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Marcus E Peter - One of the best experts on this subject based on the ideXlab platform.
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Methods to analyze the palmitoylated CD95 high molecular weight Death-Inducing Signaling Complex.
Methods in enzymology, 2008Co-Authors: Christine Feig, Marcus E PeterAbstract:Major work elucidating the structure and function of the CD95 death inducing Signaling Complex (DISC) was carried in the late 1990s. Since then the DISC has become a paradigm for multiprotein Signaling Complexes in the apoptosis literature. We analyzed the earliest events of DISC formation with a set of different techniques and found a surprising new characteristic of the CD95 DISC. Data revealed that CD95 is palmitoylated on cysteine 199. This lipid modification enhances receptor aggregation to the high molecular weight DISC.
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Isolation and analysis of components of CD95 (APO-1/Fas) Death-Inducing Signaling Complex.
Methods (San Diego Calif.), 1999Co-Authors: Carsten Scaffidi, Peter H Krammer, Marcus E PeterAbstract:CD95 (APO-1/Fas) is an apoptosis-inducing receptor belonging to the tumor necrosis factor receptor superfamily. Multimerization of CD95 leads to instant recruitment of the Signaling molecules FADD and caspase-8 to the activated receptor forming the Death-Inducing Signaling Complex (DISC). DISC formation is the first essential step of CD95 Signaling and results in activation of caspase-8 starting a Signaling cascade that leads to apoptosis. Here we describe a method for analyzing the CD95 DISC. The method is based on coimmunoprecipitation of the Signaling molecules with the activated CD95 receptor followed by Western blot detection of associated molecules. Therefore, this method can analyze the very first Signaling events during CD95-mediated apoptosis.
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bcl xl acts downstream of caspase 8 activation by the cd95 death inducing Signaling Complex
Journal of Biological Chemistry, 1998Co-Authors: Carsten Scaffidi, Jan Paul Medema, Peter H Krammer, Marcus E PeterAbstract:The Bcl-2 family member Bcl-xL has often been correlated with apoptosis resistance. We have shown recently that in peripheral human T cells resistance to CD95-mediated apoptosis is characterized by a lack of caspase-8 recruitment to the CD95 Death-Inducing Signaling Complex (DISC) and by increased expression of Bcl-xL (Peter, M. E., Kischkel, F. C., Scheuerpflug, C. G., Medema, J. P., Debatin, K.-M., and Krammer, P. H. (1997) Eur. J. Immunol. 27, 1207-1212). This raises the possibility that Bcl-xL directly prevents caspase-8 activation by the DISC. To test this hypothesis a cell line in which CD95 Signaling was inhibited by overexpression of Bcl-xL was used. In these MCF7-Fas-bcl-xL cells Bcl-xL had no effect on the recruitment of caspase-8 to the DISC. It did not affect the activity of the DISC nor the generation of the caspase-8 active subunits p18 and p10. In contrast, cleavage of a typical substrate for caspase-3-like proteases, poly(ADP-ribose) polymerase, was inhibited in comparison with the control-transfected CD95-sensitive MCF7-Fas cells. To test whether Bcl-xL would inhibit active caspase-8 subunits in the cytoplasm, a number of immunoprecipitation experiments were performed. Using monoclonal antibodies directed against different domains of caspase-8, anti-Bcl-xL antibodies, or fusion proteins of glutathione S-transferase with different domains of caspase-8, no evidence for a direct or indirect physical interaction between caspase-8 and Bcl-xL was found. Moreover, overexpression of Bcl-xL did not inhibit the activity of the caspase-8 active subunits p18/p10. Therefore, in this cell line that has become resistant to CD95-induced apoptosis due to overexpression of Bcl-xL, Bcl-xL acts independently and downstream of caspase-8.
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Mechanisms of CD95 (APO-1/Fas)-mediated apoptosis
Current opinion in immunology, 1998Co-Authors: Marcus E Peter, Peter H KrammerAbstract:This review covers recent advances of CD95 Signaling. It focuses on CD95-interacting molecules, formation of the death inducing Signaling Complex and the role of caspases, particularly caspase-8, and their death substrates. We also discuss the relevance of mitochondria in the CD95-mediated apoptotic process and how viral proteins interfere with crucial steps of this Signaling pathway.
