NORMAL GENOMIC

• Hippe D, Lytovchenko O, Schmitz I, Luder CG
• Fas/CD95-mediated apoptosis of type II cells is blocked by Toxoplasmagondii primarily via interference with the mitochondrial amplificationloop.
• Infect Immun. 2008; 76: 2905-12
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• The intracellular protozoan Toxoplasma gondii induces persistentinfections in various hosts and is an important opportunistic pathogen ofhumans with immature or deficient immune responses. The ability to surviveintracellularly largely depends on the blocking of different proapoptoticsignaling cascades of its host cell. Fas/CD95 triggers an apoptoticcascade that is crucial for immunity and the outcome of infectiousdiseases. We have determined the mechanism by which T. gondii counteractsdeath receptor-mediated cell death in type II cells that transduceFas/CD95 ligation via caspase 8-mediated activation of the mitochondrialamplification loop. The results showed that infection with T. gondiisignificantly reduced Fas/CD95-triggered apoptosis in HeLa cells byinhibiting the activities of initiator caspases 8 and 9 and effectorcaspase 3/7. Parasitic infection dose dependently diminished cleavage ofcaspase 8, the BH3-only protein Bid, and the downstream caspases 9 and 3.Importantly, interference with Fas/CD95-triggered caspase 8 and caspase3/7 activities after parasitic infection was largely dependent on thepresence of caspase 9. Within the mitochondrial amplification loop, T.gondii significantly inhibited the Fas/CD95-triggered release ofcytochrome c into the host cell cytosol. These results indicate that T.gondii inhibits Fas/CD95-mediated apoptosis in type II cells primarily bydecreasing the apoptogenic function of mitochondria.

• Yang JK et al.
• Crystal structure of MC159 reveals molecular mechanism of DISC assemblyand FLIP inhibition.
• Mol Cell. 2005; 20: 939-49
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• The death-inducing signaling complex (DISC) comprising Fas, Fas-associateddeath domain (FADD), and caspase-8/10 is assembled via homotypicassociations between death domains (DDs) of Fas and FADD and between deatheffector domains (DEDs) of FADD and caspase-8/10. Caspase-8/10 andFLICE/caspase-8 inhibitory proteins (FLIPs) that inhibit caspaseactivation at the DISC level contain tandem DEDs. Here, we report thecrystal structure of a viral FLIP, MC159, at 1.2 Angstroms resolution. Itreveals a noncanonical fold of DED1, a dumbbell-shaped structure withrigidly associated DEDs and a different mode of interaction in the DDsuperfamily. Whereas the conserved hydrophobic patch of DED1 interactswith DED2, the corresponding region of DED2 mediates caspase-8 recruitmentand contributes to DISC assembly. In contrast, MC159 cooperativelyassembles with Fas and FADD via an extensive surface that encompasses theconserved charge triad. This interaction apparently competes with FADDself-association and disrupts higher-order oligomerization required forcaspase activation in the DISC.

