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PubMed1985

Oligomeric structure of the multifunctional protein CAD that initiates pyrimidine biosynthesis in mammalian cells.

The first three steps in mammalian de novo pyrimidine biosynthesis are catalyzed by the multifunctional protein designated CAD. Regions of the single 240-kDa poly-peptide chain are folded into separate structural domains that have discrete functions. Previous studies suggested that CAD forms predominantly trimers. The trimers are found to be in slow equilibrium with hexamers and higher oligomers composed of multiples of three copies of the CAD polypeptide chain. However, quantitative chemical crosslinking studies of CAD with dimethyl suberimidate were used here to show a progressive conversion of monomer to crosslinked hexamer. High levels of the hexamer accumulate in the reaction mixture, suggesting that the major oligomeric form is hexameric, although residual amounts of smaller oligomers remain present. Larger oligomers may form by association of hexamers and are seen after longer crosslinking times. Sucrose gradient centrifugation shows a 20.8S species to be the slowest sedimenting peak, while the larger species sediments at 27.9S. Electron microscopic studies of rotary-shadowed preparations of CAD have confirmed that, while small amounts of other oligomeric forms are present, the CAD monomer is primarily associated into cyclic hexamers with an open planar appearance.

Animals↗
PubMed1987

Shared active sites in oligomeric enzymes: model studies with defective mutants of aspartate transcarbamoylase produced by site-directed mutagenesis.

Many oligomeric enzymes are functional only in the assembled form, and it is often difficult to determine unambiguously why monomers are inactive. In some cases individual monomers cannot fold into stable correct ("native") conformations without contributions from interchain interactions. For other oligomers, catalysis requires the contributions of amino acid residues at the interface between adjacent polypeptide chains, and monomers are inactive because they cannot form complete active sites. A test for the presence of shared sites was devised that is based on the formation of active hybrid oligomers from appropriate inactive parental mutants produced by site-directed mutagenesis. This approach was applied in a study of the catalytic trimer of aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, using three mutants, in which Ser-52 was replaced by His, Lys-84 by Gln, or His-134 by Ala. Hybrid trimers formed from the virtually inactive Ser and Lys mutants were 10(5) more active than the parental proteins, and the specific activities of each hybrid were about 33% that of the wild-type trimer, as expected for the scheme based on shared sites. Hybrids from the His and Lys mutants had comparable specific activities. Moreover, one hybrid with approximately 33% activity had one high-affinity binding site for a bisubstrate analog as compared to about three for wild-type trimer. As a further test, hybrids were also formed from wild-type and double-mutant (Lys-84----Gln and His-134----Ala) trimers. The hybrid containing two chains with the double mutation and one wild-type chain had very little activity, and that composed of one double mutant and two wild-type chains had 32% the specific activity of wild-type trimers. This negative complementation experiment is in quantitative accord with the scheme based on shared sites at or near the interfaces between adjacent chains. The techniques used to demonstrate shared active sites in the catalytic subunits of aspartate transcarbamoylase can be applied generally to various types of oligomers (dimers, tetramers, etc.) to determine whether the participation of amino acid residues from adjoining chains is essential for forming active sites in oligomeric enzymes.

Alleles↗
PubMed1995

Periodicity of polar and nonpolar amino acids is the major determinant of secondary structure in self-assembling oligomeric peptides.

