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We analyse viral load data of five patients under ritonavir monotherapy using a model of HIV dynamics under antiretroviral therapy that includes both drug pharmacokinetics and the intracellular delay from the time of cell infection to viral production. Using this approach we separate pharamacokinetic from intracellular delays, and obtain new estimates of intracellular delay and the antiviral efficacy of ritonavir. We find the average intracellular delay to be 1 day, in agreement with experiments. The average viral generation time is now estimated at 2 days, resulting in ~180 replication cycles per year. The model also reveals that ritonavir monotherapy is ~65% as efficacious as a recently used potent four-drug therapy, suggesting that selection for drug resistance may be facilitated by the relatively low efficacy of individual drugs, contributing in part to the inherent limitations of current therapies in combating HIV-1 infection.

Inhibitors of apoptosis (IAPs) physically interact with a variety of pro-apoptotic proteins and inhibit apoptosis induced by diverse stimuli. X-linked IAP (X-IAP) is a prototype IAP family member that inhibits several caspases, the effector proteases of apoptosis. The inhibitory activity of X-IAP is regulated by SMAC, a protein that is processed to its active form upon receipt of a death stimulus. Cleaved SMAC binds X-IAP and antagonizes its anti-apoptotic activity. Here we show that melanoma IAP (ML-IAP), a potent anti-cell death protein and caspase inhibitor, physically interacts with SMAC through its BIR (baculovirus IAP repeat) domain. In addition to binding full-length SMAC, ML-IAP BIR associates with SMAC peptides that are derived from the amino terminus of active, processed SMAC. This high affinity interaction is very specific and can be completely abolished by single amino acid mutations either in the amino terminus of active SMAC or in the BIR domain of ML-IAP. In cells expressing ML-IAP and X-IAP, SMAC coexpression or addition of SMAC peptides abrogates the ability of the IAPs to inhibit cell death. These results demonstrate the feasibility of using SMAC peptides as a way to sensitize IAP-expressing cells to pro-apoptotic stimuli such as chemotherapeutic agents.

Receptor-interacting protein (RIP), a Ser/Thr kinase component of the tumor necrosis factor (TNF) receptor-1 signaling complex, mediates activation of the nuclear factor κB (NF-κB) pathway. RIP2 and RIP3 are related kinases that share extensive sequence homology with the kinase domain of RIP. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 possesses a unique C terminus. RIP3 binds RIP through this unique C-terminal segment to inhibit RIP- and TNF receptor-1-mediated NF-κB activation. We have identified a unique homotypic interaction motif at the C terminus of both RIP and RIP3 that is required for their association. Sixty-four amino acids within RIP3 and 88 residues within RIP are sufficient for interaction of the two proteins. This interaction is a prerequisite for RIP3-mediated phosphorylation of RIP and subsequent attenuation of TNF-induced NF-κB activation.

During an immune response naive T helper (Th) cells differentiate into two functionally distinct subsets, Th1 and Th2, based on their cytokine secretion profile and immunomodulatory function. c-Jun amino terminal kinase (JNK) regulates Th cell differentiation by activating a transcriptional program required for cytokine production. We have recently identified a TNFR superfamily death domain–containing molecule, death receptor (DR)6, which potently activates JNK. T cells from DR6-deficient mice are substantially impaired in JNK activation. When DR6−/− mice were challenged with protein antigen, their T cells hyperproliferate and display a profound polarization toward a Th2 response whereas Th1 differentiation is not equivalently affected. In addition, DR6−/− T cells showed preference toward Th2 differentiation in vitro. The phenotype seen in the DR6−/− mice is not due to the apoptotic pathway. Therefore, DR6, working through JNK, rather than apoptosis, functions to attenuate the Th2 response. This is the first demonstration of a role in the activation and differentiation of Th cells by DR6 in particular and DRs in general.

Poly(ADP-ribosyl)ation is an important mechanism for the maintenance of genomic integrity in response to DNA damage. The enzyme responsible for poly(ADP-ribose) synthesis, poly(ADP-ribose) polymerase 1 (PARP-1), has been implicated in two distinct modes of cell death induced by DNA damage, namely apoptosis and necrosis. During the execution phase of apoptosis, PARP-1 is specifically proteolyzed by caspases to produce an N-terminal DNA-binding domain (DBD) and a C-terminal catalytic fragment. The functional consequence of this proteolytic event is not known. However, it has recently been shown that overactivation of full-length PARP-1 can result in energy depletion and necrosis in dying cells. Here, we investigate the molecular basis for the differential involvement of PARP-1 in these two types of cellular demise. We show that the C-terminal apoptotic fragment of PARP-1 loses its DNA-dependent catalytic activity upon cleavage with caspase 3. However, the N-terminal apoptotic fragment, retains a strong DNA-binding activity and totally inhibits the catalytic activity of uncleaved PARP-1. This dominant-negative behavior was confirmed and extended in cellular extracts where DNA repair was completely inhibited by nanomolar concentrations of the N-terminal fragment. Furthermore, overexpression of the apoptotic DBD in mouse fibroblast inhibits endogenous PARP-1 activity very efficiently in vivo, thereby confirming our biochemical observations. Taken together, these experiments indicate that the apoptotic DBD of PARP-1 acts cooperatively with the proteolytic inactivation of the enzyme to trans-inhibit NAD hydrolysis and to maintain the energy levels of the cell. These results are consistent with a model in which cleavage of PARP-1 promotes apoptosis by preventing DNA repair-induced survival and by blocking energy depletion-induced necrosis.