• Acheampong EA et al.
• Molecular interactions of human immunodeficiency virus type 1 with primaryhuman oral keratinocytes.
• J Virol. 2005; 79: 8440-53
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• Infection of the oral mucosa of human immunodeficiency virus type 1(HIV-1)-infected individuals remains an under-evaluated and somewhatenigmatic process. Nonetheless, it is of profound importance in theongoing AIDS pandemic, based on its potential as a site ofperson-to-person transmission of the virus as well as a location of HIV-1pathogenesis and potential reservoir of disease in the setting of virallysuppressive highly active antiretroviral therapy. We utilized molecularand virological techniques to analyze HIV-1 infection of primary humanmucosal cells and also evaluated the proapoptotic potential of selectedHIV-1 proteins in primary isolated human oral keratinocytes. Primaryisolated human oral keratinocytes were plated on 0.4 microMpolyethylenetetraphthalate cell culture inserts to form an in vitro oralmucosal layer. The strength of this layer in forming a barrier wasdetermined by measuring trans-epithelial electrical current passage acrossthe monolayer. The oral keratinocyte monolayers had trans-epithelialelectrical resistance of approximately 176 to 208 omega. For viralinfectivity assays, the macrophage-tropic (R5) HIV-1 strains, YU-2 andADA, and T-cell-line-tropic (X4), NL4-3 virions, incubated with or withoutdeoxynucleoside triphosphates (dNTPs) and/or the polyamines spermine andspermidine, were used to infect oral keratinocytes. Of importance,polyamines and dNTPs have been shown to enhance natural endogenous reversetranscription (NERT), a step essential for early lentiviral infection, andare abundantly present in human semen. The infectivities of HIV-1 strainsYU-2, ADA, and NL4-3 for these primary keratinocytes were dramaticallyincreased by the addition of physiological concentrations of dNTPs,spermine, and spermidine. Binding and viral internalization assay studiesshowed no differences in these oral mucosal cells, with or withoutNERT-altering agents. It was also observed that the recombinant, cell-freeHIV-1 proteins Nef, Tat, and gp120 (R5) induced apoptosis in primary oralkeratinocytes compared with the results seen with nontreated cells orcells treated with glutathione S-transferase protein as a control undersimilar conditions. Microarray analyses suggested that HIV-1 gp120 and Tatinduce apoptosis in primary human oral keratinocytes via the Fas/FasLapoptotic pathway, whereas induction of apoptosis by Nef occurs throughboth Fas/FasL and mitochondrial apoptotic pathways. Thus, these findingssuggest molecular mechanisms by which semen in particular, as well asother bodily fluids such as cervicovaginal secretions, could increase oraltransmission of HIV-1 via increasing infectivity in confluent andlow-replicating oral keratinocytes. As well, the induction of apoptosis inhuman oral keratinocytes with relevant HIV-1-specific proteins suggestsanother potential complementary mechanism by which the oral mucosa barriermay be disrupted during HIV-1 infection in vivo.

• Cheema ZF, Santillano DR, Wade SB, Newman JM, Miranda RC
• The extracellular matrix, p53 and estrogen compete to regulatecell-surface Fas/Apo-1 suicide receptor expression in proliferatingembryonic cerebral cortical precursors, and reciprocally, Fas-ligandmodifies estrogen control of cell-cycle proteins.
• BMC Neurosci. 2004; 5: 11-11
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• BACKGROUND: Apoptosis is important for normal cerebral corticaldevelopment. We previously showed that the Fas suicide receptor wasexpressed within the developing cerebral cortex, and that in vitro Fasactivation resulted in caspase-dependent death. Alterations incell-surface Fas expression may significantly influence corticaldevelopment. Therefore, in the following studies, we sought to identifydevelopmentally relevant cell biological processes that regulatecell-surface Fas expression and reciprocal consequences of Fas receptoractivation. RESULTS: Flow-cytometric analyses identified two distinctneural sub-populations that expressed Fas on their cell surface at high(FasHi) or moderate (FasMod) levels. The anti-apoptotic protein FLIPfurther delineated a subset of Fas-expressing cells with potentialapoptosis-resistance. FasMod precursors were mainly in G0, while FasHiprecursors were largely apoptotic. However, birth-date analysis indicatedthat neuroblasts express the highest levels of cell-surface Fas at the endof S-phase, or after their final round of mitosis, suggesting that Fasexpression is induced at cell cycle checkpoints or during interkineticnuclear movements. FasHi expression was associated with loss ofcell-matrix adhesion and anoikis. Activation of the transcription factorp53 was associated with induction of Fas expression, while the gonadalhormone estrogen antagonistically suppressed cell-surface Fas expression.Estrogen also induced entry into S-phase and decreased the number ofFas-expressing neuroblasts that were apoptotic. Concurrent exposure toestrogen and to soluble Fas-ligand (sFasL) suppressed p21/waf-1 and PCNA.In contrast, estrogen and sFasL, individually and together, inducedcyclin-A expression, suggesting activation of compensatory survivalmechanisms. CONCLUSIONS: Embryonic cortical neuronal precursors areintrinsically heterogeneous with respect to Fas suicide-sensitivity.Competing intrinsic (p53, cell cycle, FLIP expression), proximal(extra-cellular matrix) and extrinsic factors (gonadal hormones)collectively regulate Fas suicide-sensitivity either during neurogenesis,or possibly during neuronal migration, and may ultimately determine whichneuroblasts successfully contribute neurons to the differentiatingcortical plate.