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FLICE is predominantly expressed as two functionally active isoforms, caspase-8/a and caspase-8/b.
The Journal of biological chemistry, 1997Co-Authors: Carsten Scaffidi, Jan Paul Medema, Peter H Krammer, Marcus E PeterAbstract:Induction of apoptosis by the cell surface receptor CD95 (APO-1/Fas) has been shown to involve activation of a family of cysteine proteases (caspases). Recently, a new member of this family has been identified, designated FLICE (caspase-8/MACH/Mch5). FLICE is part of the CD95 Death-Inducing Signaling Complex and is therefore the most upstream caspase in the CD95 apoptotic pathway. A total of eight different isoforms of FLICE (caspase-8/a–h) have been described. To determine which isoforms are expressed in different cells we have generated a panel of monoclonal antibodies directed against all functional domains of FLICE. Using these antibodies we could show that only two of the FLICE isoforms (caspase-8/a and caspase-8/b) were predominantly expressed in cells of different origin. Both isoforms were recruited to the CD95 Death-Inducing Signaling Complex and were activated upon CD95 stimulation with similar kinetics. Taken together, only two of the eight published caspase-8 isoforms could be detected in significant amounts at the protein level.
Yongkeun Jung - One of the best experts on this subject based on the ideXlab platform.
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peroxiredoxin 6 interferes with trail induced death inducing Signaling Complex formation by binding to death effector domain caspase
Cell Death & Differentiation, 2011Co-Authors: Hyunwoo Choi, Jae Woong Chang, Yongkeun JungAbstract:Peroxiredoxin 6 interferes with TRAIL-induced Death-Inducing Signaling Complex formation by binding to death effector domain caspase
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Suppression of receptor-mediated apoptosis by death effecter domain recruiting domain binding peptide aptamer.
Biochemical and Biophysical Research Communications, 2006Co-Authors: Young Ae Park, Hyunwoo Choi, Yongkeun Jung, Yong Seok Choi, Yun-hee Choi, Sunjoo JeongAbstract:FLASH protein is a component of Death-Inducing Signaling Complex and might be involved in death receptor-mediated extrinsic apoptosis. Here we developed the peptide aptamer against death effecter domain recruiting domain (DRD) of FLASH protein and showed that the peptide bound to FLASH protein in vitro. Intracellular expression of the DRD-binding peptide aptamer specifically suppressed receptor-mediated extrinsic apoptosis but not intrinsic pathway, which was recapitulated by the antisense oligonucleotides for FLASH. These data suggest that DRD-binding peptide is not only a novel inhibitor modulating receptor-mediated apoptosis but also a tool for elucidating the roles of FLASH in apoptosis.
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Fas-associated Factor 1, FAF1, Is a Member of Fas Death-Inducing Signaling Complex
The Journal of biological chemistry, 2003Co-Authors: Seung-wook Ryu, Yongkeun Jung, Soojin Lee, Min Young Park, Joon Il Jun, Eunhee KimAbstract:FAF1 has been introduced as a Fas-binding protein. However, the function of FAF1 in apoptotic execution is not established. Based on the fact that FAF1 is a Fas-binding protein, we asked if FAF1 interacted with other members of the Fas-Death-Inducing Signaling Complex (Fas-DISC) such as Fas-associated death domain protein (FADD) and caspase-8. FAF1 could interact with caspase-8 and FADD in vivo as well as in vitro. The death effector domains (DEDs) of caspase-8 and FADD interacted with the amino acid 181–381 region of FAF1, previously known to have apoptotic potential. Considering that FAF1 directly binds to Fas and caspase-8, FAF1 shows similar protein-interacting characteristics to that of FADD. In the coimmunoprecipitation with an anti-Fas antibody (APO-1) in Jurkat cells, endogenous FAF1 was associated with the precipitates in which caspase-8 was present. By confocal microscopic analysis, both Fas and FAF1 were detected in the cytoplasmic membrane before Fas activation, and in the cytoplasm after Fas activation. FADD and caspase-8 colocalized with Fas in Jurkat cells validating the presence of FAF1 in the authentic Fas-DISC. Overexpression of FAF1 in Jurkat cells caused significant apoptotic death. In addition, the FAF1 deletion mutant lacking the N terminus where Fas, FADD, and caspase-8 interact protected Jurkat cells from Fas-induced apoptosis demonstrating dominant-negative phenotype. Cell death by overexpression of FAF1 was suppressed significantly in both FADD- and caspase-8-deficient Jurkat cells when compared with that in their parental Jurkat cells. Collectively, our data show that FAF1 is a member of Fas-DISC acting upstream of caspase-8.