A kinetic model for the nucleation of a crystalline phase consisting of particles experiencing short-range attractions is developed. Of particular significance is the proximity of the metastable fluid/fluid phase boundary. The model incorporates self-consistent thermodynamics, changes in gradient diffusivity, and density fluctuations in the vicinity of the critical point. Density fluctuations associated with the spinodal of this metastable phase transition greatly enhance nucleation rates, suggesting that experimental conditions may be found where rapid nucleation and slow crystal growth can be achieved by moving the metastable critical point relative to the solubility boundary.

Apoptosis inhibition rather than enhanced cellular proliferation occurs in prostate cancer (CaP), the most commonly diagnosed malignancy in American men. Therefore, it is important to characterize residual apoptotic pathways in CaP cells. When intracellular Ca2+stores are released and plasma membrane “store-operated” Ca2+entry channels subsequently open, cytosolic [Ca2+] increases and is thought to induce apoptosis. However, cells incapable of releasing Ca2+stores are resistant to apoptotic stimuli, indicating that Ca2+store release is also important. We investigated whether release of intracellular Ca2+stores is sufficient to induce apoptosis of the CaP cell line LNCaP. We developed a method to release stored Ca2+without elevating cytosolic [Ca2+]; this stimulus induced LNCaP cell apoptosis. We compared the apoptotic pathways activated by intracellular Ca2+store release with the dual insults of store release and cytosolic [Ca2+] elevation. Earlier processing of caspases-3 and -7 occurred when intracellular store release was the sole Ca2+perturbation. Apoptosis was attenuated in both conditions in stable transfected cells expressing antiapoptotic proteins BclxLand catalytically inactive caspase-9, and in both scenarios inactive caspase-9 became complexed with caspase-7. Thus, intracellular Ca2+store release initiates an apoptotic pathway similar to that elicited by the dual stimuli of cytosolic [Ca2+] elevation and intracellular store release.

The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) synthesizes poly(ADP-ribose) in response to DNA strand breaks. During almost all forms of apoptosis, PARP is cleaved by caspases, suggesting the crucial role of its inactivation. A few studies have also reported a stimulation of PARP during apoptosis. However, the role of PARP stimulation and cleavage during this cell death process remains poorly understood. Here, we measured the stimulation of endogenous poly(ADP-ribose) synthesis during VP-16-induced apoptosis in HL60 cells and found that PARP was cleaved by caspases at the time of its poly(ADP-ribosyl)ation. In vitro experiments showed that PARP cleavage by caspase-7, but not by caspase-3, was stimulated by its automodification by long and branched poly(ADP-ribose). Consistently, caspase-7 exhibited an affinity for poly(ADP-ribose), whereas caspase-3 did not. In addition, caspase-7 was activated and accumulated in the nucleus of HL60 cells in response to the VP-16 treatment. Furthermore, caspase-7 activation was concommitant with PARP cleavage in the caspase-3-deficient cell line MCF-7 in response to staurosporine treatment. These results strongly suggest that, in vivo, it is caspase-7 that is responsible for PARP cleavage and that poly(ADP-ribosyl)ation of PARP accelerates its proteolysis. Cleavage of the active form of caspase substrates could be a general feature of the apoptotic process, ensuring the rapid inactivation of stress signaling proteins.

The recombinant form of the proapoptotic caspase-9 purified following expression in Escherichia coli is processed at Asp315, but largely inactive; however, when added to cytosolic extracts of human 293 cells it is activated 2000-fold in the presence of cytochrome c and dATP. Thus, the characteristic activities of caspase-9 are context-dependent, and its activation may not recapitulate conventional caspase activation mechanisms. To explore this hypothesis we produced recombinant forms of procaspase-9 containing mutations that disabled one or both of the interdomain processing sites of the zymogen. These mutants were able to activate downstream caspases, but only in the presence of cytosolic factors. The mutant with both processing sites abolished had 10% of the activity of wild-type, and was able to support apoptosis, with equal vigor to wild-type, when transiently expressed in 293 cells. Thus caspase-9 has an unusually active zymogen that does not require proteolytic processing, but instead is dependent on cytosolic factors for expression of its activity.

RIP3 is a novel gene product containing a N-terminal kinase domain that shares extensive homology with the corresponding domain in RIP (receptor-interacting protein) and RIP2. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 has a unique C terminus. RIP3 binds RIP through its unique C-terminal segment and by virtue of this interaction is recruited to the tumor necrosis factor (TNF) receptor-1 signaling complex. Previous studies have shown that RIP mediates TNF-induced activation of the anti-apoptotic NF-kappaB pathway. RIP3, however, attenuates both RIP and TNF receptor-1-induced NF-kappaB activation. Overexpression studies revealed RIP3 to be a potent inducer of apoptosis, capable of selectively binding to large prodomain initiator caspases.

