Biomedical subjects

V M Dixit

Publications and source records attributed to V M Dixit.

At least 73 records · Page 4Linked to original sources

PubMed1997

Bik and Bak induce apoptosis downstream of CrmA but upstream of inhibitor of apoptosis.

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.

Apoptosis↗

PubMed1997

The receptor for the cytotoxic ligand TRAIL.

TRAIL (also known as Apo-2L) is a member of the tumor necrosis factor (TNF) ligand family that rapidly induces apoptosis in a variety of transformed cell lines. The human receptor for TRAIL was found to be an undescribed member of the TNF-receptor family (designated death receptor-4, DR4) that contains a cytoplasmic "death domain" capable of engaging the cell suicide apparatus but not the nuclear factor kappa B pathway in the system studied. Unlike Fas, TNFR-1, and DR3, DR4 could not use FADD to transmit the death signal, suggesting the use of distinct proximal signaling machinery. Thus, the DR4-TRAIL axis defines another receptor-ligand pair involved in regulating cell suicide and tissue homeostasis.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Target protease specificity of the viral serpin CrmA. Analysis of five caspases.

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.

Amino Acid Sequence↗

PubMed1997

Fas-associated death domain protein interleukin-1beta-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95- and p55-mediated death signaling.

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.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death.

Previous genetic studies of the nematode Caenorhabditis elegans identified three important components of the cell death machinery. CED-3 and CED-4 function to kill cells, whereas CED-9 protects cells from death. Here CED-9 and its mammalian homolog Bcl-xL (a member of the Bcl-2 family of cell death regulators) were both found to interact with and inhibit the function of CED-4. In addition, analysis revealed that CED-4 can simultaneously interact with CED-3 and its mammalian counterparts interleukin-1beta-converting enzyme (ICE) and FLICE. Thus, CED-4 plays a central role in the cell death pathway, biochemically linking CED-9 and the Bcl-2 family to CED-3 and the ICE family of pro-apoptotic cysteine proteases.

Animals↗

PubMed1997

Reciprocal expression of the Eph receptor Cek5 and its ligand(s) in the early retina.

Recent evidence suggests that Eph receptor tyrosine kinases and their ligands provide positional information in the developing visual system. We previously found that the Eph receptor Cek5 is more highly expressed in the ventral than dorsal chicken embryonic retina. We now report the identification of a chicken ligand for Cek5 (cCek5-L) that is 75% identical to the ligand LERK2. In situ hybridization experiments do not reveal a dorsoventral gradient of cCek5-L transcripts in the optic tectum at Embryonic Day 8, suggesting that this ligand is not involved in guiding Cek5-expressing axons in the tectum. Surprisingly, it is in the retina that high levels of cCek5-L mRNA are present. In the early retina, cCek5-L is more highly expressed in the dorsal than the ventral aspect. Similarly, a Cek5 Ig chimera labels dorsal but not ventral retina, indicating that even if several Cek5 ligands are present, their overall distribution is complementary to that of Cek5. Hence, Cek5 and cCek5-L may both contribute to define anatomical compartments within the early retina. In contrast, in the 11-day embryonic retina the distributions of Cek5 and its ligand(s) show considerable overlap, suggesting changing functions as development progresses. In dissociated cultures of dorsal or ventral retinal cells seeded on plates coated with either receptor or ligand Ig chimeras, the interaction between Cek5 and its ligand(s) or cCek5-L and its receptor(s) is sufficient to mediate cell adhesion and allows neurite outgrowth.

Amino Acid Sequence↗

PubMed1997

FLICE induced apoptosis in a cell-free system. Cleavage of caspase zymogens.

Engagement of CD95 or tumor necrosis factor 1 receptor (TNFR-1) by ligand or agonist antibodies is capable of activating the cell death program, the effector arm of which is composed of mammalian interleukin-1beta converting enzyme (ICE)-like cysteine proteases (designated caspases) that are related to the Caenorhabditis elegans death gene, CED-3. Caspases, unlike other mammalian cysteine proteases, cleave their substrates following aspartate residues. Furthermore, proteases belonging to this family exist as zymogens that in turn require cleavage at internal aspartate residues to generate the two-subunit active enzyme. As such, family members are capable of activating each other. Remarkably, both CD95 and TNFR-1 death receptors initiate apoptosis by recruiting a novel ICE/CED-3 family member, designated FLICE/MACH, to the receptor signaling complex. Therefore, FLICE/MACH represents the apical triggering protease in the cascade. Consistent with this, recombinant FLICE was found capable of proteolytically activating downstream caspases. Furthermore, CrmA, a pox virus-encoded serpin that inhibits Fas and tumor necrosis factor-induced cell death attenuates the ability of FLICE to activate downstream caspases.

Antigens, CD↗

PubMed1997

RAIDD is a new 'death' adaptor molecule.