The tendency of a polypeptide chain to form alpha-helical or beta-strand secondary structure depends upon local and nonlocal effects. Local effects reflect the intrinsic propensities of the amino acid residues for particular secondary structures, while nonlocal effects reflect the positioning of the individual residues in the context of the entire amino acid sequence. In particular, the periodicity of polar and nonpolar residues specifies whether a given sequence is consistent with amphiphilic alpha-helices or beta-strands. The importance of intrinsic propensities was compared to that of polar/nonpolar periodicity by a direct competition. Synthetic peptides were designed using residues with intrinsic propensities that favored one or the other type of secondary structure. The polar/nonpolar periodicities of the peptides were designed either to be consistent with the secondary structure favored by the intrinsic propensities of the component residues or in other cases to oppose these intrinsic propensities. Characterization of the synthetic peptides demonstrated that in all cases the observed secondary structure correlates with the periodicity of the peptide sequence--even when this secondary structure differs from that predicted from the intrinsic propensities of the component amino acids. The observed secondary structures are concentration dependent, indicating that oligomerization of the amphiphilic peptides is responsible for the observed secondary structures. Thus, for self-assembling oligomeric peptides, the polar/nonpolar periodicity can overwhelm the intrinsic propensities of the amino acid residues and serves as the major determinant of peptide secondary structure.

Amino Acid Sequence↗
PubMed1995

Oligomerization of CD4 is required for stable binding to class II major histocompatibility complex proteins but not for interaction with human immunodeficiency virus gp120.

Previous studies have failed to detect an interaction between monomeric soluble CD4 (sCD4) and class II major histocompatibility complex (MHC) proteins, suggesting that oligomerization of CD4 on the cell surface may be required to form a stable class II MHC binding site. To test this possibility, we transfected the F43I CD4 mutant, which is incapable of binding to class II MHC or human immunodeficiency virus (HIV) gp120, into COS-7 cells together with wild-type CD4 (wtCD4). Expression of F43I results in a dominant negative effect: no class II MHC binding is observed even though wtCD4 expression is preserved. Apparently, F43I associates with wtCD4 oligomers and interferes with the formation of functional class II MHC binding structures. In contrast, F43I does not affect the binding of gp120 to wtCD4, implying that gp120 binds to a CD4 monomer. By production and characterization of chimeric CD4 molecules, we show that domains 3 and/or 4 appear to be involved in oligomerization. Several models of the CD4-class II MHC interaction are offered, including the possibility that one or two CD4 molecules initially interact with class II MHC dimers and further associate to create larger complexes important for facilitating T-cell receptor crosslinking.

Animals↗
PubMed1997

Peptidyl prolyl cis-trans isomerase activity of cyclophilin A in functional homo-oligomeric receptor expression.

The functional expression of homo-oligomeric alpha7 neuronal nicotinic and type 3 serotonin receptors is dependent on the activity of a cyclophilin. In this paper we demonstrate that the mechanism of cyclophilin action during functional homo-oligomeric receptor expression in Xenopus oocytes is distinct from the calcineurin-dependent immunosuppressive mechanism by showing that a nonimmunosuppressive analog of cyclosporin A (CsA), SDZ 211-811, reduces functional receptor expression to the same extent as CsA. The cytoplasmic subtype of cyclophilin, cyclophilin A (CyPA), appears to be required for functional receptor expression. This is because overexpression of CyPA and a CyPA mutant that is deficient in CsA binding activity reverses CsA-induced reduction in functional receptor expression. The mechanism of action of CyPA is likely to involve its prolyl isomerase activity because a mutant CyPA with a single amino acid substitution (arginine 55 to alanine) that is predicted to produce a 1000-fold attenuation in isomerase activity fails to reverse the cyclosporin A effect. Our data also suggest that CyPA does not form a stable complex with receptor subunits.

Amino Acid Isomerases↗
PubMed1998

Oligomerization of activated D- and L-guanosine mononucleotides on templates containing D- and L-deoxycytidylate residues.

The oligomerization of activated D- and L- and racemic guanosine-5'-phosphoro-2-methylimidazole on short templates containing D- and L-deoxycytidylate has been studied. Results obtained with D-oligo(dC)s as templates are similar to those previously reported for experiments with a poly(C) template. When one L-dC or two consecutive L-dCs are introduced into a D-template, regiospecific synthesis of 3'-5' oligo(G)s proceeds to the end of the template, but three consecutive L-dCs block synthesis. Alternating D-,L-oligomers do not facilitate oligomerization of the D-, L-, and racemic 2-guanosine-5'-phosphoro-2-methylimidazole. We suggest that once a "predominately D-metabolism" existed, occasional L-residues in a template would not have led to the termination of self-replication.