The Eph family of receptor protein tyrosine kinases (RPTKs) is the largest family of RPTKs. The signal transduction pathways initiated by this family have only recently begun to be explored. Using a yeast two-hybrid screen to identify molecules that interact with the cytoplasmic domain of Eck, it was previously shown that activated Eck RPTK bound to and stimulated phosphatidylinositol 3-kinase (Pandey, A., Lazar, D.F., Saltiel, A. R., and Dixit, V.M. (1994) J. Biol. Chem. 269, 30154-30157). Also isolated from this same screen was a novel protein containing SH3 and SH2 adapter modules that had striking homology to those found in the Src family of non-receptor tyrosine kinases. However, unlike other Src family members, it lacked a catalytic tyrosine kinase domain. Hence, this protein was designated SLAP for Src-like adapter protein. Using glutathione S-transferase fusion Proteins, it was demonstrated that SLAP bound to activated Eck receptor tyrosine kinase. Therefore, SLAP is a novel candidate downstream signaling intermediate and the first member of the Src family that resembles an adapter molecule.

Purified platelet thrombospondin binds to immobilized fibrinogen if both Ca++and Mg++are present. Digestion of the purified molecule with thermolysin results in a limited number of discrete proteolytic fragments. When such digests are subjected to affinity chromatography on immobilized fibrinogen, only the fragments with Mr of 120,000 and 140,000 are specifically bound and subsequently eluted by the additon of EDTA to the column buffer. Examination by SDS-PAGE under both reducing and nonreducing conditions reveals that the fibrinogen-binding domain is derived from the region of the thrombospondin molecule containing the interchain disulfide bonds. The requirement for Ca++and Mg++for optimal binding to fibrinogen is also manifest by the Mr 120,000/140,000 thermolytic fragments.

Fas and the type I tumor necrosis factor receptor (TNF-R) are two cell surface receptors that, when stimulated with ligand or cross-linking antibody, trigger apoptotic cell death by a mechanism that has yet to be elucidated. The CrmA protein is a serpin family protease inhibitor that can inhibit interleukin-1β converting enzyme (ICE) and ICE-like proteases. We showed previously that expression of CrmA potently blocks apoptosis induced by activation of either Fas or TNF-R, implicating protease involvement in these death pathways (Tewari, M., and Dixit, V. M.(1995) J. Biol. Chem.270, 3255-3260). Here we report that the 70-kDa component of the U1 small ribonucleoprotein (U1-70 kDa) is a proteolytic substrate rapidly cleaved during both Fas- and TNF-R-induced apoptosis. This cleavage was inhibited by expression of CrmA but not by expression of an inactive point mutant of CrmA, confirming the involvement of an ICE-like protease. These data for the first time identify U1-70 kDa as a death substrate cleaved during Fas- and TNF-R-induced apoptosis and emphasize the importance of protease activation in the cell death pathway.

Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Sepharose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparin-binding proteins. Edman degradation of six different preparations revealed the amino-terminal sequence of thrombospondin (TSP) to be Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-. This sequence was obtained in initial yields as high as 85%, indicating that no blocked chains are present. Cleavage of calcium-replete TSP with thermolysin or plasmin results in the production of relatively stable fragments. Chromatography of these digests on heparin-Sepharose followed by elution with 0.6 M NaCl affords purification of an Mr 25,000 fragment from the thermolysin digest and an Mr 35,000 fragment from the plasmin digest. The binding of these fragments to heparin-Sepharose does not require divalent metal ions. Neither fragment is disulfide-bonded to other fragments present in the digests. The heparin-binding domains from both digests have similar amino acid compositions and their tryptic peptide maps on high performance liquid chromatography are identical with the exception of one peptide unique to each fragment. Automated Edman degradation in a vapor-phase sequenator of the thermolytic heparin-binding domain electroeluted from sodium dodecyl sulfate-gels indicates that the heparin-binding domain resides at the amino terminus of the Mr 180,000 TSP peptide chain.

A monoclonal antibody (A3.3) has been generated against human platelet fibronectin (FN). A3.3 reacts with human plasma FN but with no other plasma proteins. A3.3 was found to inhibit thrombin- or ionophore A23187-stimulated aggregation of gel-filtered platelets in a concentration-dependent manner in both an aggregometer assay and a sensitive well plate aggregation assay. The antibody does not block secretion of serotonin. Four other anti-FN monoclonal antibodies that recognize different epitopes on FN than A3.3 does have no effect on platelet aggregation. A3.3 does not block the adhesion of CHO cells to FN-coated surfaces, indicating that it does not bind to the identified cell-binding domain of FN. A3.3 reacts with a 160/140-kDa doublet, known to contain the cell-binding domain, that is produced by digestion of FN with elastase or thermolysin. However, the antibody does not react with lower molecular weight species that also contain the cell-binding domain or with any of the other identified domains of FN. The A3.3 epitope is extremely protease sensitive and the smallest fragment found in any digest that retains reactivity with A3.3 is a 70-kDa peptide produced in low yield by mild thermolytic cleavage of FN. These data suggest that A3.3 defines a functional site present on both the platelet and plasma FN molecule that has a direct role in platelet aggregation.

