei. The image is from a section of border region between control, non-ischemic left ventricle (upper left) and a region of the left ventricle that was ischemic for 45 minutes and reperfused for three hours (lower right). In Figure 8A, Usuφin-positive myocytes are represented by the black arrows. The Usuφin-positive immunoreactivity was in non-ischemic, control left ventricle (as well as the entire contra-lateral ventricle; not shown), whereas the region that was Usuφin negative (darker non- fluorescent region) was within the ischemic and reperfused ventricle. In Figure 8B, TUNEL-positive myocyte nuclei are indicated by the white arrows and do not co-localize with Usuφin-positive cells. The width of each panel corresponds to 0.2 mm.

Figure 9 A shows the nucleotide sequence of a cDNA encoding Usuφin-α (SEQ.ID.NO. :1). Figure 9B shows the amino acid sequence of Usuφin-α (SEQ.ID.NO. :4).

Figure 10A shows the nucleotide sequence of a cDNA encoding Usuφin-β (SEQ.ID.NO.:2). Figure 10B shows the amino acid sequence of Usuφin-β (SEQ.ID.NO.:5). Figure 11 A shows the nucleotide sequence of a cDNA encoding

Usuφin-γ (SEQ.ID.NO. :3). Figure 1 IB shows the amino acid sequence of Usuφin-γ (SEQ.ID.NO.:6).

DETAILED DESCRIPTION OF THE INVENTION For the puφoses of this application:

"Substantially free from other proteins" means at least 90%), preferably 95%, and even more preferably 99%, free of other proteins. Thus, a Usuφin protein preparation that is substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, and even more preferably no more than 1%, of non-Usuφin proteins. Whether a given Usuφin protein preparation is substantially free from other proteins can be determined by such conventional techniques of assessing protein purity as, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with appropriate staining methods, e.g., silver staining.

"Substantially free from other nucleic acids" means at least 90%, preferably 95%, and even more preferably 99%, free of other nucleic acids. Thus, a Usuφin DNA preparation that is substantially free from other nucleic acids will contain, as a percent of its total nucleic acid, no more than 10%, preferably no more than 5%, and even more preferably no more than 1 %, of non-Usuφin nucleic acids. Whether a given Usuφin DNA preparation is substantially free from other nucleic acids can be determined by such conventional techniques of assessing nucleic acid purity as, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) combined with appropriate staining methods, e.g. , silver staining. A polypeptide has "substantially the same biological activity" as

Usuφin if that polypeptide can prevent the binding off FADD/MORTl to pro- caspase-8 with an IC50 value of no more than 5 fold greater than the IC50 value of full-length (480 amino acid) Usuφin-α (650 pM) as determined in vitro.

A "conservative amino acid substitution" refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid). A "specific binding interaction" between a Usuφin polypeptide and pro-caspase-8 refers to a binding interaction that has a Kd that is no more than 5-fold greater than the Kd of full-length (480 amino acid) Usuφin-α for pro-caspase-8.

The present invention provides a protein that is an endogenous mammalian regulator of CD95 (Fas/APO-1 )-mediated cell death. This protein, named "Usuφin" (since it usurps caspase-8 function, by precluding recruitment of the caspase-8 proenzyme to the death-signaling complex, and inhibits apoptotic cell death), resembles other DED caspases except that it lacks critical residues that are necessary for substrate recognition, binding, and subsequent caspase proteolytic activity. Usuφin is implicated in the regulation of Fas-mediated cell death and thus is likely to play a role in lymphocyte homeostasis, memory T-cell formation, immune privilege, and tumor immuno-evasion. Accordingly, the Usuφin polypeptides of the present invention will be useful in modulating these processes. In addition, Usuφin appears to play a protective role in cardiac tissue and may do so in other organ systems as well. Usuφin may also regulate the pro-apoptotic signaling of other CD95 (Fas/APO-l)/TNF-Rl-like death receptors.

An emerging theme in the conversion of catalytically dormant caspase proenzymes to their functionally mature counteφarts is a mechanism of auto- activation which is initiated by "facilitated" proenzyme dimerization. In this model, the driving force for protease activation would be the dimerization of caspase proenzymes. This hypothesis is supported by several lines of experimental and physical evidence (reviewed in Nicholson and Thornberry, (1997)). First, when caspase proenzymes are generated at sufficiently high concentrations by overexpression in heterologous systems, such as bacteria, they become proteolytically activated. That this process is autolytic has been confirmed with active site mutants which do not become processed under identical conditions. In the few cases that have been examined, the Asp-X maturation sites are the same as those found in the purified active enzymes from human cells. Similar auto-activation events can be reconstituted in vitro when caspase proenzymes (e.g. caspase- 1 and -3) are concentrated by ultrafiltration or by chromatographic accumulation on anion-exchange beads.

Collectively, these observations demonstrate that proenzyme aggregation is sufficient to initiate caspase self-maturation and that the resulting enzyme is indistinguishable from the enzyme in vivo.

The most compelling evidence linking proenzyme dimerization (or multimerization) events with caspase activation comes from the CD95 (Fas/APO-1) system where ligand-mediated receptor oligomerization at the cell surface leads to the recruitment of multiple caspase-8 proenzymes to a common site (Boldin et al, 1996; Medema et al, 1997; Muzio et al, 1996). The resulting activation of pro-caspase-8 presumably occurs through intermolecular autoproteolysis as it does in vitro. Although other possibilities have not yet been excluded (e.g. co-recruitment of as-yet- unidentified activation proteases), this appears to be a reasonably likely inteφretation of the sequence of events that occur in response to receptor ligation. Supporting evidence that this mechanism is feasible comes from fusion constructs in which the prodomain of caspase-8 was replaced with the FK-506 binding protein (FK506-BP), a protein that dimerizes in the presence of the immunosuppressant FK-506. Cells transfected with this chimera were induced to undergo apoptosis when the protein was dimerized with a cell permeable FK-506 dimer (FK-1012) (Muzio et al, 1998). A third line of evidence indicating the importance of caspase proenzyme dimerization in protease maturation is suggested by the physical arrangement of subunits in the caspase- 1 and -3 three-dimensional structures (Rotonda et al, 1996; Walker et al, 1994; Wilson et al, 1994). Both enzymes exist as [large subunit/small subunit]2 tetramers and the orientation of each functional large subunit/small subunit heterodimer is consistent with them originating from separate polypeptides following the interdigitation of two homologous proenzymes. In this model, the catalytic activity of both enzymes involved in the interdigitated proenzyme dimer would be necessary for the generation of functional, mature caspases. If Usuφin were interdigitated with pro-caspase-8, the large subunit of caspase-8 would be paired with the small subunit of Usuφin, and vice versa, and a proteolytically- competent caspase could not arise from this hybrid.

Together, these features provide an attractive model for the modulation of caspase activation and also account for the attenuation of caspase-8 activation by Usuφin in the CD95 (Fas/APO-1) system. The sensitivity of cells bearing components of this receptor system can be regulated at several levels including, now, the relative abundance of Usuφin. Usuφin heterodimerizes with pro-caspase-8 and prevents i) its recruitment via FADD/MORTl to the CD95 (Fas/APO-1) receptor complex, ii) the homodimerization of pro-caspase-8 which would otherwise lead to autoactivation, and iii) the activation of caspase-8 within the putative interdigitated Usuφin/pro-caspase-8 heterodimer. The presence of Usuφin thus confers resistance to Fas-ligand cell death, a process which plays an important role in memory T cell persistence, resistance of tumors to CTL killing and other key physiological processes which rely on pro-caspase-8 dimerization to launch a cell death response. The importance of Usuφin is exemplified by its presence in cardiac tissue that is protected from apoptosis following ischemia/reperfusion injury and its absence from areas where prominent apoptotic cell death occurs.

The present invention provides recombinant DNA molecules encoding Usuφin. The present invention provides a DNA molecule substantially free from other nucleic acids having the nucleotide sequence of SEQ.ID.NO. :1 and encoding Usuφin-α. (GenBank AF015450). Analysis of SEQ.ID.NO.:! revealed that it contains a 480 amino acid-long open reading frame at positions 505-1,947. Thus, the present invention also provides a DNA molecule substantially free from other nucleic acids having the nucleotide sequence of positions 505-1,947 of SEQ.ID.NO.: 1.