Hyunwoo Choi - One of the best experts on this subject based on the ideXlab platform.
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peroxiredoxin 6 interferes with trail induced death inducing Signaling Complex formation by binding to death effector domain caspase
Cell Death & Differentiation, 2011Co-Authors: Hyunwoo Choi, Jae Woong Chang, Yongkeun JungAbstract:Peroxiredoxin 6 interferes with TRAIL-induced Death-Inducing Signaling Complex formation by binding to death effector domain caspase
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Peroxiredoxin 6 interferes with TRAIL-induced Death-Inducing Signaling Complex formation by binding to death effector domain caspase
Cell Death & Differentiation, 2011Co-Authors: Hyunwoo Choi, Jae Woong Chang, Y.-k. JungAbstract:Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent with cancer-selective apoptogenic activity. It evokes the canonical caspase-mediated cell death pathway through Death-Inducing Signaling Complex (DISC) formation. We identified that Peroxiredoxin 6 (Prx6) interacts with caspase-10 and caspase-8 via the death effector domain (DED). Prx6 suppresses TRAIL-mediated cell death in human cancer cells, but not that induced by intrinsic apoptosis inducers such as etoposide, staurosporine, or A23187. Among Prx1–6 members, only Prx6 binds to DED caspases and is most effective in suppressing TRAIL or DED caspase-induced cell death. The antiapoptotic activity of Prx6 against TRAIL is not likely associated with its peroxidase activity but is associated with its ability to bind to DED caspases. Increased expression of Prx6 enhances the binding of Prx6 to caspase-10 but reduces TRAIL-induced DISC formation and subsequently caspase activation. Interestingly, Prx6 is highly upregulated in metastatic gastric cancer cells, which are relatively resistant to TRAIL as compared with primary cancer cells. Downregulation of Prx6 sensitizes the metastatic cancer cells to TRAIL-induced cell death. Taken together, these results suggest that Prx6 modulates TRAIL Signaling as a negative regulator of caspase-8 and caspase-10 in DISC formation of TRAIL-resistant metastatic cancer cells.
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Suppression of receptor-mediated apoptosis by death effecter domain recruiting domain binding peptide aptamer.
Biochemical and Biophysical Research Communications, 2006Co-Authors: Young Ae Park, Hyunwoo Choi, Yongkeun Jung, Yong Seok Choi, Yun-hee Choi, Sunjoo JeongAbstract:FLASH protein is a component of Death-Inducing Signaling Complex and might be involved in death receptor-mediated extrinsic apoptosis. Here we developed the peptide aptamer against death effecter domain recruiting domain (DRD) of FLASH protein and showed that the peptide bound to FLASH protein in vitro. Intracellular expression of the DRD-binding peptide aptamer specifically suppressed receptor-mediated extrinsic apoptosis but not intrinsic pathway, which was recapitulated by the antisense oligonucleotides for FLASH. These data suggest that DRD-binding peptide is not only a novel inhibitor modulating receptor-mediated apoptosis but also a tool for elucidating the roles of FLASH in apoptosis.
Peter H Krammer - One of the best experts on this subject based on the ideXlab platform.
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stoichiometry of the cd95 death inducing Signaling Complex experimental and modeling evidence for a death effector domain chain model
Molecular Cell, 2012Co-Authors: Kolja Schleich, Nicolai Fricker, Selcen Ozturk, Kerstin Kammerer, Petra Richter, Peter H Krammer, Uwe Warnken, Martina Schnölzer, Inna N. LavrikAbstract:Summary The CD95 (Fas/APO-1) Death-Inducing Signaling Complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.