We have previously described the cloning of a group of novel cellular immediate-early response genes whose expression in human umbilical vein endothelial cells is induced by tumor necrosis factor alpha in the presence of cycloheximide. These genes are likely to participate in mediating the response of the vascular endothelium to proinflammatory cytokines. In this study, we further characterized one of these novel gene products named B61. Sequence analysis of cDNA clones encoding B61 revealed that its protein product has no significant homology to previously described proteins. Southern analysis suggested that B61 is an evolutionarily conserved single-copy gene. B61 is primarily a hydrophilic molecule but contains both a hydrophobic N-terminal and a hydrophobic C-terminal region. The N-terminal region is typical of a signal peptide, which is consistent with the secreted nature of the protein. The mature form of the predicted protein consists of 187 amino acid residues and has a molecular weight of 22,000. Immunoprecipitation of metabolically labeled human umbilical vein endothelial cell preparations revealed that B61 is a 25-kilodalton secreted protein which is markedly induced by tumor necrosis factor.

We explored the role that thrombospondin (TSP). a multifunctional extracellular matrix protein, plays in hematopoietic cell-cell and cell-matrix interactions. Thrombospondin synthesis is differentially regulated in human long-term bone marrow cultures. Consistent with this, human hematopoietic progenitor cells of all three lineages (erythrocyte. megakaryocyte, and granulocyte) use TSP as an attachment protein. However, terminally differentiated cells (erythrocytes and neutrophils) show absent or reduced attachment to TSP. The region within the TSP molecule that mediates cell attachment (cell binding domain) was delineated by examining both attachment to proteolytic fragments of TSP and by inhibition of cytoadhesion using monoclonal antibodies directed against TSP domains. The cell binding domain resides toward the C-terminus of a 140 Kd chymotryptic fragment of TSP. We conclude that thrombospondin functions as a hematopoietic cytoadhesion molecule, capable of binding primary hematopoietic progenitor cells, and may, therefore, be important in blood cell development.

Thrombospondin (TSP) is a homotrimeric extracellular glycoprotein with a subunit molecular mass of 140 kD. The subunits have a modular or domain-like structure and are held together by interchain disulphide bonds. A number of domains have been identified including those for the binding of collagen, fibrinogen, and heparin. Due to the trimeric form of the TSP molecule, the various domains are trivalent in nature and this contributes to the ability of TSP to mediate cell-substrate interactions. Indeed, TSP has recently been shown not only to promote cell adhesion but also to be intimately involved in cell growth and migration. The adhesive function of TSP is attributable to the "solid-phase" or matrix-bound form of the molecule. There is some evidence that the heparin-binding domain mediates incorporation of soluble TSP into the insoluble matrix form. The heparin-binding domain of TSP is a compact globular amino-terminal moiety that contains two clusters of basic amino acids and a single intrachain disulphide bond. To delineate the role of the heparin-binding domain in matrix assembly and to define further the precise region of interchain disulphide bonding that results in trimer formation, we have expressed deleted forms of the cDNA encoding TSP in SV-40-transformed. African green monkey kidney cells. The proteins synthesized from the various deleted TSP cDNAs were examined for (a) secretion into the culture medium and incorporation into the extracellular matrix; (b) binding to heparin-Sepharose; (c) immunoprecipitability by a conformation-specific monoclonal antibody; and (d) ability to form trimers. This analysis allowed us to draw the following conclusions. (a) A 218 amino acid NH2-terminal protein that preserves the intrachain disulphide bridge of the heparin-binding domain is capable of binding to heparin-Sepharose and incorporating into the extracellular matrix. (b) A shorter 164 amino acid NH2-terminal peptide that does not contain the intrachain disulphide bridge of the heparin-binding domain is neither able to bind to heparin-Sepharose nor able to incorporate into the extracellular matrix. (c) The region of interchain disulphide bridging necessary for trimer assembly resides within a cluster of seven cysteine residues immediately adjacent to the heparin-binding domain.

The apoptotic machinery of Caenorhabditis elegans includes three core interacting components: CED-3, CED-4, and CED-9. CED-3 is a death protease composed of a prodomain and a protease domain. CED-4 is a P-loop-containing, nucleotide-binding molecule that complexes with the single polypeptide zymogen form of CED-3, promoting its activation by autoprocessing. CED-9 blocks death by complexing with CED-4 and suppressing its ability to promote CED-3 activation. A naturally occurring alternatively spliced form of CED-4 that contains an insertion within the nucleotide-binding region (CED-4L) functions as a dominant negative inhibitor of CED-3 processing and attenuates cell death. Domain mapping studies revealed that distinct regions within CED-4 bind to the CED-3 prodomain and protease domain. Importantly, the CED-4 P-loop was involved in prodomain binding. Disruption of P-loop geometry because of mutation of a critical lysine (K165R) or insertional inactivation (CED-4L) abolished prodomain binding. Regardless, K165R and CED-4L still retained CED-3 binding through the protease domain but were unable to initiate CED-3 processing. Therefore, the P-loop-prodomain interaction is critical for triggering CED-4-mediated CED-3 processing. Underscoring the importance of this interaction was the finding that CED-9 contacted the P-loop and selectively inhibited its interaction with the CED-3 prodomain. These results provide a simple mechanism for how CED-9 functions to block CED-4-mediated CED-3 processing and cell death.