The present invention also provides a DNA molecule substantially free from other nucleic acids having the nucleotide sequence of positions 793-1,947 of SEQ.ID.NO.: 1. Such a DNA molecule encodes a Usuφin polypeptide consisting of amino acids 97-480 of full-length Usuφin-α and represents a Usuφin polypeptide lacking the first (i.e., amino terminal) DED region.

The present invention also provides a DNA molecule substantially free from other nucleic acids having the nucleotide sequence of positions 1,096-1,947 of SEQ.ID.NO. : 1. Such a DNA molecule encodes a Usuφin polypeptide consisting of amino acids 198-480 of full-length Usuφin-α and represents a Usuφin polypeptide lacking the prodomain.

The present invention also provides a DNA molecule substantially free from other nucleic acids having the nucleotide sequence of SEQ.ID.NO. :2 encoding Usuφin-β (GenBank AF015451). Analysis of SEQ.ID.NO. :2 revealed that it contains a 462 amino acid-long open reading frame at positions 1-1,389. Thus, the present invention also provides a DNA molecule substantially free from other nucleic acids having the nucleotide sequence of positions 1-1,389 of SEQ.ID.NO. :2. The present invention also provides a DNA molecule substantially free from other nucleic acids having the nucleotide sequence of SEQ.ID.NO.:3 encoding Usuφin-γ (GenBank AF015452). Analysis of SEQ.ID.NO.:3 revealed that it contains a 292 amino acid-long open reading frame at positions 1-879. Thus, the present invention also provides a DNA molecule substantially free from other nucleic acids having the nucleotide sequence of positions 1-879 of SEQ.ID.NO. :3.

The novel DNA sequences of the present invention encoding Usuφin can be linked with other DNA sequences, i.e., DNA sequences to which Usuφin is not naturally linked, to form "recombinant DNA molecules" containing Usuφin. Such other sequences can include DNA sequences that control transcription or translation such as, e.g., translation initiation sequences, promoters for RNA polymerase II, transcription or translation termination sequences, enhancer sequences, sequences that control replication in microorganisms, or that confer antibiotic resistance. The novel DNA sequences of the present invention can be inserted into vectors such as plasmids, cosmids, viral vectors, or yeast artificial chromosomes. Included in the present invention are DNA sequences that hybridize to SEQ.ID.NO.:l, SEQ.ID.N0.:2, or SEQ.ID.NO.:3 under stringent conditions. By way of example and not limitation, a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA is carried out for 2 hr. to overnight at 65°C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65°C in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X lθ6 cpm of 32p-i_aDeled probe. Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0.1 X SSC, 0.1 % SDS at 50°C for 45 min. before autoradiography.

Other procedures using conditions of high stringency would include either a hybridization carried out in 5XSSC, 5X Denhardt's solution, 50% formamide at 42°C for 12 to 48 hours or a washing step carried out in 0.2X SSPE, 0.2% SDS at 65°C for 30 to 60 minutes. Reagents mentioned in the foregoing procedures for carrying out high stringency hybridization are well known in the art. Details of the composition of these reagents can be found in, e.g., Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press. In addition to the foregoing, other conditions of high stringency which may be used are well known in the art.

Another aspect of the present invention includes host cells that have been engineered to contain and/or express DNA sequences encoding Usuφin. Such recombinant host cells can be cultured under suitable conditions to produce Usuφin. An expression vector containing DNA encoding Usuφin can be used for expression of Usuφin in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which are suitable for recombinant expression of Usuφin and which are commercially available, include but are not limited to, Jurkat cells (ATCC TJJB-152), L cells L-M(TK') (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NTH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS- C-l (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

A variety of mammalian expression vectors can be used to express recombinant Usuφin in mammalian cells. Commercially available mammalian expression vectors which are suitable include, but are not limited to, pMClneo (Stratagene), pSG5 (Stratagene), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1 (Invitrogen), EBO-pSV2-neo (ATCC 37593), pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), and pSV2-dhfr (ATCC 37146). Following expression in recombinant cells, Usuφin can be purified by conventional techniques to a level that is substantially free from other proteins.

Accordingly, the present invention includes Usuφin protein substantially free from other proteins. The amino acid sequence of the full-length 480 amino acid-long Usuφin protein is given in SEQ.ID.NO. :4 and is shown in Figure 1 A. This 480 amino acid-long Usuφin protein is known as Usuφin-α. Thus, the present invention includes Usuφin protein substantially free from other proteins having the amino acid sequence SEQ.ID.NO. :4.

The present invention also includes a Usuφin-β protein substantially free from other proteins having the amino acid sequence SEQ.ID.NO. :5. The present invention also includes a Usuφin-γ protein substantially free from other proteins having the amino acid sequence SEQ.ID.NO. :6.

The present invention also includes polypeptides having the amino acid sequence of positions 97-480 of SEQ.ID.NO. :4. The present invention also includes polypeptides having the amino acid sequence of positions 198-480 of SEQ.ID.NO. :4. The present invention also includes polypeptides having the amino acid sequence MetAla followed by positions 198-480 of SEQ.ID.NO.:4.

The present invention also includes polypeptides comprising the amino acid sequence of positions 2-73 of SEQ.ID.NO. :4, as well as DNA encoding positions 2-73 of SEQ.ID.NO. :4. Such polypeptides comprise the DED-A domain of Usuφin- α.

The present invention also includes polypeptides comprising the amino acid sequence of positions 93-170 of SEQ.ID.NO. :4, as well as DNA encoding positions 93-170 of SEQ.ID.NO. :4. Such polypeptides comprise the DED-B domain ofUsuφin-α. It will be recognized by those of skill in the art that the polypeptides of the present invention may have short amino acid sequences appended to their amino or carboxy-termini in order to aid in, e.g., purification or immunological detection of the polypeptides. Such amino acid "tags" are, e.g., the FLAG epitope tag (DYKDDDDK) (SEQ.ID.NO. : 18), or the StrepTag streptavidin recognition decapeptide (SAWRHPQFGG) (SEQ.ID.NO.: 19).

As with many proteins, it is possible to modify many of the amino acids of Usuφin and still retain substantially the same biological activity in the modified protein as in the original protein. Thus this invention includes modified Usuφin polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as Usuφin. It is generally accepted that single amino acid substitutions do not usually alter the biological activity of a protein (see, e.g., Molecular Biology of the Gene, Watson et al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co., Inc., page 226; and Cunningham & Wells, 1989, Science 244:1081-1085). Accordingly, the present invention includes polypeptides where one amino acid substitution has been made in SEQ.ID.NO.:4, SEQ.JD.NO.:5, SEQ.ID.NO.:6, positions 97-480 of SEQ.ID.NO. :4, or positions 198-480 of SEQ.ID.NO. :4 wherein the polypeptides still retain substantially the same biological activity as the unmodified polypeptide. The present invention also includes polypeptides where two or more amino acid substitutions have been made in SEQ.ID.NO.:4, SEQ.JD.NO.:5, SEQ.ED.NO.:6, positions 97-480 of SEQ.ID.NO. :4, or positions 198-480 of SEQ.ID.NO. :4 wherein the polypeptides still retain substantially the same biological activity as the unmodified polypeptide. In particular, the present invention includes embodiments where the above-described substitutions are conservative substitutions.

The present invention includes methods for the identification of inhibitors of the interaction between Usuφin and pro-caspase-8. Such methods comprise:

(a) providing a Usuφin polypeptide; (b) providing pro-caspase-8 ; under conditions such that a specific binding interaction between the Usuφin polypeptide and pro-caspase-8 will occur in the absence of an inhibitor of the interaction between Usuφin and pro-caspase-8; and measuring the amount of specific binding interaction between the Usuφin polypeptide and pro-caspase-8 in the presence and in the absence of a substance suspected of being an inhibitor of the specific binding interaction between Usuφin and pro-caspase-8; where a decrease in the amount of specific binding interaction between

Usuφin and pro-caspase-8 in the presence of the substance as compared to the absence of the substance indicates that the substance is an inhibitor of the interaction between Usuφin and pro-caspase-8.