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Dynamics within the CD95 Death-Inducing Signaling Complex decide life and death of cells
Molecular systems biology, 2010Co-Authors: Leo Neumann, Nicolai Fricker, Inna N. Lavrik, Peter H Krammer, Carina Pforr, Joël Beaudouin, Alexander Pappa, Roland EilsAbstract:This study explores the dilemma in cellular Signaling that triggering of CD95 (Fas/APO-1) in some situations results in cell death and in others leads to the activation of NF-κB. We established an integrated kinetic mathematical model for CD95-mediated apoptotic and NF-κB Signaling. Systematic model reduction resulted in a surprisingly simple model well approximating experimentally observed dynamics. The model postulates a new link between c-FLIPL cleavage in the Death-Inducing Signaling Complex (DISC) and the NF-κB pathway. We validated experimentally that CD95 stimulation resulted in an interaction of p43-FLIP with the IKK Complex followed by its activation. Furthermore, we showed that the apoptotic and NF-κB pathways diverge already at the DISC. Model and experimental analysis of DISC formation showed that a subtle balance of c-FLIPL and procaspase-8 determines life/death decisions in a nonlinear manner. We present an integrated model describing the Complex dynamics of CD95-mediated apoptosis and NF-κB Signaling.
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cd95 stimulation results in the formation of a novel death effector domain protein containing Complex
Journal of Biological Chemistry, 2008Co-Authors: Inna N. Lavrik, Julia C Hoffmann, Simone Baumann, Alexander Golks, Thomas Mock, Peter H KrammerAbstract:Stimulation of CD95 (APO-1/Fas) by its natural ligand CD95L (APO-1L/FasL) leads to the formation of the Death-Inducing Signaling Complex. Here we report that upon CD95 stimulation in several T and B cell lines, a novel Signaling Complex is formed, which we term Complex II. Complex II is composed of the death effector domain proteins as follows: procaspase-8a/b, three isoforms of c-FLIP (c-FLIPL, c-FLIPS, c-FLIPR), and FADD. Notably, Complex II does not contain CD95. Based on our findings we suggest that CD95 Signaling includes two steps. The first step involves formation of the Death-Inducing Signaling Complex at the cell membrane. The second step involves formation of the cytosolic death effector domain protein-containing Complex that may play an important role in amplification of caspase activation.
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Caspase-2 is activated at the CD95 Death-Inducing Signaling Complex in the course of CD95-induced apoptosis.
Blood, 2006Co-Authors: Inna N. Lavrik, Simone Baumann, Alexander Golks, Peter H KrammerAbstract:Caspase-2 was reported to be involved in a number of apoptotic pathways triggered by various stimuli. However, the molecular mechanism of procaspase-2 activation in the course of apoptosis remains poorly defined. In this report, we demonstrate that procaspase-2 is recruited to the CD95 (Fas/APO-1) Death-Inducing Signaling Complex (DISC) in human T- and B-cell lines. We show that procaspase-2 is activated at the DISC on CD95 stimulation. Despite its presence at the DISC, caspase-2 does not initiate apoptosis on CD95 stimulation in caspase-8–deficient cell lines. Taken together, our data reveal that caspase-2 is activated at the DISC but does not play an initiating role in the CD95-induced apoptosis.
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Glutathione dependence of caspase-8 activation at the Death-Inducing Signaling Complex.
The Journal of biological chemistry, 2001Co-Authors: Hannes Hentze, Peter H Krammer, Ingo Schmitz, Andreas Krueger, Markus Latta, Albrecht WendelAbstract:Apoptosis triggered by the death receptor CD95 (APO-1 or Fas) is pivotal for the homeostasis of the immune system. We investigated differential effects of glutathione depletion on CD95-triggered apoptosis in T and B cell lines as well as the glutathione dependence of caspase-8 activation. In B lymphoblastoid SKW6.4 cells, CD95-mediated apoptosis was prevented upstream of caspase-8 activation and caspase-3-like activity after acute glutathione depletion by diethyl maleate or cis-chloro-dinitrobenzene. Immunoprecipitation of the Death-Inducing Signaling Complex (DISC) revealed that the DISC was still formed in the glutathione-depleted state. The first cleavage step of procaspase-8 activation at the DISC, however, was inhibited. Accordingly, under cell-free conditions, radiolabeled procaspase-8 was processed at the immunoprecipitated DISC only after the addition of exogenous dithiothreitol or reduced glutathione. We also observed suppression of CD95-mediated apoptosis in glutathione-depleted CEM and H9 cells. Notably, Jurkat cells still died upon CD95 engagement under this condition, displaying incomplete nuclear fragmentation and a partial switch to necrosis; this may be explained by reduced cytochrome c/dATP-mediated caspase activation observed in cytosol from glutathione-depleted Jurkat cytosol. Our data indicate that the activation of caspase-8 at the DISC and hence CD95-mediated apoptosis induction shows a cell-specific requirement for intracellular glutathione.