Through specific interactions with members of the tumor necrosis receptor (TNFR) family, adapter molecules such as the serine/threonine (Ser/Thr) kinase RIP mediate divergent signaling pathways including NF-kappaB activation and cell death. In this study, we have identified and characterized a novel 61-kDa protein kinase related to RIP that is a component of both the TNFR-1 and the CD40 signaling complexes. Receptor interacting protein-2 (RIP2) contains an N-terminal domain with homology to Ser/Thr kinases and a C-terminal caspase activation and recruitment domain (CARD), a homophilic interaction motif that mediates the recruitment of caspase death proteases. Overexpression of RIP2 signaled both NF-kappaB activation and cell death. Mutational analysis revealed the pro-apoptotic function of RIP2 to be restricted to its C-terminal CARD domain, whereas the intact molecule was necessary for NF-kappaB activation. RIP2 interacted with other members of the TNFR-1 signaling complex, including inhibitor of apoptosis protein cIAP1 and with members of the TNFR-associated factor (TRAF) family, specifically TRAF1, TRAF5, and TRAF6, but not with TRAF2, TRAF3, or TRAF4. These TRAF interactions mediate the recruitment of RIP2 to receptor signaling complexes.

Exposure to ultraviolet light (UV) can induce apoptosis in mammalian cells. The mechanism by which UV radiation engages the suicide apparatus is unclear. Here we demonstrate that UV radiation can activate the Fas pathway via receptor aggregation and subsequent recruitment of the death adaptor molecule FADD/MORT1. UV radiation-induced apoptosis was inhibited by both a dominant negative version of FADD (FADD-DN) and the caspase inhibitor CrmA. Thus, activation of the Fas pathway represents a physiologic mechanism by which UV-damaged cells are eliminated.

Molluscum contagiosum virus proteins MC159 and MC160 and the equine herpesvirus 2 protein E8 share substantial homology to the death effector domain present in the adaptor molecule Fas-associated death domain protein (FADD) and the initiating death protease FADD-like interleukin-1beta-converting enzyme (FLICE) (caspase-8). FADD and FLICE participate in generating the death signal from both tumor necrosis factor receptor-1 (TNFR-1) and the CD-95 receptor. The flow of death signals from TNFR-1 occurs through the adaptor molecule tumor necrosis factor receptor-associated death domain protein (TRADD) to FADD to FLICE, whereas for CD-95 the receptor directly communicates with FADD and then FLICE. MC159 and E8 inhibited both TNFR-1- and CD-95-induced apoptosis as well as killing mediated by overexpression of the downstream adaptors TRADD and FADD. Neither viral molecule, however, inhibited FLICE-induced killing, consistent with an inhibitory action upstream of the active death protease. These data suggest the existence of a novel strategy employed by viruses to attenuate host immune killing mechanisms. Given that bovine herpesvirus 4 protein E1.1 and Kaposi's sarcoma associated-herpesvirus protein K13 also possess significant homology to the viral inhibitory molecules MC159, MC160, and E8, it may be that this class of proteins is used ubiquitously by viruses to evade host defense.

Recent studies have identified a number of cell death pathway components. In this study, we describe the role that two such components, Bik and Bak, play in initiating the apoptotic program. These Bcl-2 family members engage the death pathway downstream of the block imposed by the serpin CrmA, but upstream of the block initiated by cellular inhibitors of apoptosis, which are a family of molecules characterized by a conserved baculovirus inhibitor of apoptosis repeat motif. Distal death pathway components activated by Bik and Bak are similar to those activated by the CD-95 (Fas/Apo1) and tumor necrosis factor death receptors.

Phylogenetic analysis of the CED-3/ICE family of cysteine proteases suggests the existence of a subfamily most related to the Caenorhabditis elegans death gene ced-3 and includes Yama (CPP32, apopain), LAP3 (Mch3, CMH1), and Mch2. Here, we show that Mch2 is processed from its zymogen form to a proteolytically active dimeric species during execution of the apoptotic program and by the cytotoxic T cell death protease granzyme B. Additionally, like Yama and LAP3, Mch2 functions downstream of the death inhibitors Bcl-2, Bcl-xL, and CrmA. Importantly, Mch2, but not Yama or LAP3, is capable of cleaving lamin A to its signature apoptotic fragment, indicating that Mch2 is an apoptotic laminase.

Members of the ICE/Ced-3 gene family are likely effector components of the cell death machinery. Here, we characterize a novel member of this family designated ICE-LAP6. By phylogenetic analysis, ICE-LAP6 is classified into the Ced-3 subfamily which includes Ced-3, Yama/CPP32/apopain, Mch2, and ICE-LAP3/Mch3/CMH-1. Interestingly, ICE-LAP6 contains an active site QACGG pentapeptide, rather than the QACRG pentapeptide shared by other family members. Overexpression of ICE-LAP6 induces apoptosis in MCF7 breast carcinoma cells. More importantly, ICE-LAP6 is proteolytically processed into an active cysteine protease by granzyme B, an important component of cytotoxic T cell-mediated apoptosis. Once activated, ICE-LAP6 is able to cleave the death substrate poly(ADP-ribose) polymerase into signature apoptotic fragments.