In a particular embodiment of the above-described method, the Usuφin polypeptide has an amino acid sequence selected from the group consisting of: SEQ.ID.NO.:4, SEQ.JD.NO.:5, SEQ.ΓD.NO.:6, positions 97-480 of SEQ.ID.NO.:4, positions 198-480 of SEQ.ID.NO. :4, and the amino acid sequence MetAla followed by positions 198-480 of SEQ.ID.NO. :4.

In a particular embodiment of the above-described method, pro- caspase-8 has an amino acid sequence shown in SEQ.ID.NO. :7.

In particular embodiments of the above-described method, the method is practiced in vitro and the conditions are conditions that are typically used in the art for the study of protein-protein interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS; a temperature of about 4°C to about 55°C. The presence of commonly used non-ionic detergents, e.g., NP-40®, sarcosyl, Triton X-100®, is optional. Other suitable conditions include, e.g., 20 mM Tris/HCl (pH 7.5), 100 mM NaCl, 2 mM EDTA, 0.1% (w/v) Nonidet P- 40, 2 mM dithiothreitol, 0.05% (w/v) BSA plus 5% (v/v) glycerol; or 50 mM Tris/HCl (pH 8.0), 2 mM EDTA. In particular embodiments of the above-described method, the method is practiced in vitro and either the Usuφin polypeptide or pro-caspase-8 is labeled, e.g., enzymatically, radioactively, or the like, and either the Usuφin polypeptide or pro-caspase-8 is immobilized, e.g., by being bound to a solid phase such as agarose beads or the wells of a tissue culture plate. In embodiments of this type, the specific binding interaction between the Usuφin polypeptide and pro-caspase-8 is measured by contacting a labeled Usuφin polypeptide in solution with pro-caspase-8 bound to a solid phase and determining the amount of labeled Usuφin polypeptide that is bound to the solid phase. Alternatively, the pro-caspase-8 could be labeled and would therefore be in solution and the Usuφin polypeptide could be bound to a solid phase. In other embodiments of the above-described method, the method is practiced in vivo, i.e., in living cells, and either the Usuφin polypeptide, pro-caspase- 8, or both, are introduced into the cells by, e.g., transfection of an expression vector that directs the production of the Usuφin polypeptide or pro-caspase-8, or both, in the cells. In such embodiments, the specific binding interaction between the Usuφin polypeptide and pro-caspase-8 is measured by measuring the amount of apoptosis that the cells undergo. In the absence of an inhibitor of the specific binding interaction, the specific binding interaction between the Usuφin polypeptide and pro-caspase-8 will take place and apoptosis will not occur or will be limited. In the presence of inhibitor, the specific binding interaction between the Usuφin polypeptide and pro- caspase-8 will not take place, or will take place to a lesser extent, and apoptosis will be correspondingly greater.

Accordingly, the present invention includes methods for the identification of inhibitors of the interaction between Usuφin and pro-caspase-8 where such methods comprise:

(a) providing a cell that contains a Usuφin polypeptide and pro- caspase-8;

(b) contacting the cell with an apoptotic stimulus in the presence and in the absence of a substance suspected of being an inhibitor of the specific binding interaction between Usuφin and pro-caspase-8; and

(c) measuring the amount of apoptosis the cells undergo in the presence and in the absence of the substance; where an increase in the amount of apoptosis measured in the presence of the substance indicates that the substance is an inhibitor of the specific binding interaction between Usuφin and pro-caspase-8.

Of course, if the above-described method were practiced and it were determined that the amount of apoptosis increased in the presence of the substance, then that substance would be a possible activator of the specific binding interaction between Usuφin and pro-caspase-8. Such activators would be useful in the modulation of apoptosis.

It is widely believed in the art that aberrant apoptosis plays a key role in various diseases, (e.g., autoimmune diabetes, cancer, Parkinson's disease; see Hetts, 1998) and that therefore the modulation of apoptosis is likely to be useful in the treatment of such diseases. Accordingly, activators and inhibitors of the interaction between Usuφin and pro-caspase-8 are likely to be useful in the treatment of disease.

In a particular embodiment of the above-described method, the Usuφin polypeptide has an amino acid sequence selected from the group consisting of: SEQ.ID.NO. :4, SEQ.ID.NO. :5, SEQ.ID.NO. :6, positions 97-480 of

SEQ.ID.NO. :4, positions 198-480 of SEQ.ID.NO. :4, and the amino acid sequence MetAla followed by positions 198-480 of SEQ.ID.NO. :4.

In a particular embodiment of the above-described method, pro- caspase-8 has an amino acid sequence shown in SEQ.ID.NO.:7. In particular embodiments of the above-described method, the cells are selected from the group consisting of Jurkat cells (ATCC TIB-152, L cells L-M(TK^') (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 cells (ATCC CRL 1573), Raji cells (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 cells (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

An apoptotic stimulus can be delivered by treating the cells with, e.g., an anti-CD95 (Fas/APO-1) monoclonal antibody, e.g., CHI 1, or the Fas ligand.

Apoptosis can be measured by, e.g. , measuring the increase in surface PS (phosphatidylserine) exposure accompanying apoptosis as measured by the binding of biotinylated Annexin V and visualization with streptavidin PE by Facs analysis; measuring by the degree of DNA fragmentation by propidium iodide staining of trypsin-permeabilized cells followed by FACS analysis; or measuring the retention of activity in the cells that can convert thiazolyl blue tetrazolium Br to the corresponding formazan.

The present invention includes methods of inhibiting apoptosis by introducing into cells a Usuφin polypeptide selected from the group consisting of: SEQ.ID.NO.:4, SEQ.ID.N0.:5, SEQ.ID.NO.^, positions 97-480 of SEQ.ID.NO. :4, positions 198-480 of SEQ.ID.NO. :4, and the amino acid sequence MetAla followed by positions 198-480 of SEQ.ID.NO. :4. In preferred embodiments, the method involves the introduction of the Usuφin polypeptide into the cells by transfection of the cells with an expression vector encoding the Usuφin polypeptide. In certain embodiments, the expression vector is a viral vector. In other embodiments, the viral vector is a retroviral vector. In preferred embodiments, the cells are mammalian cells, preferably human cells. In certain embodiments, the expression vector comprises a DNA sequence selected from the group consisting of: SEQ.ID.NO.: 1, SEQ.ID.NO.:2, SEQ.ID.NO. :3, positions 505-1,947 of SEQ.ID.NO.: 1, positions 793-1,947 of SEQ.ED.NO.:l, and positions 1,096-1,947 of SEQ.ID.NO.: 1. In certain embodiments, the expression vector comprises a DNA sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ.ID.NO. :4, SEQ.ID.NO.:5, SEQ.ID.NO.:6, positions 97-480 of SEQ.ID.NO. :4, positions 198-480 of SEQ.ID.NO. :4, and the amino acid sequence MetAla followed by positions 198- 480 of SEQ.ID.NO. :4. The present invention also includes antibodies to the Usuφin protein.

Such antibodies may be polyclonal antibodies or monoclonal antibodies. The antibodies of the present invention are raised against the entire Usuφin protein or against suitable antigenic fragments of the protein that are coupled to suitable carriers, e.g., serum albumin or keyhole limpet hemocyanin, by methods well known in the art. Methods of identifying suitable antigenic fragments of a protein are known in the art. See, e.g., Hopp & Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824-3828; and Jameson & Wolf, 1988, CABIOS (Computer Applications in the Biosciences) 4:181- 186.