The effector arm of the cell-death pathway is composed of cysteine proteases belonging to the ICE/CED-3 family. In metazoan cells these exist as inactive polypeptide precursors (zymogens), each composed of a prodomain, which is cleaved to activate the protease, and a large and small catalytic subunit. The coupling of these 'death' proteases to signalling pathways is probably mediated by adaptor molecules that contain protein-protein interaction motifs such as the death domain. Here we describe such an adaptor molecule, RAIDD, which has an unusual bipartite architecture comprising a carboxy-terminal death domain that binds to the homologous domain in RIP, a serine/threonine kinase component of the death pathway. The amino-terminal domain is surprisingly homologous with the sequence of the prodomain of two ICE/CED-3 family members, human ICH-1 (ref. 5) and Caenorhabditis elegans CED-3 (ref. 6). This similar region mediates the binding of RAIDD to ICH-1 and CED-3, serving as a direct link to the death proteases, indicating that the prodomain may, through homophilic interactions, determine the specificity of binding of ICE/CED-3 zymogens to regulatory adaptor molecules. Finally, alternations in the sequence of the N-terminal domain that are equivalent to inactivating mutations in the C. elegans ced-3 gene prevent homophilic binding, highlighting the potentially primordial nature of this interaction.

Amino Acid Sequence↗

PubMed1997

Portrait of an executioner: the molecular mechanism of FAS/APO-1-induced apoptosis.

Fas/APO-1 is a member of the TNF receptor family and plays a dominant role in the immune system, down-regulating the immune response and contributing to T-cell-mediated cytotoxicity. Activation of Fas/APO-1 by either natural ligand or agonist antibody initiates apoptosis. Until recently, however, the mechanism by which Fas/APO-1 engages the cell death machinery has been an enigma. Here we describe a proposed molecular mechanism by which this cell surface death receptor signals the suicide response.

Animals↗

PubMed1997

The inhibition of pro-apoptotic ICE-like proteases enhances HIV replication.

Accelerated programmed cell death, or apoptosis, contributes to the CD4+ T-cell depletion characteristic of infection by human immunodeficiency virus (HIV). It has therefore been proposed that limiting apoptosis may represent a therapeutic modality for HIV infection. We found, however, that T leukemia cells or peripheral blood mononuclear cells (PBMCs) exposed to HIV-1 underwent enhanced viral replication in the presence of the cell death inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-AVD-fmk). Furthermore, z-VAD-fmk, which targets the pro-apoptotic interleukin-1 beta-converting enzyme (ICE)-like proteases, stimulated endogenous virus production in activated PBMCs derived from HIV-1-infected asymptomatic individuals. These findings suggest that programmed cell death may serve as a beneficial host mechanism to limit HIV spread and that strategies to inhibit it may have deleterious consequences for the infected host.

Amino Acid Chloromethyl Ketones↗

PubMed1997

Thrombospondin-1 suppresses tumorigenesis and angiogenesis in serum- and anchorage-independent NIH 3T3 cells.

Thrombospondin-1 (TSP1) is a multifunctional matrix protein that influences the growth and function of a variety of normal and neoplastic epithelial and mesenchymal cell types. In vivo, TSP1 has shown potent antitumor activity in suppressing tumor neovascularization. Paradoxically, however, as we have reported, NIH 3T3 fibroblasts overexpressing TSP1 acquire the transformation-associated phenotypes of serum and anchorage independence in vitro but fail to form tumors in nude mice. To investigate these divergent results, and to determine the functional domains in TSP1 that confer serum and anchorage independence as well as antitumor and antiangiogenic activities, we transfected a series of deletion constructs of TSP1 into NIH 3T3 cells and into a v-src-transformed NIH 3T3 line. The antiangiogenic activity of TSP1-expressing, v-src-transformed NIH 3T3 cells was examined by assaying the conditioned media for inhibition of endothelial cell chemotaxis and suppression of basic fibroblast growth factor-mediated angiogenesis in the rat cornea. The link between TSP1 antitumor and antiangiogenic activities was assessed by measuring the rate of tumor growth and counting factor VIII-stained microvessels in the solid tumors developing in nude mice. Our results indicate that v-src NIH 3T3 cells transfected with a 449-amino acid N-terminal domain of TSP1 exhibit a dose-dependent suppression of tumor growth and neovascularization in nude mice. Truncated forms of TSP1 containing the type 1 properdin domain suppressed both endothelial cell chemotaxis and comeal neovascularization. Furthermore, when full-length TSP1 and deletion constructs containing the antiangiogenic type I properdin domain were transfected into highly tumorigenic v-src-transformed NIH 3T3 cells, they were able to confer transdominant suppression of tumorigenicity and angiogenesis of these cells in nude mice. These results confirm the role of TSP1 as a potent inhibitor of angiogenesis and provide support for the notion that alterations in the net balance between inducers and inhibitors of angiogenesis are largely responsible for the sustained growth of solid tumors in vivo.