Dimerization↗
PubMed1998

The crystal structure of Pyrococcus furiosus ornithine carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures.

The Pyrococcus furiosus (PF) ornithine carbamoyltransferase (OTCase; EC 2.1.3.3) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100 degrees C. To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 A and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A striking exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme. We now report that the thermostable PF OTCase (420 kDa) presents the same 23-point group symmetry. The enzyme displays Michaelis-Menten kinetics. A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif. Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.

Allosteric Site↗
PubMed2000

Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral.

DsRed is a recently cloned 28-kDa fluorescent protein responsible for the red coloration around the oral disk of a coral of the Discosoma genus. DsRed has attracted tremendous interest as a potential expression tracer and fusion partner that would be complementary to the homologous green fluorescent protein from Aequorea, but very little is known of the biochemistry of DsRed. We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching. In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met. However, DsRed has major drawbacks, such as strong oligomerization and slow maturation. Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro, and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells. Also, DsRed takes days to ripen fully from green to red in vitro or in vivo, and mutations such as Lys-83 to Arg prevent the color change. Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.

Amino Acid Sequence↗
PubMed1995

Oligomerization of a trans-Golgi/trans-Golgi network retained protein occurs in the Golgi complex and may be part of its retention.

The mouse hepatitis virus M protein is a triple spanning membrane glycoprotein that, when expressed independently, localizes to trans-Golgi as well as to the trans-Golgi network (TGN). Passage of this protein from the endoplasmic reticulum through the intermediate compartment to the late Golgi and TGN can be conveniently followed by analyzing its O-linked sugars. Using pulse-chase analyses we studied the oligomerization of the M protein in sucrose gradients. The Golgi and TGN forms migrated as large heterogeneous complexes, whereas the endoplasmic reticulum and intermediate compartment forms of the protein appeared to migrate as monomer. Moreover, a mutant of the M protein lacking the 22 COOH-terminal amino acids, that is transported to the plasma membrane, gave rise to similar complexes, albeit smaller in size, that persisted at the plasma membrane. We propose that the trans-Golgi/TGN retention of the MHV-M protein is governed by two mechanisms: oligomerization possibly mediated by the transmembrane domains and binding of its cytoplasmic tail to cellular factors in trans Golgi/TGN.

Animals↗
PubMed1996

Function of the duplicated IIB domain and oligomeric structure of the fructose permease of Escherichia coli.

The fructose permease of Escherichia coli, the fructose-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), contains a duplicated IIB domain. The protein therefore consists of three distinct domains, B', B, and C (N-terminal to C-terminal), joined by flexible linkers and is thus designated FruB'BC. The N-terminal B' domain was removed using molecular genetic techniques, and the truncated Enzyme II (FruBC) was characterized relative to the wild-type enzyme both in vivo and in vitro. In vivo, FruBC exhibited depressed fermentation characteristics at low fructose concentrations. [14C]Fructose uptake measurements revealed reduced rates only when the permease was rate-limiting for transport. In vitro, FruBC exhibited a 10-fold lower affinity for its phosphoryl donating protein, the IIA-FPr diphosphoryl transfer protein (DTP), than was observed with the wild-type enzyme, and the maximal velocity of fructose phosphorylation was 7-fold depressed. Because the fructose-1-phosphate:[14C]fructose transphosphorylation reaction appeared normal, we conclude that the loss of the B' domain primarily affected phosphoryl transfer between the IIA and IIB domains of the permease. A mutant FruBC derivative with cysteine 112 replaced by serine (C112S FruBC) was inactive as a phosphoryl carrier and a sugar transport protein. Expression of the plasmid-encoded mutant protein inhibited the in vivo activity of the chromosomally encoded wild-type fructose permease, but it did not observably affect the activities of the mannitol or glucitol PTS permeases or of non-PTS sugar permeases. Further, the presence of the detergent extracted mutant protein inhibited the activity of the detergent solubilized wild-type or FruBC enzyme. In contrast, the wild-type FruB BC permease was apparently epistatic over the truncated FruBC permease in vivo. The experiments reported 1) show that the B' domain of the fructose permease functions to facilitate phosphoryl transfer between DTP and the permease, 2) establish the essentiality of cysteine 112 in the B domain of the permease, 3) provide evidence that a functional fructose permease consists of an oligomer in which both IIB domains must be active for the enzyme to catalyze normal rates of phosphoryl transfer and transport, 4) suggest that a single B' domain in the oligomeric Enzyme II is sufficient to allow high efficiency phosphoryl transfer between the IIA domain of DTP and the IIB domain of the permease, and 5) show that the B' domain is not important for oligomerization.