For the production of polyclonal antibodies, Usuφin protein or an antigenic fragment, coupled to a suitable carrier, is injected on a periodic basis into an appropriate non-human host animal such as, e.g., rabbits, sheep, goats, rats, mice. The animals are bled periodically and sera obtained are tested for the presence of antibodies to the injected antigen. The injections can be intramuscular, intraperitoneal, subcutaneous, and the like, and can be accompanied with adjuvant. For the production of monoclonal antibodies, Usuφin protein or an antigenic fragment, coupled to a suitable carrier, is injected into an appropriate non- human host animal as above for the production of polyclonal antibodies. In the case of monoclonal antibodies, the animal is generally a mouse. The animal's spleen cells are then immortalized, often by fusion with a myeloma cell, as described in Kohler & Milstein, 1975, Nature 256:495-497. For a fuller description of the production of monoclonal antibodies, see Antibodies: A Laboratory Manual Harlow & Lane, eds., Cold Spring Harbor Laboratory Press, 1988.

The following non-limiting examples are presented to better illustrate the invention. EXAMPLE 1

Cloning of Usuφin and Splice Variants

Unless otherwise indicated, all nucleotide numbering refers to the Usuφin-α isoform, GenBank accession AF015450. cDNA clones encoding Usuφin polypeptides were generated from brain, liver, spleen and placenta Marathon-ready cDNA templates (Clontech) by 5' and 3' RACE amplification using as primers the reverse-complement of bp 1093-1114 (5'-GGA GCC TGA AGT TAT TTG AAG G- 3') (SEQ.ID.NO. :20) plus the API primer (Clontech) in the primary PCR amplification reaction with Taq polymerase (Boehringer Mannheim), then the reverse-complement of bp 1067-1090 (5'-CCT TGA GAC TCT TTT GGA TTG CTG-3') (SEQ.ID.NO. :21) plus the AP2 primer in the nested secondary reaction. Amplification products were ligated into the vector pCR 2.1 by TA cloning (Invitrogen) and then sequenced. The longest 5' RACE products extended to bp 735, which was sufficient to encode ΔDED-A Usuφin (using Mef97 as a start codon) but did not include the first "death-effector domain." 3' RACE amplification was performed to attempt to recover functionally-competent splice variants if they were to exist. The primary 3' RACE PCR amplification of the same Marathon-ready cDNAs as described above was performed using the primer 5'-CAA GTT AC A GGA ATG TTC TCC AAG-3' (bp 1043-1066) (SEQ.ID.NO.:22) plus API, then the nested primer 5'-CAG CAA TCC AAA AGA GTC TCA AG-3' (bp 1067-1089) (SEQ.ID.NO. :23) plus AP2 in the secondary reaction. Amplification products were ligated into the vector pCR 2.1 by TA cloning (Invitrogen) and then sequenced, but no clones were revealed containing putatively functional caspase sequences. Full length cDNA clones of Usuφin (Usuφin-α) and splice variants (Usuφin-β and -γ) were identified by screening a human fetal kidney, lambda g/11 cDNA library (Clontech 5' StretchPlus) on nylon membranes (Colony/Plaque Screen Hybridization membranes, NEN). The probe for library screening was generated by reverse-transcriptase PCR (corresponding to bp 792-1947 which encodes Mef97 . Thr480 of Usuφin-α) and was radiolabeled using [α-32p]dATP (RediPrime, Amersham). Hybridization was conducted overnight at 65°C in ExpressHyb solution (Clontech). Positive clones, identified by autoradiography after multiple rounds of dilution subcloning, were sequenced by PCR amplification of the phage inserts followed by direct DNA sequencing of the amplification products. Multiple clones of the three splice variants described herein were identified by this procedure (GenBank accession numbers AF015450 (Usuφin-α), AF015451 (Usuφin-β) and AF015452 (Usuφin-γ)). The absence of a splice variant that could encode a caspase with functional substrate binding and catalytic determinants was verified by sequencing of a genomic Usuφin clone.

EXAMPLE 2

Molecular Organization of Usuφin and Absence of Caspase Catalytic Determinants

The Usuφin polypeptide contains both functionally important similarities and differences when compared to the two known DED caspase proenzymes (Fig. 1). First, all three contain a long prodomain (approximately 200 amino acids) that harbors tandem "death effector domains" (Fig. IB). Second, the prodomains are followed by caspase protease regions which, in each case, contain both large and small subunit counteφarts. Third, the P4-P1 tetrapeptide motif at the activation cleavage sites that separate the large subunits from the small subunits (Fig. 1 A, box d) perfectly correspond to the substrate recognition sequence for group III caspases ([P4](I,V,L)ExD[P ]). Finally, the high overall primary sequence homology between these three polypeptides substantiates their molecular relatedness.

In contrast to caspases-8 and -10, however, Usuφin lacks key features that are necessary for substrate binding and catalysis (Fig. 1 A). First, the proteolytic machinery of all caspases requires a catalytic diad composed of the sulfhydryl group of Cys[ICE:285] and the imidazole of ffi_s[ICE:237]. The counteφarts of both of these residues in Usuφin are substituted with other amino acids (Tyr360 (Fig. 1 A, box c) and Arg31 (box b), respectively) that would render Usuφin non- functional as a protease. Second, the QACxG (SEQ.ID.NO. : 16) pentapeptide motif containing the catalytic Cys residue (box c), that is conserved in all functional caspase family members across species, contains only the Gin residue (QNYVV) (SEQ.ID.NO.:35). Third, all caspases have a strict requirement for Asp in P and this near absolute specificity is conferred by four residues, two from each subunit, which stabilize the Asp carboxylate side chain within the S subsite (Arg[ICE:179]_j Gln[ICE:283]₅ Arg[ICE:341] and Ser[ICE:347]). in Usuφin, both of the Arg residues are substituted with uncharged alternates (Cys259_s box a and Gln415₅ box e) whereas the Gln[ICE:283] and Ser[ICE:347] counteφarts (Gln358, box c and Ser421, box d, respectively) remain unaltered. Overall, these changes would affect both the chemical nature (loss of 3 hydrogen bond donors) and geometry of the Si subsite, resulting in a predicted loss of affinity for Pi Asp.

Collectively, these four key amino acid changes should render Usuφin catalytically incompetent. This was confirmed experimentally by two approaches: i) recombinant Usuφin was unable to cleave tetrapeptide-aminomethylcoumarin substrates; and ii) Usuφin that was overexpressed in bacteria under an inducible

(LacZ) promoter (either with or without the prodomain) did not undergo autocatalytic maturation as is normally observed with functional caspase proenzymes. Furthermore, catalytic activity could not be restored by retro-mutation of these residues in Usuφin to their counteφarts in functional caspases (Usuφin:C259R; R315H; QNYVV358-362QACQG; Q415R), indicating that other important catalytic determinants are also lacking in Usuφin.

EXAMPLE 3

Usuφin Tissue Distribution

Consistent with the multiple cDNA variants found by library screening, Northern blot analysis indicated that Usuφin is expressed as multiple poly(A)⁺ transcripts in both fetal (Fig. 3A) and adult tissues (data not shown). Three major transcripts (6, 7 and 10 Kb) as well as less abundant smaller transcripts were observed in a variety of human organs. Usuφin transcripts were particularly abundant in cardiac and skeletal muscle, differing from the distribution of caspase-8 and -10 transcripts (Boldin et al, 1996; Fernandes-Alnemri et al, 1996; Muzio et al, 1996; Vincenz and Dixit, 1997). Western blot analysis of human tissue homogenates confirmed the abundance of Usuφin polypeptides in heart and skeletal muscle as well as its presence in other organs with the exception of colon, placenta, and testis (Fig. 3B). Although Usuφin transcripts could not be detected in brain by Northern blot analysis, the protein was clearly detectable on Western blots. The 55.3 kDa Usuφin- α isoform was the dominant polypeptide in all organs containing Usuφin, with the exception of spleen. A smaller protein, corresponding approximately in mass to the 33.3 kDa Usuφin-γ isoform, was present with a highly restricted distribution in lymphoid tissues (thymus, lymph node, and spleen). Usuφin-β polypeptides (52.6 kDa), on the other hand, could not be detected in any of the tissues tested.