3T3 Cells↗

PubMed1996

Type I insulin-like growth factor receptor activation regulates apoptotic proteins.

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.

Apoptosis↗

PubMed1996

CD95 (APO-1/Fas) induces activation of SAP kinases downstream of ICE-like proteases.

Triggering of CD95 (APO-1/Fas) on different T- and B-cell lines resulted in the induction of a number of kinases (35 kDa, 38 kDa, 46 kDa and 54 kDa) that phosphorylate c-Jun and to a lesser extent Histone H1. Activation of these kinases was independent of protein biosynthesis and preceded apoptotic DNA degradation. The kinase activation pattern was specific for CD95 triggering since a variety of physical or chemical inducers of T- and B-cell apoptosis activated different kinases. The kinase activities at 46 and 54 kDa contained members of the stress-activated family of protein kinases (JNK/SAPK). Activation of the CD95-specific set of kinases was prevented by treating cells with the ICE-inhibiting peptide N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) or by overexpression of the cow pox virus serpin CrmA. However, despite inhibition of ICE-like proteases the death signal was readily initiated at the cell membrane since a CD95 death-inducing signaling complex (DISC) was formed. Thus, our results demonstrate that ICE-like proteases in the CD95 pathway function downstream of the DISC but upstream of SAP kinases.

Animals↗

PubMed1996

New paradigm for lymphocyte granule-mediated cytotoxicity. Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis.

Lymphocyte granule-mediated apoptosis is postulated to entail the formation of membrane pores by perforin. Then soluble granzyme reaches the cytosol either through these pores or by reparative pinocytosis. We demonstrate here that Jurkat cells bind and internalize granzyme B via high affinity binding sites without toxic consequence. Apoptosis occurs, however, if sublytic perforin is added to targets washed free of soluble granzyme B. We suggest that granule-mediated apoptosis mimics viral strategies for cellular entry. Accordingly, co-internalization of granzyme B with adenovirus, a virus that escapes endosomes to reach the cytosol, also induced apoptosis. Poly(ADP-ribose) polymerase cleavage and processing of CPP32, ICE-LAP3, and Mch2 were detected at 30 min, while cytosolic acidification and DNA fragmentation occurred at 60 min. Annexin V binding and membrane permeabilization arose at 4 h. The concurrent activation of the Ced-3 proteases differed from the rate at which each cysteine protease is cleaved in vitro by granzyme B. Thus, granzyme B may not directly process these proteases in whole cells but rather may function by activating a more proximal enzyme. These results indicate that adenovirus-mediated delivery of granzyme B is suitable for elucidating biochemical events that accompany granule-mediated apoptosis.

Apoptosis↗

PubMed1996

Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95.

Tumor necrosis factor receptor-1 (TNFR-1) and CD95 (also called Fas or APO-1) are cytokine receptors that engage the apoptosis pathway through a region of intracellular homology, designated the "death domain." Another death domain-containing member of the TNFR family, death receptor 3 (DR3), was identified and was shown to induce both apoptosis and activation of nuclear factor kappaB. Expression of DR3 appears to be restricted to tissues enriched in lymphocytes. DR3 signal transduction is mediated by a complex of intracellular signaling molecules including TRADD, TRAF2, FADD, and FLICE. Thus, DR3 likely plays a role in regulating lymphocyte homeostasis.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Molecular ordering of apoptotic mammalian CED-3/ICE-like proteases.

Apoptosis is executed by cysteine proteases belonging to the CED-3/ICE family, which, unlike other mammalian cysteine proteases, cleave their substrates following aspartate residues. Proteases belonging to this family exist in the cytosol as zymogens that require accurate processing at internal aspartate residues to generate the two-chain active enzymes. As such, CED-3/ICE family members are capable of activating each other in a manner analogous to the protease zymogens of the coagulation or complement cascades. At present, it is unknown whether such mutual processing exists in vivo, and if so whether it is sequential, implying an order to the death pathway. Using a cell-free apoptosis system, recombinant ICE proteases and both biochemical and morphological criteria, we demonstrate an ordering of the mammalian ICEs that are most related to the Caenorhabditis elegans death protease CED-3.

Animals↗

PubMed1996

14-3-3 proteins associate with A20 in an isoform-specific manner and function both as chaperone and adapter molecules.

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 eta14-3-3 isoform, whereas the beta/zeta isoforms co-immunoprecipitated much less efficiently, and epsilon14-3-3 had an intermediate affinity. Importantly, c-Raf, a previously described 14-3-3-interacting protein, also preferentially bound the eta 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.

14-3-3 Proteins↗

PubMed1996

ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B.

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.

Amino Acid Sequence↗