• Westphal S, Kalthoff H
• Apoptosis: targets in pancreatic cancer.
• Mol Cancer. 2003; 2: 6-6
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• Pancreatic adenocarcinoma is characterized by poor prognosis, because oflate diagnosis and lack of response to chemo- and/or radiation therapies.Resistance to apoptosis mainly causes this insensitivity to conventionaltherapies. Apoptosis or programmed cell death is a central regulator oftissue homeostasis. Certain genetic disturbances of apoptotic signalingpathways have been found in carcinomas leading to tumor development andprogression. In the past few years, the knowledge about the complexpathways of apoptosis has strongly increased and new therapeuticapproaches based on this knowledge are being developed. This review willfocus on the role of apoptotic proteins contributing to pancreatic cancerdevelopment and progression and will demonstrate possible targets toinfluence this deadly disease.

• Li JH, Kluger MS, Madge LA, Zheng L, Bothwell AL, Pober JS
• Interferon-gamma augments CD95(APO-1/Fas) and pro-caspase-8 expression andsensitizes human vascular endothelial cells to CD95-mediated apoptosis.
• Am J Pathol. 2002; 161: 1485-95
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• We have examined the effects of interferon (IFN)-gamma on expression andfunction of CD95 (APO-1/Fas) and associated proteins in cultured humanumbilical vein and dermal microvascular endothelial cells (HUVEC andHDMEC, respectively). Unstimulated cells express only low levels of CD95;IFN-gamma produces a time- and concentration-dependent increase of CD95 inboth cell types at the mRNA and cell surface protein levels. IFN-gammaalso produces an increase in expression of pro-caspase-8 (FLICE/MACH) butdoes not significantly change expression of either Fas-associated deathdomain (FADD) protein or cellular FLICE inhibitory protein (cFLIP), otherproteins associated with the CD95 death-inducing signaling complex (DISC).Neither resting nor IFN-gamma-treated EC express detectable CD95L mRNA orprotein. Untreated HUVEC and HDMEC show minimal apoptosis when transducedto express CD95L. Treatment of CD95L-transduced cells with IFN-gammacauses apoptosis within 24 to 36 hours that can be blocked by antagonisticanti-CD95 antibody or by the caspase-inhibitory peptide zVAD-FMK. Theextent of apoptosis is increased by co-treatment with either the proteinsynthesis inhibitor cycloheximide or the phosphatidylinositol 3-kinaseinhibitor LY294002. Untransduced HUVEC treated with IFN-gamma also undergoCD95-initiated apoptosis when mixed with CD95L-transduced HUVEC or whenincubated with pharmacologically activated cytolytic T lymphocytes.Overexpression of CD95 in HUVEC confers sensitivity to CD95L in theabsence of IFN-gamma-treatment. We conclude that IFN-gamma inducessensitivity of endothelium to CD95L-mediated apoptosis, and that thisresponse may result from increased expression of CD95 and/orpro-caspase-8.

• Cottin V, Van Linden AA, Riches DW
• Phosphorylation of the tumor necrosis factor receptor CD120a (p55)recruits Bcl-2 and protects against apoptosis.
• J Biol Chem. 2001; 276: 17252-60
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• Ligation of the tumor necrosis factor alpha receptor CD120a initiatesresponses as diverse as apoptosis and the expression ofNF-kappaB-dependent pro-survival genes. How these opposing responses arecontrolled remains poorly understood. Here we demonstrate thatphosphorylation by p42(mapk/erk2) inhibits the apoptotic activity ofCD120a while preserving its ability to activate NF-kappaB. PhosphorylatedCD120a is re-localized from the Golgi complex to tubular structures of theendoplasmic reticulum wherein it recruits Bcl-2. Antisense-mediateddown-regulation of Bcl-2 antagonized the localization of CD120a to tubularstructures and reversed the protection from apoptosis conferred byreceptor phosphorylation. We propose that phosphorylation of CD120arepresents a novel, Bcl-2-dependent mechanism by which the apoptoticactivity of the receptor may be regulated. Thus, oncogenic activation ofp42(mapk/erk2) may serve to inhibit the apoptotic activity of this deathreceptor while preserving NF-kappaB-dependent responses and may thusindirectly contribute to a failure to eliminate cells bearing oncogenes ofthe Ras-Raf-MEK-p42(mapk/erk2) pathway.