Adapter proteins containing Src homology 2 (SH2) domains link transmembrane receptor protein-tyrosine kinases to downstream signal transducing molecules. A family of SH2 containing adapter proteins including Grb7 and Grb10 has been recently identified. We had previously shown that Grb10 associates with Ret via its SH2 domain in an activation-dependent manner (Pandey, A., Duan, H., Di Fiore, P.P., and Dixit, V.M. (1995) J. Biol, Chem. 270, 21461-21463). We now demonstrate that the related adapter molecule Grb7 also associates with Ret in vitro and in vivo, and that the binding of the SH2 domain of Grb7 to Ret is direct. This binding is dependent upon Ret autophosphorylation since Grb7 is incapable of binding a kinase-defective mutant of Ret. Thus two members of the Grb family, Grb7 and Grb10, likely relay signals emanating from Ret to other, as yet, unidentified targets within the cell.

crmAis a cowpox virus gene that encodes a protease inhibitor of the serpin family. The only reported target for the CrmA protein is the cysteine protease interleukin-1β converting enzyme (ICE). ICE, by virtue of its homology to the Caenorhabditis eleganscell death protein Ced-3, has been suggested to play a fundamentally important role in mammalian apoptosis. We hypothesized that a function of crmAmay be to inhibit cell death, since a major mechanism of viral clearance is the immune system-mediated induction of apoptosis of infected cells. The tumor necrosis factor receptor and the Fas antigen are two cytokine receptors which, by engaging and activating the death pathway, can eliminate virus-infected cells. Remarkably, crmAwas found to be an exceptionally potent inhibitor of apoptosis induced by both these receptors, capable of blocking the cell death program even at pharmacological doses of the death stimulus. Therefore, an important new function for crmAis the inhibition of cytokine-induced apoptosis. Further, the data suggest that a protease, either ICE or a related crmA-inhibitable protein, is a component of the Fas- and tumor necrosis factor-induced cell death pathways.

Genetic analysis of apoptosis in the nematodeCaenorhabditis eleganshas revealed the cell death machine to be composed of three core interacting components. CED-4 (equivalent to mammalian Apaf-1) is a nucleotide binding molecule that complexes with the zymogen form of the death protease CED-3, leading to its autoactivation and cell death. CED-9 blocks death by complexing with CED-4 and attenuating its ability to promote CED-3 activation. An equivalent ternary complex was found to be present in mammalian cells involving Apaf-1, the mammalian death protease caspase-9, and Bcl-XL, an anti-apoptotic member of the Bcl-2 family. Consistent with a central role for caspase-9, a dominant negative form effectively inhibited cell death initiated by a wide variety of inducers.

Programmed cell death, or apoptosis, is a process of fundamental importance to cellular homeostasis in metazoan organisms (Ellis, R. E., Yuan, J., and Horvitz, H. R. (1991) Annu. Rev. Cell Biol. 7, 663-698). The caspase family of mammalian proteases, related to the nematode death protein CED-3, plays a crucial role in apoptosis and inflammation. We report here the isolation and characterization of a new caspase, tentatively termed ERICE (Evolutionarily Related Interleukin-1beta Converting Enzyme). Based on phylogenetic analysis, ERICE (caspase-13) is a member of the ICE subfamily of caspases which includes caspase-1 (ICE), caspase-4 (ICErel-II, TX, ICH-2), and caspase-5 (ICErel-III, TY). Overexpression of ERICE induces apoptosis of 293 human embryonic kidney cells and MCF7 breast carcinoma cells. Like other members of the subfamily, ERICE is not activated by the serine protease granzyme B, a caspase-activating component of cytotoxic T cell granules. Therefore, ERICE most likely does not play a role in granzyme B-induced cell death. ERICE, however, was activated by caspase-8 (FLICE, MACH, Mch-5), the apical caspase activated upon engagement of death receptors belonging to the tumor necrosis factor family. This is consistent with a potential role for ERICE in this receptor-initiated death pathway.

When ectopically expressed in animal cells, cytokine response modifier A (CrmA), a product of the cowpox virus, prevents programmed cell death initiated by a variety of stimuli. Since CrmA is a proteinase inhibitor, its target is probably a protease that promotes cell death. The identification of this target is crucial in delineating essential regulation points that modulate the apoptotic program. We have compared the kinetics of interaction of CrmA with five proteases that may play a role in apoptosis. Four of the proteases, all members of the caspase family, are inhibited with widely different rates and affinities ranging over 5 orders of magnitude. One is not inhibited at all under the experimental conditions. CrmA is quite selective in its ability to inhibit caspases, showing the highest affinity for interleukin-1beta-converting enzyme and the second highest for the caspase FLICE (Ki = 0.95 nM), identified as a component of the intracellular signaling complex recruited by ligation of the death receptor Fas. On the basis of comparative inhibitor kinetics, we propose that CrmA is unlikely to inhibit the caspases Yama, Mch2, or LAP3 in vivo but that its inhibition of FLICE is of a magnitude for this protease to be a key target of CrmA during Fas-mediated apoptosis. Therefore, our results support the hypothesis that FLICE catalyzes a crucial step in the promotion of cell death.

The pivotal discovery that Fas-associated death domain protein (FADD) interleukin-1beta-converting enzyme (FLICE)/MACH was recruited to the CD95 signaling complex by virtue of its ability to bind the adapter molecule FADD established that this protease has a role in initiating the death pathway (Boldin, M. P., Goncharov, T. M. , Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803-815; Muzio, M., Chinnaiyan, A. M., Kischkel, K. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827). In this report, we describe the cloning and characterization of a new member of the caspase family, a homologue of FLICE/MACH, and Mch4. Since the overall architecture and function of this molecule is similar to that of FLICE, it has been designated FLICE2. Importantly, the carboxyl-terminal half of the small catalytic subunit that includes amino acids predicted to be involved in substrate binding is distinct. We show that the pro-domain of FLICE2 encodes a functional death effector domain that binds to the corresponding domain in the adapter molecule FADD. Consistent with this finding, FLICE2 is recruited to both the CD95 and p55 tumor necrosis factor receptor signaling complexes in a FADD-dependent manner. A functional role for FLICE2 is suggested by the finding that an active site mutant of FLICE2 inhibits CD95 and tumor necrosis factor receptor-mediated apoptosis. FLICE2 is therefore involved in CD95 and p55 signal transduction.