EXAMPLE 4

Usuφin Binds pro-Caspase-8 and Prevents its Association with FADD/MORTl

Death effector domains (DEDs) have been shown to modulate intermolecular dimerization events through DEDDED associations. In the case of the CD95 (Fas/APO-1) system, the DEDs contained within the prodomain of pro- caspase-8 bind to the DED contained within the FADD/MORTl adapter protein and this facilitates the recruitment of pro-caspase-8 to the CD95 (Fas/APO-1) receptor complex where caspase-8 is subsequently activated and launches the cell death pathway. The two DEDs contained within Usuφin might also participate in these interactions. Caspases also homodimerize. This may in some cases be mediated by prodomains (e.g., ICE/caspase-1) (Van Criekinge et al, 1996), but the catalytic subunits of the enzymes can also associate, as demonstrated by the three-dimensional arrangement of caspases- 1 and 3 (Rotonda et al, 1996; Walker et al, 1994; Wilson et al, 1994). In both cases, two large subunits and two small subunits are intimately associated in a compact tetramer comprised of two large subunit/small subunit heterodimers. This arrangement suggests that mature caspase tetramers arise from the association of two homologous caspase proenzymes. Heterologous association of different caspase family members has not been shown to occur in vivo, suggesting that stringent structural features disfavor non-homologous proenzyme interactions. It is possible, however, that highly related caspase-like polypeptides, such as Usuφin, sufficiently mimic these determinants enabling association with other proteins such as pro-caspase-8. These associations were examined using an in vitro binding assay where FLAG-epitope tagged recombinant Usuφin-α, or a prodomainless version (Δpro-Usuφin), was immuno-immobilized on agarose beads and used as bait to harvest [35S] proteins generated by coupled in vitro transcription translation (Fig. 4A). Usuφin-α was able to homodimerize in vitro and this interaction occurred in both the presence and absence of the DED-containing prodomain (Fig. 4A, first and second panels). Removal of the prodomain from the recombinant bait protein slightly decreased the efficiency of harvesting of [35s]Usuφin but improved the efficiency of harvesting of [35s]Δpro-Usuφin (lanes 3 versus 4). These results demonstrate that determinants from both the prodomain and the catalytically non- functional caspase component of Usuφin contribute to homodimerization, and that the caspase-like component, without the prodomain, is itself sufficient for homodimerization.

Similar interactions were observed with the association of Usuφin-α and pro-caspase-8 (Fig. 4A, third panel). Both full-length Usuφin-α and Δpro- Usuφin could harvest [35s]pro-caspase-8, with the binding efficiency in the former case (in the presence of the prodomain) being marginally better in vitro than in the absence of the prodomain. This demonstrates that Usuφin can bind pro-caspase-8 in vitro, that this heterodimerization is most efficient with intact Usuφin containing both the prodomain and caspase-like components, and that the caspase-like domain of Usuφin is alone sufficient for heterodimerization with the caspase-8 proenzyme. Despite the presence of two DEDs within the Usuφin prodomain,

Usuφin did not interact with the FADD/MORTl adapter protein in vitro (Fig. 4 A, bottom panel). This indicates, at least within the CD95 (Fas/APO-1) receptor system, that Usuφin interactions are confined to pro-caspase-8 but probably do not include FADD/MORTl. Because of the ability of Usuφin to bind pro-caspase-8, but not

FADD/MORTl, Usuφin is likely to interfere with pro-caspase-8 recruitment by FADD/MORTl to the CD95 (Fas/APO-1) receptor complex. This was tested by determining whether Usuφin affected FADD/MORTl :pro-caspase- 8 association in vitro (Fig. 4B). StrepTag-FADD/MORTl was immobilized on streptavidin agarose and then used to harvest [ 5s]pro-caspase-8 in the absence or presence of various Usuφin constructs. In a competition assay (Fig. 4B, upper panel), Usuφin-α effectively inhibited the ability of FADD/MORTl to harvest [35s]pro-caspase-8 (83%> inhibition, lane 3 versus 2; IC50 value = 650 pM, titration depicted in graph) whereas neither Usuφin lacking the first DED (ΔDED-A Usuφin, lane 4) nor prodomainless Usuφin (Δpro-Usuφin, lane 5) were able to. In a displacement assay, where the FADD/MORTl : [35S]pro-caspase-8 interaction was allowed to form prior to the addition of Usuφin-α (Fig. 4B lower panel), Usuφin-α was less effective at inhibiting their association (60% inhibition; lane 3 versus 2). Collectively, these experiments demonstrate that Usuφin precludes pro-caspase-8 association with the FADD/MORTl adapter protein, but that once an interaction is formed between these polypeptides, it is relatively resistant to Usuφin interference.

EXAMPLE 5

Usuφin Attenuates Cell Death Resulting from CD95 (Fas/APO-1) Receptor Ligation

Jurkat T lymphocytes are a well established cell line that is responsive to Fas ligand-mediated apoptosis. Jurkats contain multiple caspase family members including the group III activator protease, caspase-8 (17 pM), the group II apoptosis- effector protease, caspase-3 (86 pM), as well as Usuφin (6 pM) (Fig. 5A). When induced to undergo apoptosis by CD95 (Fas/APO-1) receptor ligation, all of the caspase-8 proenzyme was processed from its 55 kDa (p55) precursor form (Fig. 5A, left panel; concomitant appearance of the mature form (pi 7) was not detected by immunoblotting owing to putative high turnover of the mature form, a feature common to several caspases following activation). Similarly, more than 60% of the 32 kDa caspase-3 proenzyme (p32) was converted to the catalytically active mature forms (Fig. 5 A, middle panel; p20 and pi 7 being the large subunit with or without the 28 amino acid prodomain, respectively). The full length Usuφin-α isoform, however, was unaffected during apoptosis (Fig. 5A, right panel, band a). In contrast, a faster- migrating Usuφin-γ band was present in apoptotic extracts (band b and b ' putatively being Usuφin-γ and its cleaved form, respectively). A similar faster-migrating

Usuφin-γ band was generated when healthy Jurkat cell extracts were incubated with granzyme B (Fig. 5B). Based on fragment sizes, the cleavage site within the smaller Usuφin-γ polypeptide was predicted to be LITD258/c259_{5 a} consensus site for group III caspases, including caspase-8, as well as for granzyme B (Thornberry et al., 1997). These experiments demonstrate that the endogenous levels of Usuφin-α in Jurkat cells (6 pM) are insufficient to block caspase-8 activation and subsequent caspase-3 mediated apoptosis. (This is consistent with the concentration of Usuφin required in vitro to inhibit pro-caspase-8 association with FADD/MORTl (IC50 = 650 pM; see above and Fig. 4B).) Furthermore, since Usuφin-α contains several excellent caspase consensus sites which do not become cleaved during apoptosis (e.g. LITD258/C259 _at the prodomain/large subunit counteφart junction, and LEVD376/G377 _at the large subunit/small subunit counteφart junction), it is possible that homodimerization masks them from caspase proteolysis. The shorter Usuφin-γ isoform, which cannot benefit from homodimerization endowed by the caspase-like component of Usuφin- α, is probably monomeric in Jurkat cells and thus susceptible to cleavage.

Despite the presence of Usuφin (albeit at lower molar concentration versus pro-caspase-8), Jurkat cells are clearly sensitive to CD95 (Fas/APO-1) - mediated cell death. To determine whether elevated levels of Usuφin would favor Usuφin heterodimerization with pro-caspase-8 (as demonstrated above in vitro) and prevent CD95 (Fas/APO-1 )-mediated cell death, Usuφin-α was transfected into healthy Jurkat cells by electroporation then treated with the CD95 cross-linking antibody, CHI 1 (Fig. 6A). Cells harboring Usuφin-α (indicated by co-transfection with Aequorea victoria green fluorescent protein) were substantially resistant to CD95 ligation-induced apoptosis. In contrast, Usuφin-α transfected cells remained susceptible to TNFa-induced cell death (Fig. 6B). Similar results were observed when apoptosis was induced by either caspase-8 or FADD/MORTl overexpression followed by CD95 (Fas/APO-1)- ligation (Fig. 6C). In these cases, the presence of a Usuφin construct lacking the first 'death effector domain' (ΔDED-A Usuφin) was still sufficient to afford protection against cell death initiated by these proteins. Although the presence of Usuφin DED-A appears to be important for preventing pro- caspase-8 recruitment to FADD/MORTl in vitro (as described above in Fig. 4B), ΔDED-A Usuφin was still able to impede the activation of caspase-8 and resulting cell death. This was also observed in a stable HeLa cell line bearing ΔDED-A Usuφin (Fig. 6D) which was also resistant to cell death following CD95 (Fas/APO-1) ligation.