• Hu S, Yang X
• dFADD, a novel death domain-containing adapter protein for the Drosophilacaspase DREDD.
• J Biol Chem. 2000; 275: 30761-4
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• Apoptotic cell death occurs through activation of procaspases, theprecursors of a group of aspartate-specific cysteine proteases known ascaspases. Procaspase activation is mediated by death adapter proteins suchas the mammalian proteins FADD and Apaf-1 and the Caenorhabditis elegansprotein CED-4. These adapters bind to procaspases and facilitateoligomerization and subsequent auto-proteolytic processing of thezymogens. Here we report cloning and characterization of dFADD, a FADDhomologue in Drosophila. dFADD contains a death domain that is highlyhomologous to the FADD death domain, and it also shares a novel domainwith a Drosophila caspase DREDD, which we call death-inducing domain.dFADD binds to DREDD through the death-inducing domain and enhances thecell death activity and proteolytic processing of DREDD. dFADD and DREDDare stabilized by their interaction. The structural and functionalsimilarities between dFADD and FADD suggest the existence of a FADD-likeapoptosis pathway in Drosophila.

• Guicciardi ME et al.
• Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis bypromoting mitochondrial release of cytochrome c.
• J Clin Invest. 2000; 106: 1127-37
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• TNF-alpha-induced apoptosis is thought to involve mediators from acidicvesicles. Cathepsin B (cat B), a lysosomal cysteine protease, has recentlybeen implicated in apoptosis. To determine whether cat B contributes toTNF-alpha-induced apoptosis, we exposed mouse hepatocytes to the cytokinein vitro and in vivo. Isolated hepatocytes treated with TNF-alpha in thepresence of the transcription inhibitor actinomycin D (AcD) accumulatedcat B in their cytosol. Further experiments using cell-free systemsindicated that caspase-8 caused release of active cat B from purifiedlysosomes and that cat B, in turn, increased cytosol-induced release ofcytochrome c from mitochondria. Consistent with these observations, theability of TNF-alpha/AcD to induce mitochondrial release of cytochrome c,caspase activation, and apoptosis of isolated hepatocytes was markedlydiminished in cells from CatB(-/-) mice. Deletion of the CatB generesulted in diminished liver injury and enhanced survival after treatmentin vivo with TNF-alpha and an adenovirus construct expressing the IkappaBsuperrepressor. Collectively, these observations suggest thatcaspase-mediated release of cat B from lysosomes enhances mitochondrialrelease of cytochrome c and subsequent caspase activation inTNF-alpha-treated hepatocytes.

• Reed JC
• Mechanisms of apoptosis.
• Am J Pathol. 2000; 157: 1415-30
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• Programmed cell death plays critical roles in a wide variety ofphysiological processes during fetal development and in adult tissues. Inmost cases, physiological cell death occurs by apoptosis as opposed tonecrosis. Defects in apoptotic cell death regulation contribute to manydiseases, including disorders where cell accumulation occurs (cancer,restenosis) or where cell loss ensues (stroke, heart failure,neurodegeneration, AIDS). In recent years, the molecular machineryresponsible for apoptosis has been elucidated, revealing a family ofintracellular proteases, the caspases, which are responsible directly orindirectly for the morphological and biochemical changes that characterizethe phenomenon of apoptosis. Diverse regulators of the caspases have alsobeen discovered, including activators and inhibitors of these cell deathproteases. Inputs from signal transduction pathways into the core of thecell death machinery have also been identified, demonstrating ways oflinking environmental stimuli to cell death responses or cell survivalmaintenance. Knowledge of the molecular mechanisms of apoptosis isproviding insights into the causes of multiple pathologies where aberrantcell death regulation occurs and is beginning to provide new approaches tothe treatment of human diseases.