Activation of the type I insulin-like growth factor receptor (IGF-IR) blocks osmotic mediated programmed cell death (PCD) in neurons. We speculated that IGF-IR activation could afford neuroprotection either by effecting the negative regulators of the death pathway, Bcl-2 and Bcl-xL, or by altering activity of the ced-3/ICE-like proteases. Here we report that osmotic stress decreases total neuronal Bcl-2 by 4-fold and that hyperosmotic PCD correlates with proteolytic processing of neuronal ced-3/ICE-like proteases. IGF-IR activation maintains normal Bcl-2 levels, and signaling via the IGF-IR:phosphatidylinositol 3-kinase pathway prevents ICE/LAP-3 and Yama/CPP32 processing. Finally, increased neuronal IGF-IR expression enhances the negative death regulator Bcl-xL. We suggest that IGF-IR signaling exerts its short-term inhibitory effects upon PCD "upstream" of both Bcl proteins and ced-3/ICE-like proteases, while chronic increased IGF-IR expression may modulate susceptibility to death signals by mediating the negative death regulator, Bcl-xL.

Cytotoxic T-lymphocytes (CTLs), by virtue of their ability to recognize and induce apoptotic death of virus-infected cells, comprise a major antiviral defense mechanism. The induction of apoptosis by CTLs can be completely accounted for by two mechanisms: (i) a Ca(2+)-dependent component that involves the exocytotic release of serine proteases known as granzymes from CTL granules and their subsequent insertion into the target cell to induce apoptosis and (ii) a Ca(2+)-independent component that involves the activation of Fas, a receptor on the target cell membrane that triggers apoptosis. Although viruses have evolved several indirect mechanisms for evading the CTL response, direct inhibition of the apoptotic cascade has never been described. We now show for the first time that the cowpox virus protein CrmA, a protease inhibitor of the serpin family, is capable of inhibiting CTL-mediated cytolysis. The inhibitory effect is largely the result of blockade of the Ca(2+)-independent (i.e. Fas-mediated) component of CTL killing. CrmA thus represents the first example of a viral gene product capable of directly blocking CTL-mediated cell death.

Monoclonal antibodies (mAbs) raised against reduced and alkylated thrombospondin (TSP) were screened for the ability to react with Ca2+-replete TSP versus EDTA-treated TSP. Two mAbs designated A6.1 and D4.6 were found to react much more strongly with TSP after EDTA treatment. The dissociation constants for these mAbs were measured in 5 mM EDTA and found to be 6 X 10(-10) M for A6.1 and 7 X 10(-9) M for D4.6. Binding to A6.1 was undetectable in the presence of 1 mM Ca2+ while binding of D4.6 occurred with about 100-fold lower affinity. The Ca2+ concentration dependence of A6.1 binding was broad with a midpoint near 50 microM free Ca2+ while that of D4.6 showed a sharp transition below 0.1 microM. Upon dialysis of EDTA-treated TSP into Ca2+ containing buffer, the binding of the mAbs was prevented or decreased, indicating reversibility of the conformational transition induced by the initial removal of Ca2+ . Mg2+ can compete with the Ca2+ binding sites involved in mAb binding, but TSP dialyzed from Ca2+ into Mg2+ binds the two mAbs as well as EDTA-treated TSP, indicating that Mg2+ cannot maintain the Ca2+-replete structure of TSP. The proteolytic fragments of TSP with which the two mAbs react were determined by probing Western blots of digests of TSP with the mAbs. A6.1 reacts with the 70-kDa fragment generated by chymotrypsin in EDTA which contains the interchain disulfide bonds of TSP and the binding site(s) for type V collagen (Mumby, S. M., Raugi, G. J., and Bornstein, P. (1984) J. Cell Biol. 98, 646-652). D4.6 reacts with fragments of 140 and 120 kDa found in digests of Ca2+-replete TSP which are absent from digests in EDTA. Electron microscopy of rotary shadowed, carbon-coated replicas of TSP mAb complexes confirms the Ca2+ sensitivity of mAb binding and has been used to localize the epitopes for both mAbs on the three-dimensional structure of TSP.

A 1 or 2% crosslinked divinylbenzene‐styrene copolymer, incorporating benzyl alcohol groups (1) was treated with p‐nitrophenyl chloroformate to give a polymer‐bound benzyl p‐nitrophenyl carbonate (2). The reaction of (2) with an excess of the symmetrical diamines 1,4‐diaminobutane (3a), 1,6‐diaminohexane (3b), 1,8‐diaminooctane (3c), 1,10‐diaminodecane (3d) and 1,12‐diaminododecane (3e) gave the respective monoprotected polymer‐bound aminocarbamates (4a‐e). Benzoylation or tosylation of (4a‐e) gave the polymer‐bound N‐benzoyl or N‐tosyl carbamates (7a‐e) or (8a‐e) respectively. Cleavage of (7a‐e) with trifluoroacetic acid liberated the N‐benzoyl‐N'‐trifluoroacetyl‐1, ω‐diaminoalkanes (9a‐e) in 80% yield. Cleavage of (8a‐e) with HCl in benzene, followed by acetylation yielded N‐acetyl‐N'‐tosyl‐1, ω‐diaminoalkanes (11a‐e) in 80% yield. From both cleavage reactions about 20% of the symmetrical diamines were recovered as their N,N'‐ditrifluoroacetyl or N,N'‐diacetyl derivatives (10a‐e) or (12a‐e) respectively.