Collectively, these results demonstrate that Usuφin is able to attenuate cell death mediated by caspase-8 activation, such as that which normally occurs following ligation of CD95 (Fas/APO-1). The molar ratio of Usuφin versus that of caspase-8 proenzyme probably plays a critical role in determining whether Usuφin homodimerization is favored and caspase-8 activation can occur unabated, or whether sufficient Usuφin is present to heterodimerize with pro-caspase-8 and preclude both its recruitment to the CD95 (Fas/APO-1) receptor complex and its autolytic activation (Fig- 7). EXAMPLE 6

Usuφin Deficit in Cardiac Myocytes Undergoing Apoptosis Following Ischemia/Reperfusion Injury in vivo

Owing to the prominence of Usuφin in cardiac tissue, its distribution was examined in rat heart before and after ischemia/reperfusion injury which has recently been shown to result in cardiac myocyte apoptosis (Gottlieb et al, 1994; Kajstura et al, 1996; Sharov et al, 1996) and concomitant regional up-regulation of caspase-3 (apopain, CPP32, Yama) expression levels (Black et al., 1998). Usuφin (represented by the black arrows in Fig. 8 A) was present in the non-ischemic, control region of the heart, while there was limited Usuφin immunoreactivity in the ischemic and reperfused region of the heart. Usuφin immunoreactivity was uniformly distributed in all non-ischemic regions of the rat heart, including the right ventricular free wall and the interventricular septal region of the left ventricle (data not shown). To determine the possible relationship between ischemia/reperfusion-induced apoptosis and cardiac Usuφin localization, co-localization studies with the Usuφin antibody and TUNEL positive nuclei were performed (Fig. 8B). Myocytes that were TUNEL positive, and thus presenting nuclear changes associated with the apoptotic phenotype, were evident in the region of the infarcted tissue where there was a clear attenuation in the Usuφin signal. In contrast, there were no TUNEL-positive myocyte nuclei in the non-ischemic right ventricle and the interventricular septal region where Usuφin immunoreactivity was high. The distribution of Usuφin immunoreactivity was the reciprocal of that of the apoptosis effector protease, caspase-3, which was abundant in the TUNEL-positive infarcted tissue and low in unaffected regions of the heart (not shown). Collectively these data suggest that the vulnerability of cardiac myocytes to apoptotic cell death following ischemia/reperfusion injury may result from the simultaneous gain of the pro- apoptotic protease, caspase-3, and loss of the protective contribution of Usuφin. EXAMPLE 7

Usuφin engineering and expression

All variants of Usuφin were engineered by PCR-directed template modification using appropriate full-length clones as templates and Pwo polymerase (Boehringer Mannheim) for the amplification reaction. Once ligated into appropriate vectors, all clones were fully sequenced to ensure that no errors were introduced by the PCR reaction. Clone designations below are in the following format: [construct] : [vector] : [insert site] : [sense orientation] : [identifier] . Constructs for in vitro transcription translation: Full length Usuφin-α, ΔDED-A Usuφin and Δpro-Usuφin fragments were generated using synthetic oligonucleotide primers containing flanking, nested restriction sites suitable for ligation into various commercial vectors. All three clones were made as Xbal-Xhol-Ncol-(Usuτpin construct)- Ncol- Xhol- Xbal fragments that were subsequently purified from preparative agarose gels, trimmed with Xbal (Boehringer Mannheim) then ligated into the Xbal site of pBluescript II SK+ and transformed into E. coli XL2-blue cells

(Stratagene). Amplimers for the full-length Usuφin-α construct were 5'-GCG GAT CCT CTA GAC TCG AGC CAT GGC TGC TGA AGT CAT CCA TCA GGT TGA AGA AGC AC-3' (forward amplimer) (SEQ.ID.NO.:24) and 5'-GCT CTA GAC TCG AGC CAT GGT TAT GTG TAG GAG AGG ATA AGT TTC-3' (reverse amplimer) (SEQ.ID.NO.:25) resulting in the construct designated [Usuφin, Met - Thr480];[_pBII SK+]:[ ^bαI]:[T7]:[MF-UMP#4556]. Amplimers for ΔDED-A Usuφin were 5'-GCT CTA GAC TCG AGC CAT GGC AGA GAT TGG TGA GGA TTT GG-3' (SEQ.JD.NO.:26) and the reverse amplimer described above, resulting in the construct designated [ΔDED-A Usuφin, Met97-Thr480]_:[_pBII SK+]:[ føzI]:[T7]:[MF-UMP#3712]. Amplimers for Δpro-Usuφin were 5'-GCT CTA GAC TCG AGC CAT GGC TTC AAA TAA CTT CAG GCT CCA TAA TGG-3' (SEQ.ID.NO. :27) and the reverse amplimer described above, resulting in the construct designated [Δpro-Usuφin, MetAlaSerl98-Thr480]_:[_pBπ SK+]:[ -YbαI]:[T7]:[MF-UMP#4225]. A similar approach was used to generate pro-caspase-8 (MACH/FLICE/Mch5) beginning with the amplimer 5 '-GCT CTA GAG GAT CCA TGG ACT TCA GCA GAA ATC TTT ATG ATA TTG-3' (forward amplimer) (SEQ.ID.NO.:28) plus 5'-GCT CTA GAG GAT CCA CAT GTT CAA TCA GAA GGG AAG ACA AGT TTT TTT CTT AG-3' (reverse amplimer) (SEQ.ID.NO. :29) and resulting in the construct [pro-Caspase-8, Met -Asp479]_:[ Xbal]:[T7]:[MF- UMP#4091]. For the generation of radiolabeled polypeptides, purified plasmid DNA (Qiagen) was used to drive transcription (T7 polymerase) and coupled translation in rabbit reticulocyte lysates (Promega TnT) in the presence of [35]methionine (>1000 Ci/mmol, Amersham).

Constructs for expression in bacteria: Full length Usuφin-α, ΔDED-A Usuφin and Δpro-Usuφin constructs that were generated for recombinant expression in bacteria were identical to those described above except that each contained the FLAG epitope tag (DYKDDDDK) (SEQ.ID.NO. : 18) at the carboxy terminus for purification and for immuno-immobilization in harvesting experiments. The forward amplimers in each case were identical to those described in the preceding section whereas a common reverse amplimer was used for all three constructs to introduce the FLAG epitope and the nested restriction sites (5'-CGT CTA GAC CAT GGT CAC TTG TCA TCG TCG TCC TTG TAG TCT GTG TAG GAG AGG ATA AGT TT-3') (SEQ.ID.NO. :30). All three clones were made as .¥bαI-Λ7?oI-NcøI-(Usuφin construct-FLAG)- Ncol- Xhol- Xbal fragments that were subsequently purified from preparative agarose gels, trimmed with Xbal (Boehringer Mannheim) then ligated into the Xbal site of pBluescript II SK+ and transformed into E. coli XL2-blue cells (Stratagene). After sequence verification, the constructs were excised with Ncol then ligated into the

Ncol site of pETl Id and transformed into BL21(DE3)pLysS E. Coli cells (Novagen) after orientation and sequence re-verification. The resulting constructs were [Usuφin (Metl-Thr480)_FLAG]:[pETl ld]:[ NcoI]:[T7/lac]:[MF-UMP#4711], [ΔDED-A Usuφin (Met97-Thr480)-FLAG]:[pETl ld]:[ NcoI]:[T7/lac]:[MF-UMP#4320] and [Δpro-Usuφin (MetAlaSerl98-Thr480)-FLAG]:[pETl ld]:[ NcoI]:[T7/lac]:[MF-