• Izquierdo M et al.
• Blocked negative selection of developing T cells in mice expressing thebaculovirus p35 caspase inhibitor.
• EMBO J. 1999; 18: 156-66
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• Clonal deletion in the thymus by apoptosis is involved in purging theimmune system of self-reactive T lymphocytes (negative selection).Cysteine proteases (caspases) belonging to the CPP32 family are activatedduring this process. We have produced transgenic mice expressingbaculovirus p35, a broad-range caspase inhibitor. Thymocytes from p35transgenic mice were resistant in vitro to several apoptosis-inducingagents; this resistance correlated with the inhibition of CPP32-likeactivity. Negative selection in vivo of thymocytes triggered by twoexogenous antigens, staphylococcal enterotoxin B superantigen and anantigenic peptide in the F5 T-cell receptor transgenic model, wasspecifically inhibited in p35 transgenic mice. Our results provide directevidence for caspase involvement in negative selection during thymocytedevelopment.

• Cohen O et al.
• DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and itsfunction requires the death domain.
• J Cell Biol. 1999; 146: 141-8
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• Death-associated protein (DAP)-kinase is a calcium/calmodulin regulatedserine/threonine kinase that carries ankyrin repeats, a death domain, andis localized to the cytoskeleton. Here, we report that this kinase isinvolved in tumor necrosis factor (TNF)-alpha and Fas-induced apoptosis.Expression of DAP-kinase antisense RNA protected cells from killing byanti-Fas/APO-1 agonistic antibodies. Deletion of the death domainabrogated the apoptotic functions of the kinase, thus, documenting for thefirst time the importance of this protein domain. Overexpression of afragment encompassing the death domain of DAP-kinase acted as a specificdominant negative mutant that protected cells from TNF-alpha, Fas, andFADD/MORT1-induced cell death. DAP-kinase apoptotic function was blockedby bcl-2 as well as by crmA and p35 inhibitors of caspases, but not by thedominant negative mutants of FADD/MORT1 or of caspase 8. Thus, itfunctions downstream to the receptor complex and upstream to othercaspases. The multidomain structure of this serine/threonine kinase,combined with its involvement in cell death induced by several differenttriggers, place DAP-kinase at one of the central molecular pathwaysleading to apoptosis.

• Zhou P, Chou J, Olea RS, Yuan J, Wagner G
• Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD:a structural basis for specific adaptor/caspase interaction.
• Proc Natl Acad Sci U S A. 1999; 96: 11265-70
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• Direct recruitment and activation of caspase-9 by Apaf-1 through thehomophilic CARD/CARD (Caspase Recruitment Domain) interaction is criticalfor the activation of caspases downstream of mitochondrial damage inapoptosis. Here we report the solution structure of the Apaf-1 CARD domainand its surface of interaction with caspase-9 CARD. Apaf-1 CARD consistsof six tightly packed amphipathic alpha-helices and is topologicallysimilar to the RAIDD CARD, with the exception of a kink observed in themiddle of the N-terminal helix. By using chemical shift perturbation data,the homophilic interaction was mapped to the acidic surface of Apaf-1 CARDcentered around helices 2 and 3. Interestingly, a significant portion ofthe chemically perturbed residues are hydrophobic, indicating that inaddition to the electrostatic interactions predicted previously,hydrophobic interaction is also an important driving force underlying theCARD/CARD interaction. On the basis of the identified functional residuesof Apaf-1 CARD and the surface charge complementarity, we propose a modelof CARD/CARD interaction between Apaf-1 and caspase-9.