Bacterial Proteins↗
PubMed1996

Covalent dimerization of CD28/CTLA-4 and oligomerization of CD80/CD86 regulate T cell costimulatory interactions.

T lymphocyte receptors CD28 and CTLA-4 bind costimulatory molecules CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells and regulate T cell activation. While distinct functional roles have been ascribed to each of these molecules, little is known about how they interact. To better characterize these interactions, we have used surface plasmon resonance to perform equilibrium and kinetic binding analyses of extracellular fragments of CD28/CTLA-4/CD80/CD86. We show that CTLA-4 and CD28 binding are both characterized by rapid kinetic on-rates and rapid dissociation rates. Native disulfide-linked homodimers of CD28 and CTLA-4 bound with two kinetically distinct binding sites, one of high avidity and slow dissociation and one of low avidity and more rapid dissociation. Monomeric CTLA-4 bound only with low affinity and rapid dissociation. Therefore, covalent dimerization of CTLA-4 is required for its high avidity binding. Oligomerization of CD80/CD86 is also required for high avidity CTLA-4 binding since CTLA-4 bound with low avidity to monomeric CD86. This contrasts with the ability of CD80/CD86 on antigen-presenting cells to bind CTLA4Ig with high avidity and predicts their organization as oligomers or clusters that permit multivalent binding. Thus, covalent receptor dimerization and ligand oligomerization are two key features of the CD28/CTLA-4/CD80/CD86 receptor system that control ligand binding and may regulate signal transduction by controlling the duration of receptor occupancy.

Abatacept↗
PubMed1996

Negative dominance studies demonstrate the oligomeric structure of EmrE, a multidrug antiporter from Escherichia coli.

EmrE, the smallest known ion-coupled transporter, is an Escherichia coli 12-kDa protein 80% helical and soluble in organic solvents. EmrE is a polyspecific antiporter that exchanges hydrogen ions with aromatic toxic cations such as methyl viologen. Since it is many times smaller than the classical consensus 12 transmembrane segments transporters, it was particularly interesting to determine its oligomeric state. For this purpose, a series of nonfunctional mutants has been generated and characterized to test their effect on the activity of the wild-type protein upon mixing. As opposed to the wild type, these mutants do not confer resistance to methyl viologen, ethidium bromide, or a series of other toxicants. Co-expression of each of the nonfunctional mutants with the wild-type protein results in a reduction in the ability of the functional transporter to confer resistance to several toxicants. To perform mixing experiments in vitro, all the mutants have been purified by extraction with organic solvents, reconstituted in proteoliposomes, and found to be inactive. When co-reconstituted with wild-type protein, they inhibit the activity of the latter in a dose-dependent form up to full inhibition. We assume that this inhibition is due to the formation of mixed oligomers in which the presence of one nonfunctional subunit causes full inactivation. A binomial analysis of the results based on the latter assumptions do not provide statistically significant answers but suggests that the oligomer is composed of three subunits. The results described provide the first in vitro demonstration of the functional oligomeric structure of an ion-coupled transporter.