UMP#4329]. A similar approach was used to generate recombinant FADD/MORTl (containing the C-terminal streptavidin binding motif, SAYRHPQFGG) beginning with the amplimers 5'-CGT CTA GAC CAT GGA CCC GTT CCT GGT GCT GCT GCA CTC-3' (SEQ.ID.ΝO.:31) (forward amplimer) plus 5'-CGT CTA GAC CAT GGT CAA CCA CCG AAC TGC GGG TGA CGC CAA GCG CTG GAC GCT TCG GAG GTA GAT GCG TCT GAG-3' (SEQ.ID.NO. :32) (reverse amplimer) and resulting in the construct [FADD/MORTl (Metl-Ser256)_StrepTag]:[ pETl Id]: [T7/lac]:[MF-UMP#4337]. In all cases, protein expression was initiated (in cells grown in M9 medium to an OD600nm = 0.6) with 1 mM IPTG for 2 hr at 30°C. Constructs for transfections into human cells: Full length Usuφin-α and ΔDED-A Usuφin were generated by PCR as BamRl-Xbal-Xhol-Ncol-(lJs p )-Ncol-Xhol- Xbal and Xbal-Xhol-(ADED-A Usuφin)-.Y7zøI-.¥bαI fragments, respectively, using the amplimers 5'-GCG GAT CCT CTA GAC TCG AGC CAT GGC TGC TGA AGT CAT CCA TCA GGT TGA AGA AGC AC-3' (SEQ.ID.NO. :24) (Usuφin forward amplimer) plus 5'-GCT CTA GAC TCG AGC CAT GGT TAT GTG TAG GAG AGG ATA AGT TTC-3' (SEQ.ID.NO.:25) (Usuφin reverse amplimer), and 5'-GCT CTA GAC TCG AGG TGA TGG CAG AGA TTG GTG AGG ATT TG-3' (SEQ.ID.NO. :33) (ΔDED-A Usuφin forward amplimer) plus 5'-GCT CTA GAC TCG AGT TAT GTG TAG GAG AGG ATA AGT TTC TTT CTC-3 '

(SEQ.ID.NO.:34) (ΔDED-A Usuφin reverse amplimer). The fragments were subsequently purified from preparative agarose gels, trimmed with Xbal (Boehringer Mannheim) then ligated into the Xbal site of pBluescript II SK+ and transformed into E. coli XL2-blue cells (Stratagene). After sequence verification, the constructs were excised with Xbal (Usuφin) or Xhol (ΔDED-A Usuφin) and ligated into the same restriction sites in pcDNA3 (Invitrogen). The resulting constructs were [Usuφin, Metl-Thr480]:[_pcDNA3]:[ ^bαI]:[T7/CMV]:[MF-UMP#4601] and [ΔDED-A Usuφin, Met97-Thr480] _: [_pcDNA3] : [ Xhol] : [T7/CMV] : [MF-UMP#3466] .

EXAMPLE 8

In vitro binding experiments.

Buffers used in these experiments include TE (50 mM Tris/HCl (pH 8.0), 2 mM EDTA); TENT (50 mM Tris/HCl (pH 8.0), 2 mM EDTA, 150 mM NaCl, 1% (v/v) Triton X-100); and SRsBB (20 mM Tris/HCl (pH 7.5), 100 mM NaCl, 2 mM EDTA, 0.1 % (v/v) Nonidet P-40, 5% (v/v) glycerol, 2 mM dithiothreitol, 0.05% (w/v) bovine serum albumin). Recombinant proteins were generated by soluble expression in bacteria as described above. [35s]-Polypeptides were generated by coupled transcription/translation in reticulocyte lysates as described above but were spun for 30 min at 100,000 x g prior to use to remove particulate matter. Binding of [35s]-proteins to immobilized Usuφin constructs: Anti-FLAG M2 affinity resin (anti-FLAG-epitope monoclonal antibodies covalently linked to agarose beads; 25 nmol/ml binding capacity; Kodak) was washed extensively with TENT buffer then combined with saturating quantities of bacterial lysates containing the indicated FLAG -epitope tagged Usuφin protein and incubated with constant shaking for 30 min at 4°C. The beads were then washed by dilution/centrifugation three times with 10 volumes of TENT buffer then three times with SRsBB buffer. The Usuφin loaded beads were suspended as a 50% slurry in SRsBB buffer then 125 μl of the 50% slurry was combined with 25 μl of the indicated reticulocyte lysate and incubated for 2 hr at 10°C with gentle mixing. At the end of the binding incubation, non-bound proteins were removed by washing the beads twice with 1 ml (15 volumes) SRsBB buffer then three times with SRsBB containing 150 mM NaCl. After removal of the final wash buffer, proteins were eluted from the beads with 40 μl of Laemmli SDS-containing sample buffer followed by denaturation for 5 min at 95°C. The resulting supernatants were resolved on SDS-polyacrylamide gels and the [35 S] -proteins were visualized by fluorography of the dried gel. Binding of [ 5 S] -proteins to immobilized FADD/MORTl : Streptavidin-agarose beads (Pierce) were washed extensively with TE buffer then combined with

FADD/MORTl -StrepTag generated in bacteria, as described above, for 30 min at 4°C with constant shaking. The FADD/MORTl -loaded beads were then washed by dilution/centrifugation three times with 10 volumes of TE buffer and suspended as a 50% slurry in TE. Binding reaction mixtures (250 μl total volume) were prepared to contain 125 μl of the 50% slurry of FADD/MORTl -loaded beads, 25 μl reticulocyte lysate containing [35s]pro-caspase-8 and 100 μl of the indicated amounts of recombinant Usuφin proteins in phosphate-buffered saline (pH 7.4), 1% Triton X- 100, 100 mM EDTA, 5 mM dithiothreitol. In a competition format, the [35s]pro- caspase-8 was combined with the recombinant Usuφin for 15 min at 4°C before the addition of the FADD/MORTl -loaded beads. In a displacement format, the

FADD/MORTl -loaded beads were first combined with the [35s]pro-caspase-8 for 15 min at 4°C prior to the addition of the recombinant Usuφin. All mixtures were then incubated for 2 hr at 4°C with gentle mixing and subsequently washed three times with 1 ml (15 volumes) TE buffer. After removal of the final wash supernatant, the complexes were eluted by the addition of 30 μl of 1 mM d-biotin (in TE buffer) to the 62.5 μl bed volume of beads. After 30 min on ice, the resulting supernatants were recovered following centrifugation and combined with SDS-containing Laemmli sample buffer, resolved on SDS-polyacrylamide gels and the [ 5s]pro-caspase-8 that was harvested was visualized by fluorography of the dried gel. For quantitative analysis, band volumes on the fluorographs were quantified by laser densitometry.

EXAMPLE 9

Transfections, stable cell lines, and apoptosis measurement.

Stable transfection of HeLa cells: Adherent HeLa cells were plated at 5x105 cells per 60 mm dish in Dulbecco's Modified Eagle's Media supplemented with L-glutamine, penicillin, streptomycin, and 10% (v/v) fetal bovine serum and grown overnight at 37°C with 5% CO2- Following this re-plating incubation, the cells were transfected using calcium phosphate with 15 μg of either vector DNA (pcDNA3) or the vector harboring ΔDED-A Usuφin. Bulk-stable cell lines were established by placing cells under G418 selection (at 0.4 mg/ml) 48 hrs post-transfection, and maintaining the selection continuously for 6 weeks prior to assaying for anti-apoptotic effects. Stably transfected cells were plated in 96-well plates at lxl 04 cells/well and grown overnight. Cells were then treated with anti-CD95 (Fas/APO-1) monoclonal antibody (clone CH-11; MBL International) at concentrations ranging from 0-0.1 mg/ml (done in triplicate for each cell line) and incubated 16 hrs. The number of viable cells remaining was determined using the Cell Titre-96 (Promega) MTT reagent (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Viable cells were indicated by their ability to convert the MTT tetrazolium salt into its formazan counteφart which absorbs at 570 nm.