• Scaffidi C et al.
• Two CD95 (APO-1/Fas) signaling pathways.
• EMBO J. 1998; 17: 1675-87
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• We have identified two cell types, each using almost exclusively one oftwo different CD95 (APO-1/Fas) signaling pathways. In type I cells,caspase-8 was activated within seconds and caspase-3 within 30 min ofreceptor engagement, whereas in type II cells cleavage of both caspaseswas delayed for approximately 60 min. However, both type I and type IIcells showed similar kinetics of CD95-mediated apoptosis and loss ofmitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering,all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xLoverexpression in both cell types. However, in type II but not type Icells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3activation as well as apoptosis. In type I cells, induction of apoptosiswas accompanied by activation of large amounts of caspase-8 by thedeath-inducing signaling complex (DISC), whereas in type II cells DISCformation was strongly reduced and activation of caspase-8 and caspase-3occurred following the loss of DeltaPsim. Overexpression of caspase-3 inthe caspase-3-negative cell line MCF7-Fas, normally resistant toCD95-mediated apoptosis by overexpression of Bcl-xL, converted these cellsinto true type I cells in which apoptosis was no longer inhibited byBcl-xL. In summary, in the presence of caspase-3 the amount of activecaspase-8 generated at the DISC determines whether amitochondria-independent apoptosis pathway is used (type I cells) or not(type II cells).

• Faris M, Latinis KM, Kempiak SJ, Koretzky GA, Nel A
• Stress-induced Fas ligand expression in T cells is mediated through a MEKkinase 1-regulated response element in the Fas ligand promoter.
• Mol Cell Biol. 1998; 18: 5414-24
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• T lymphocytes undergo apoptosis in response to a variety of stimuli,including exposure to UV radiation and gamma-irradiation. While themechanism by which stress stimuli induce apoptosis is not well understood,we have previously shown that the induction of Fas ligand (FasL) geneexpression by environmental stress stimuli is dependent on c-JunN-terminal kinase (JNK) activation. Using inducible dominant-active (DA)JNK kinase kinase (MEKK1) expression in Jurkat cells, we map a specificMEKK1-regulated response element to positions -338 to -316 of the Fasligand (FasL) promoter. Mutation of that response element abrogatedMEKK1-mediated FasL promoter activation and interfered in stress-inducedactivation of that promoter. Using electrophoretic mobility shift assays,we demonstrate that activator protein 1 (AP-1) binding proteins, namely,activating transcription factor 2 (ATF2) and c-Jun, bind to the MEKK1response element. Transient transfection of interfering c-Jun and ATF2mutants, which lack the consensus JNK phosphorylation sites, abrogated thetranscriptional activation of the FasL promoter, demonstrating theinvolvement of these transcription factors in the regulation of the FasLpromoter. Taken together, our data indicate that MEKK1 and transcriptionfactors regulated by the JNK pathway play a role in committing lymphocytesto undergo apoptosis by inducing FasL expression via a novel responseelement in the promoter of that gene.

• Hara H et al.
• Inhibition of interleukin 1beta converting enzyme family proteases reducesischemic and excitotoxic neuronal damage.
• Proc Natl Acad Sci U S A. 1997; 94: 2007-12
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• The interleukin 1beta converting enzyme (ICE) family plays a pivotal rolein programmed cell death and has been implicated in stroke andneurodegenerative diseases. During reperfusion after filamentous middlecerebral artery occlusion, ICE-like cleavage products and tissueimmunoreactive interleukin 1beta (IL-1beta) levels increased in ischemicmouse brain. Ischemic injury decreased after intracerebroventricularinjections of ICE-like protease inhibitors,N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.FMK),acetyl-Tyr-Val-Ala-Asp-chloromethylketone, or a relatively selectiveinhibitor of CPP32-like caspases,N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, but not acathepsin B inhibitor, N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone.z-VAD.FMK decreased ICE-like cleavage products and tissue immunoreactiveIL-1beta levels in ischemic mouse brain and reduced tissue damage whenadministered to rats as well. Only z-VAD.FMK andacetyl-Tyr-Val-Ala-Asp-chloromethylketone reduced brain swelling, andN-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone did not attenuatethe ischemia-induced increase in tissue IL-1beta levels. The threecysteine protease inhibitors significantly improved behavioral deficits,thereby showing that functional recovery of ischemic neuronal tissue canfollow blockade of enzymes associated with apoptotic cell death. Finally,we examined the effect of z-VAD.FMK on excitotoxicity and found that itprotected against alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-induced or to a lesser extent N-methyl-D-aspartate-inducedexcitotoxic brain damage. Thus, ICE-like and CPP32-like caspasescontribute to mechanisms of cell death in ischemic and excitotoxic braininjury and provide therapeutic targets for stroke and neurodegenerativebrain damage.