Antiporters↗
PubMed1997

Multiple molecular chaperones complex with misfolded large oligomeric glycoproteins in the endoplasmic reticulum.

Thyroglobulin (Tg), the major protein secreted by thyroid epithelial cells and precursor of thyroid hormones, is a large dimeric glycoprotein with multiple disulfide bonds. The folding and assembly of this complex molecule begins in the endoplasmic reticulum (ER) and is likely to involve a variety of reactions catalyzed by molecular chaperones (Kuznetsov, G., Chen, L. B., and Nigam, S. K. (1994) J. Biol. Chem. 269, 22990-22995). By coimmunoprecipitation in rat thyroid cells, we were able to demonstrate that BiP, grp94, ERp72, and grp170, four proteins believed to function as specific molecular chaperones, complex with Tg during its maturation. The same complex of the four putative chaperones with Tg was observed in cells treated with tunicamycin, indicating that these four ER chaperones stably associate with Tg when it is misfolded/misassembled due to inhibition of its glycosylation. BiP, grp94, and ERp72 were also found to associate with Tg in cells in which misfolding was induced by perturbing ER calcium stores. To determine if the assembly of a complex between the four chaperones and Tg under conditions of misglycosylation was unique to the maturation of this particular secretory protein or a more general phenomenon, adenovirus-transformed rat thyroid cells that do not synthesize Tg were analyzed. In these transformed cells, the only protein these same four chaperones were found to complex with was a protein of approximately 200 kDa. This protein was subsequently identified as thrombospondin, which, like Tg, is a large oligomeric secreted glycoprotein with multiple disulfide bonds. We therefore propose that these ER chaperones complex together with a variety of large oligomeric secretory glycoproteins as they fold and assemble in the ER.

Animals↗
PubMed1997

Assembly of immunoglobulin light chains as a prerequisite for secretion. A model for oligomerization-dependent subunit folding.

Oligomeric proteins usually have to assemble into their final quartenary structure to be secreted. However, most immunoglobulin (Ig) light (L) chains can be exported as free chains, whereas only a few Ig L chains, here referred to as export-incompetent, have to assemble with Ig heavy (H) chains into antibody molecules to be secreted. In the absence of Ig H chain expression, these export-incompetent Ig L chains remain bound to BiP as partially folded monomers with only one of the two internal disulfide bonds being formed. To understand the apparent discrepancy in Ig L chain export, we performed assembly studies with chimeric Ig chains and found that the variable (V) domain of the export-incompetent NS1 kappa chain cannot mediate homodimer formation. Conversely, the V domain of the export-competent J558L lambda1 chain supports homodimer formation and, concordantly, these Ig L chains are secreted as noncovalently or covalently linked homodimers. We show that the export-incompetent mutant lambda1 FS62 chain forms disulfide bonds in both domains only upon pairing with Ig H chain and is secreted as part of an antibody. Therefore, Ig L chain assembly seems to be a prerequisite for complete folding, indicating that Ig L chain secretion generally depends on either homo- or heterodimer formation. We discuss a mechanism that controls oligomerization by monitoring the conformation of individual subunits that cannot proceed in folding prior to successful assembly.

Animals↗
PubMed1998

A revised model for the oligomeric state of the N-ethylmaleimide-sensitive fusion protein, NSF.

The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking. Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions. However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers. We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical techniques. Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer. Sedimentation equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains. The sedimentation coefficient, s20,w0, of 13.4 (+/-0. 1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape. The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions.

Adenosine Triphosphatases↗
PubMed1998

Domains mediating intramolecular folding and oligomerization of MxA GTPase.