Transient transfection of Jurkat cells: Cultured Jurkat cells were harvested by centrifugation then suspended in medium at 4 x 106 cells/0.4 ml and co-transfected by electroporation (1050 μF, 720 Ω , 260 V) with 1 μg of a. Aequorea victoria green fluorescent protein (GFP) expression vector (GFP:pcDNA3) plus 5 μg of either Usuφin in pcDNA3 or the pcDNA3 vector alone. Three to six independent transfections were typically pooled and used 12 hr later for experiments. Transfected cells were purified on a Ficol gradient immediately prior to testing and were screened for transfection efficiency (typically 30-45%) by FACS analysis (FL-1 positive cells indicating the presence of GFP). The cells were aliquoted into wells in 96-well plates (5 x 104 cells per 200 μl well) then treated with anti-CD95 (Fas APO-1) monoclonal antibody (clone CH-11; MBL international) at 0.5-1 μg ml for the indicated length of time. After stimulation, cells of duplicate wells were harvested and resuspended in FACS buffer for analysis of either propidium iodide staining or Annexin-V binding as indicated.

EXAMPLE 10

Generation of Usuφin antisera.

The ΔDED-A Usuφin fragment harbored within the construct [ΔDED- A Usuφin, Met97-Thr480]:[pBII SK+]:[ AZ>αI]:[T7]:[MF-UMP#3712] (described above) was excised from nested Ncol sites and ligated into pETl Id as described above for other bacterial expression constructs. (The resulting construct is designated [ΔDED-A Usuφin (Met97-Thr480)]_:[_pETl ld]:[ Ncol] :[T7/lac]:[MF-UMP#3827] and differs from those described above in that there is no FLAG epitope tag.). Expression of ΔDED-A Usuφin and accumulation in inclusion bodies was induced by overnight culture at 37°C of BL21(DE3)pLysS cells (harboring this vector) in M9 medium containing 1 mM IPTG. The inclusion bodies (in which ΔDED-A Usuφin was the only identifiable protein) were purified, denatured in 6 M guanidine HC1, 25 mM Tris (pH 7.4) and used directly for immunization of rabbits.

EXAMPLE 11

Immunohistochemical localization of Usuφin in rat heart-

Tissue preparation: Experiments were approved by the Animal Care Committee at the Merck Frosst Centre for Therapeutic Research in accordance with the guidelines established by the Canadian Council on Animal Care. Male Sprague Dawley rats (250-400 g) were used and were anesthetized with intraperitoneal pentobarbital (65 mg kg). Heart rate, aortic pressure and a Lead II electrocardiograph were continuously recorded. A left thoracotomy was performed in the region overlying the heart and a suture passed under the left coronary artery (LCA) and used to occlude the artery. The LCA was occluded for 45 minutes and reperfused for 3 hours following release of the suture. At the end of reperfusion, rats were euthanized by pentobarbital overdose, the heart rapidly excised and flushed with 20 ml 0.9% sodium chloride. The heart was slowly (over 3-4 minutes) infused with 40 ml of 10% buffered formalin phosphate and fixed overnight (18 hours) in 10% buffered formalin phosphate, after which the heart was transferred to 70% ethanol. After paraffin embedding, serial cross sections from heart blocks were cut and fixed to glass slides for immunohistochemical study.

Immunohistochemical detection of Usuφin: A primary polyclonal antibody raised against recombinant human Usuφin (described above) was used to study the localization of rat cardiac Usuφin. The primary antibody was visualized indirectly using an ABC technique, whereby positive Usuφin immunoreactivity was evident by a red fluorescent signal. Apoptotic myocytes were identified with a commercially available TUNEL (terminal deoxynucleotidyl-transferase nick-end labeling) labeling kit (Oncor). The TUNEL reaction labels apoptotic nuclei by means of a terminal dideoxynucleotidyl transferase (TdT)-catalyzed addition of a digoxigenin-tagged nucleotide to the free 3' -OH end of nicked DNA. Labeled DNA was visualized with an FITC coupled anti-digoxigenin secondary antibody, yielding bright yellow green nuclei.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which are incoφorated by reference in their entireties.

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Claims

WHAT IS CLAIMED:

1. A recombinant DNA molecule encoding a polypeptide having an amino acid sequence selected from the group consisting of: SEQ.ID.NO. :4;

Positions 97-480 of SEQ.ID.NO. :4; Positions 198-480 of SEQ.ID.NO. :4; SEQ.ID.NO.:5; and SEQ.ID.NO. :6.

2. A recombinant DNA molecule comprising a nucleotide sequence selected from the group consisting of:

SEQ.ID.NO.: 1;

Positions 793-1,947 of SEQ.ID.NO.: 1; Positions 1,096-1,947 of SEQJD.NO.:l;

SEQ.ID.NO. :2; and SEQJD.NO.:3.

3. A DNA molecule that hybridizes under stringent conditions to the DNA of claim 2.

4. An expression vector comprising the DNA of claim 1.

5. A recombinant host cell comprising the DNA of claim 1.

6. A Usuφin polypeptide, substantially free from other proteins, comprising an amino acid sequence selected from the group consisting of: SEQ.ID.NO. :4; Positions 97-480 of SEQ.ID.NO. :4;

Positions 198-480 of SEQJD.NO.:4; positions 2-73 of SEQ.ID.NO. :4; positions 93-170 of SEQ.ID.NO. :4; SEQ.ID.NO.:5; and SEQ.ID.NO. :6.

7. The Usuφin polypeptide of claim 6 containing a single amino acid substitution.

8. The Usuφin polypeptide of claim 6 containing two or more amino acid substitutions.

9. The Usuφin polypeptide of claim 8 where the substitutions are conservative substitutions.

10. A method for the identification of inhibitors of the interaction between Usuφin and pro-caspase-8 comprising:

(a) providing a Usuφin polypeptide; (b) providing pro-caspase-8; under conditions such that a specific binding interaction between the Usuφin polypeptide and pro-caspase-8 will occur in the absence of an inhibitor of the interaction between Usuφin and pro-caspase-8; and measuring the amount of specific binding interaction between the Usuφin polypeptide and pro-caspase-8 in the presence and in the absence of a substance suspected of being an inhibitor of the specific binding interaction between Usuφin and pro-caspase-8; where a decrease in the amount of specific binding interaction between Usuφin and pro-caspase-8 in the presence of the substance as compared to the absence of the substance indicates that the substance is an inhibitor of the interaction between Usuφin and pro-caspase-8; where the Usuφin polypeptide has an amino acid sequence selected from the group consisting of:

SEQ.ID.NO.:4; Positions 97-480 of SEQ.ID.NO. :4;

Positions 198-480 of SEQ.ID.NO. :4; positions 2-73 of SEQ.ID.NO. :4; positions 93-170 of SEQ.ID.NO. :4;

SEQ.ID.NO. :5; and SEQ.E).NO.:6.

11. A method for the identification of inhibitors of the interaction between a Usuφin polypeptide and pro-caspase-8 comprising (a) providing a cell that contains the Usuφin polypeptide and pro- caspase-8;

(b) contacting the cell with an apoptotic stimulus in the presence and in the absence of a substance suspected of being an inhibitor of the specific binding interaction between the Usuφin polypeptide and pro-caspase-8; and (c) measuring the amount of apoptosis the cells undergo in the presence and in the absence of the substance; where an increase in the amount of apoptosis measured in the presence of the substance indicates that the substance is an inhibitor of the specific binding interaction between the Usuφin polypeptide and pro-caspase-8; where the Usuφin polypeptide has an amino acid sequence selected from the group consisting of:

SEQ.ID.NO. :4;

Positions 97-480 of SEQ.ID.NO. :4;

Positions 198-480 of SEQJD.NO.:4; positions 2-73 of SEQ.ID.NO.:4; positions 93-170 of SEQ.ID.NO. :4;

SEQ.ID.NO.:5; and

SEQ.ID.NO.:6.

12. An antibody to a Usuφin polypeptide where the Usuφin polypeptide has an amino acid sequence selected from the group consisting of:

SEQ.ID.NO. :4;

Positions 97-480 of SEQ.ID.NO. :4;

Positions 198-480 of SEQJD.NO.:4; positions 2-73 of SEQ.ID.NO.:4; positions 93-170 of SEQ.ID.NO. :4;

SEQ.ID.NO. :5; and

SEQ.ID.NO. :6.