• Li Y, Kang J, Horwitz MS
• Interaction of an adenovirus 14.7-kilodalton protein inhibitor of tumornecrosis factor alpha cytolysis with a new member of the GTPasesuperfamily of signal transducers.
• J Virol. 1997; 71: 1576-82
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• The adenovirus (Ad) 14.7-kDa E3 protein (E3-14.7K), which can inhibittumor necrosis factor alpha (TNF-alpha) cytolysis, was used to screen HeLacell cDNA libraries for interacting proteins in the yeast two-hybridsystem. A new member of the low-molecular-weight (LMW) GTP-binding proteinfamily with Ras and ADP-ribosylation factor homology was discovered bythis selection and has been named FIP-1 (14.7K-interacting protein). FIP-1colocalized with Ad E3-14.7K in the cytoplasm especially near the nuclearmembrane and in discrete foci on or near the plasma membrane. Itsinteraction with E3-14.7K was dependent on the FIP-1 GTP-binding domain.The stable expression of FIP-1 antisense message partially protected thecells from TNF-alpha cytolysis. FIP-1 was associated transiently withseveral unknown phosphorylated cellular proteins within 15 min aftertreatment with TNF-alpha. FIP-1 mRNA was expressed ubiquitously but athigher levels in human skeletal muscle, heart, and brain. In addition tohomology to other LMW GTP-binding proteins, FIP-1 has regions of homologyto two prokaryotic metalloproteases. However, there was no homologybetween FIP-1 and any of the recently isolated death proteins in theTNF-alpha or Fas/APO1 cytolytic pathway and no interaction with severalmembers of the Bcl-2 family of inhibitors of apoptosis. These data suggestthat FIP-1, as a cellular target for Ad E3-14.7K, is either a newintermediate on a previously described pathway or part of a novelTNF-alpha-induced cell death pathway. FIP-1 has two consensus sequencesfor myristoylation which would be expected to facilitate membraneassociation and also has sequences for Ser/Thr as well as Tyrphosphorylation that could affect its function.

• Kondo T, Yokokura T, Nagata S
• Activation of distinct caspase-like proteases by Fas and reaper inDrosophila cells.
• Proc Natl Acad Sci U S A. 1997; 94: 11951-6
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• The cytoplasmic region of Fas, a mammalian death factor receptor, shares alimited homology with reaper, an apoptosis-inducing protein in Drosophila.Expression of either the Fas cytoplasmic region (FasC) or of reaper inDrosophila cells caused cell death. The death process induced by FasC orreaper was inhibited by crmA or p35, suggesting that its death process ismediated by caspase-like proteases. Both Ac-YVAD aldehyde and Ac-DEVDaldehyde, specific inhibitors of caspase 1- and caspase 3-like proteases,respectively, inhibited the FasC-induced death of Drosophila cells.However, the cell death induced by reaper was inhibited by Ac-DEVDaldehyde, but not by Ac-YVAD aldehyde. A caspase 1-like protease activitythat preferentially recognizes the YVAD sequence gradually increased inthe cytosolic fraction of the FasC-activated cells, whereas the caspase3-like protease activity recognizing the DEVD sequence was observed in thereaper-activated cells. Partial purification and biochemicalcharacterization of the proteases indicated that there are at least threedistinct caspase-like proteases in Drosophila cells, which aredifferentially activated by FasC and reaper. The conservation of theFas-death signaling pathway in Drosophila cells, which is distinct fromthat for reaper, may indicate that cell death in Drosophila is controllednot only by the reaper suicide gene, but also by a Fas-like killer gene.