CD40 activation is critical for B-cell function, leading to activation and expression of cell surface markers, proliferation, immunoglobulin class switching and inhibition of programmed cell death (PCD). Germinal center B-cells, for example, can be prevented from undergoing PCD by CD40 activation. The mechanism by which PCD is inhibited has been an enigma. A potential role for A20, a novel zinc finger protein, in inhibiting B-cell apoptosis was suggested by our previous finding that it is induced by the Epstein-Barr virus LMP-1 gene product, a potent cell death inhibitor. We now show that CD40 activation induces A20 and that expression of A20 renders B-cell lines resistant to PCD. Additionally, we show that CD40 activation of A20 expression is mediated by inducible binding of NF-κB complexes to the A20 promoter and provide evidence for a critical role for Thr234(in the CD40 cytoplasmic domain) in activating NF-κB.

A20, a novel zinc finger protein, is an inhibitor of tumor necrosis factor-induced apoptosis. The mechanism by which A20 exerts its protective effect is currently unknown. Several isoforms of the 14-3-3 proteins were found to interact with A20 in a yeast two-hybrid screen. A20 bound several 14-3-3 isoforms in vitro. Moreover, transfected A20 was found to preferentially bind the endogenous η14-3-3 isoform, whereas the β/ζ isoforms co-immunoprecipitated much less efficiently, and ϵ14-3-3 had an intermediate affinity. Importantly, c-Raf, a previously described 14-3-3-interacting protein, also preferentially bound the η isoform. The cellular localization and subcellular fractionation of A20 was dramatically altered by co-transfected 14-3-3, providing the first experimental evidence for the notion that 14-3-3 can function as a chaperone. Furthermore, c-Raf and A20 co-immunoprecipitated in a 14-3-3-dependent manner, suggesting that 14-3-3 can function as a bridging or adapter molecule.

B61 was originally described as a novel secreted tumor necrosis factor-α-inducible gene product in endothelial cells (Holzman, L. B., Marks, R. M., and Dixit, V. M.(1990) Mol. Cell. Biol. 10, 5830-5838). It was recently discovered that soluble recombinant B61 could serve as a ligand for the Eck receptor protein-tyrosine kinase, a member of the Eph/Eck subfamily of receptor protein-tyrosine kinases (Bartley, T. D., Hunt, R. W., Welcher, A. A., Boyle, W. J., Parker, V. P., Lindberg, R. A., Lu, H. S., Colombero, A. M., Elliott, R. L., Guthrie, R. A., Holst, P. L., Skrine, J. D., Toso, R. J., Zhang, M., Fernandez, E., Trail, G., Yarnum, B., Yarden, Y., Hunter, T., and Fox, G. M.(1994) Nature368, 558-560). We now show that B61 can also exist as a cell surface glycosylphosphatidylinositol-linked protein that is capable of activating the Eck receptor protein-tyrosine kinase, the first such report of a receptor protein-tyrosine kinase ligand that is glycosylphosphatidylinositol-linked. In addition, the expression patterns of B61 and Eck during mouse ontogeny were determined by in situhybridization. Both were found to be highly expressed in the developing lung and gut, while Eck was preferentially expressed in the thymus. Finally, the gene for B61 was localized to a specific position on mouse chromosome 3 by interspecific backcross analysis.

Previous genetic studies have established Reaper and Grim as central regulators of apoptosis in Drosophila melanogaster.Reaper and Grim induce extensive apoptosis in Drosophila, yet share no homology to known vertebrate proteins. In this study, we show for the first time that ectopic expression of Reaper or Grim induced substantial apoptosis in mammalian cells. Reaper- or Grim-induced apoptosis was inhibited by a broad range of caspase inhibitors and by human inhibitor of apoptosis proteins cIAP1 and cIAP2. Additionally, in vivobinding studies demonstrated that both Reaper and Grim physically interacted with human IAPs through a homologous 15-amino acid N-terminal segment. Deletion of this segment from either Reaper or Grim abolished binding to cIAPs.In vitrobinding experiments indicated that Reaper and Grim bound specifically to the BIR domain-containing region of cIAPs as deletion of this region resulted in loss of binding. The physical interaction was further confirmed by immunolocalization. When co-expressed, Reaper or Grim co-localized with cIAP1. However, deletion of the N-terminal 15 amino acids of Reaper or Grim abolished co-localization with cIAP1, suggesting that this homologous region can serve as a protein-protein interacting domain in regulating cell death. Moreover, by virtue of this interaction, we demonstrate that cIAPs can regulate Reaper and Grim by abrogating their ability to activate caspases and thereby inhibit apoptosis. This is the first function attributed to this 15-amino acid N-terminal domain that is the only region having significant homology between these Drosophiladeath inducers.