MxA is an interferon-induced GTPase of human cells that inhibits the multiplication of several RNA viruses by a still poorly understood mechanism. Previous biochemical studies indicated that the C terminus of MxA folds back to form a functional GTP-binding pocket, and that an internal fragment contains a domain required for oligomerization. Using the yeast two-hybrid system, we have now mapped these domains. MxA sequences located downstream of amino acid 564 were found to strongly interact with an internal domain that includes amino acids 372 to 540. This interaction was abolished by mutating phenylalanine 382 or leucine 612, which is part of a leucine zipper motif. Neither the C-terminal nor the internal MxA fragments formed homo-oligomers. Using a mammalian nuclear transport assay that can detect protein-protein interactions, we further found that full-length MxA forms complexes with MxA fragments that include amino acids 372 to 540. This interaction was not observed when phenylalanine 382 was exchanged for alanine or arginine. Furthermore, interaction of two full-length MxA molecules occurred only if at least one of them carried a functional C-terminal leucine zipper motif. These results suggest that C-terminal back-folding and oligomerization are two alternative outcomes of the same type of interaction between the C-terminal and the internal domains of MxA. Intramolecular interaction is believed to result in the formation of MxA monomers, whereas intermolecular interaction may induce the formation of large MxA oligomers.

Binding Sites↗
PubMed1998

A transmembrane domain-derived peptide inhibits D1 dopamine receptor function without affecting receptor oligomerization.

In this study, we show that a peptide based on the sequence of transmembrane domain 6 of the D1 dopamine receptor (D1DR) specifically inhibited D1DR binding and function, without affecting receptor oligomerization. It has been shown that an analogous peptide from the beta2-adrenergic receptor disrupted dimerization and adenylyl cyclase activation in the beta2-adrenergic receptor (Hebert, T. E., Moffett, S., Morello, J. P., Loisel, T. P., Bichet, D. G., Barret, C., and Bouvier, M. (1996) J. Biol. Chem. 271, 16384-16392). Treatment of D1DR with the D1DR transmembrane 6 peptide resulted in a dose-dependent, irreversible inhibition of D1DR antagonist binding, an effect not seen in D1DR with peptides based on transmembrane domains of other G protein-coupled receptors. Incubation with the D1DR transmembrane 6 peptide also resulted in a dose-dependent attenuation of both dopamine-induced [35S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and receptor-mediated dopamine stimulation of adenylyl cyclase activity. Notably, GTPgammaS binding and cAMP production were reduced to levels below baseline, indicating blockade of ligand-independent, intrinsic receptor activity. Immunoblot analyses of the D1DR revealed the receptor existed as monomers, dimers, and higher order oligomers and that these oligomeric states were unaffected after incubation with the D1DR transmembrane 6 peptide. These findings represent the first demonstration that a peptide based on the transmembrane 6 of the D1DR may represent a novel category of noncompetitive D1DR antagonists.

Animals↗
PubMed1998

Membrane oligomerization and cleavage activates the caspase-8 (FLICE/MACHalpha1) death signal.

Many forms of apoptosis, including that caused by the death receptor CD95/Fas/APO-1, depend on the activation of caspases, which are proteases that cleave specific intracellular proteins to cause orderly cellular disintegration. The requirements for activating these crucial enzymatic mediators of death are not well understood. Using molecular chimeras with either CD8 or Tac, we find that oligomerization at the cell membrane powerfully induces caspase-8 autoactivation and apoptosis. Death induction was abrogated by the z-VAD-fmk, z-IETD-fmk, or p35 enzyme inhibitors or by a mutation in the active site cysteine but was surprisingly unaffected by death inhibitor Bcl-2. Amino acid substitutions that prevent the proteolytic separation of the caspase from its membrane-associated domain completely blocked apoptosis. Thus, oligomerization at the membrane is sufficient for caspase-8 autoactivation, but apoptosis could involve a death signal conveyed by the proteolytic release of the enzyme into the cytoplasm.

Apoptosis↗