• Duan H et al.
• ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by thecytotoxic T cell protease granzyme B.
• J Biol Chem. 1996; 271: 16720-4
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• Members of the ICE/Ced-3 gene family are likely effector components of thecell death machinery. Here, we characterize a novel member of this familydesignated ICE-LAP6. By phylogenetic analysis, ICE-LAP6 is classified intothe Ced-3 subfamily which includes Ced-3, Yama/CPP32/apopain, Mch2, andICE-LAP3/Mch3/CMH-1. Interestingly, ICE-LAP6 contains an active site QACGGpentapeptide, rather than the QACRG pentapeptide shared by other familymembers. Overexpression of ICE-LAP6 induces apoptosis in MCF7 breastcarcinoma cells. More importantly, ICE-LAP6 is proteolytically processedinto an active cysteine protease by granzyme B, an important component ofcytotoxic T cell-mediated apoptosis. Once activated, ICE-LAP6 is able tocleave the death substrate poly(ADP-ribose) polymerase into signatureapoptotic fragments.

• Rothe M, Pan MG, Henzel WJ, Ayres TM, Goeddel DV
• The TNFR2-TRAF signaling complex contains two novel proteins related tobaculoviral inhibitor of apoptosis proteins.
• Cell. 1995; 83: 1243-52
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• The 75 kDa tumor necrosis factor receptor (TNFR2) transduces extracellularsignals via receptor-associated cytoplasmic proteins. Two of these signaltransducers, TRAF1 and TRAF2, were isolated and characterized previously.We report here the biochemical purification and subsequent molecularcloning of two novel TNFR2-associated proteins, designated c-IAP1 andc-IAP2, that are closely related mammalian members of the inhibitor ofapoptosis protein (IAP) family originally identified in baculoviruses. Theviral and cellular IAPs contain N-terminal baculovirus IAP repeat (BIR)motifs and a C-terminal RING finger. The c-IAPs do not directly contactTNFR2, but rather associate with TRAF1 and TRAF2 through their N-terminalBIR motif-comprising domain. The recruitment of c-IAP1 or c-IAP2 to theTNFR2 signaling complex requires a TRAF2-TRAF1 heterocomplex.

• Schulze-Osthoff K, Krammer PH, Droge W
• Divergent signalling via APO-1/Fas and the TNF receptor, two homologousmolecules involved in physiological cell death.
• EMBO J. 1994; 13: 4587-96
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• Tumor necrosis factor receptor (TNF-R) and APO-1/Fas (CD95) are members ofthe tumor necrosis factor/nerve growth factor receptor superfamilyinvolved in various forms of physiological cell death. Due to thestructural homology between these receptors and their ligands, it has beensuggested that APO-1/Fas and TNF-R kill cells by similar mechanisms. Here,we compared the killing pathways mediated by each receptor molecule inTNF-sensitive L929 cells stably transfected with APO-1/Fas cDNA.Morphological analysis revealed that TNF-induced cell death resemblesnecrosis, while APO-1/Fas-mediated cell killing shows an apoptoticpattern, evident by the appearance of membrane blebbing, nuclearcondensation and non-random DNA degradation. Studies with inhibitors ofseveral intracellular pathways further demonstrate that the mechanisms ofTNF- and APO-1/Fas-mediated cell killing are substantially different. TNFcytotoxicity is mediated by reactive oxygen intermediates generated duringmitochondrial respiration. However, these mediators are not involved inAPO-1/Fas-mediated cell death as neither mitochondrial inhibitors norantioxidants exert a protecting effect. Moreover, several inhibitors ofcalcium metabolism, ADP ribosylation and phospholipase action suppress TNFcytotoxicity, but not APO-1/Fas-mediated apoptosis. Additional differencesbetween the two molecules were observed at the transcriptional level.Whereas transcription factor NF-kappa B was readily activated by TNF,activation was not induced by triggering APO-1/Fas. These data suggestthat the two molecules, though structurally related, utilize distinctsignal transduction pathways, even in a single cell type. Hence, cells mayundergo different programs of cell death depending on the activatingstimulus.