Janet A Houghton - One of the best experts on this subject based on the ideXlab platform.
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Cyclooxygenase-2 inhibition sensitizes human colon carcinoma cells to TRAIL-induced apoptosis through clustering of DR5 and concentrating Death-Inducing Signaling Complex components into ceramide-enriched caveolae.
Cancer research, 2005Co-Authors: Sophie Martin, Darren C. Phillips, Kinga Szekely-szucs, Lynda Elghazi, Fabienne Desmots, Janet A HoughtonAbstract:Cyclooxygenase-2 (COX-2) is up-regulated in human colon carcinomas, and its inhibition is associated with a reduction in tumorigenesis and a promotion of apoptosis. However, the mechanisms responsible for the antitumor effects of COX-2 inhibitors and how COX-2 modulates apoptotic Signaling have not been clearly defined. We have shown that COX-2 inhibition sensitizes human colon carcinoma cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis by inducing clustering of the TRAIL receptor DR5 at the cell surface and the redistribution of the Death-Inducing Signaling Complex components (DR5, FADD, and procaspase-8) into cholesterol-rich and ceramide-rich domains known as caveolae. This process requires the accumulation of arachidonic acid and sequential activation of acid sphingomyelinase for the generation of ceramide within the plasma membrane outer leaflet. The current study highlights a novel mechanism to circumvent colorectal carcinoma cell resistance to TRAIL-mediated apoptosis using COX-2 inhibitors to manipulate the lipid metabolism within the plasma membrane. (Cancer Res 2005; 65(24): 11447-58)
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Casein kinase II (CK2) enhances Death-Inducing Signaling Complex (DISC) activity in TRAIL-induced apoptosis in human colon carcinoma cell lines.
Oncogene, 2005Co-Authors: Kamel Izeradjene, Leslie Douglas, Addison Delaney, Janet A HoughtonAbstract:Casein kinase II (CK2) enhances Death-Inducing Signaling Complex (DISC) activity in TRAIL-induced apoptosis in human colon carcinoma cell lines
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casein kinase i attenuates tumor necrosis factor related apoptosis inducing ligand induced apoptosis by regulating the recruitment of fas associated death domain and procaspase 8 to the death inducing Signaling Complex
Cancer Research, 2004Co-Authors: Kamel Izeradjene, Leslie Douglas, Addison Delaney, Janet A HoughtonAbstract:Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide variety of malignant cell lines, in contrast to normal cells, but with considerable heterogeneity in response. Death receptor–mediated apoptosis may be attenuated by a variety of different mechanisms, including phosphorylation-based Signaling pathways. We have demonstrated that casein kinase I can attenuate TRAIL-induced apoptosis in human cell lines derived from colon adenocarcinoma (HT29 and HCT8) and pediatric rhabdomyosarcoma (JR1). Inhibition of casein kinase I (CKI) phosphorylation events in HT29, HCT8, and JR1 cells by CKI-7 dramatically increased apoptosis after exposure to TRAIL, in the absence of apoptosis induced by TRAIL treatment alone. CKI inhibition enhanced the recruitment of Fas-associated death domain and procaspase-8 to the Death-Inducing Signaling Complex after TRAIL treatment and enhanced cleavage of procaspase-8 at the Death-Inducing Signaling Complex. In HT29 cells studied further, rapid cleavage of caspase-8, caspase-3, Bid, and the caspase substrate poly(ADP-ribose) polymerase occurred when CKI-7 and TRAIL were combined. Overexpression of Bcl-2, Bcl-xL, or mutant DN-Fas-associated death domain protected HT29 cells from TRAIL-induced apoptosis in the presence of the CKI inhibitor. In addition, TRAIL combined with CKI-7 promoted the release of cytochrome c , Smac/DIABLO, HtrA2/Omi, and AIF from the mitochondria and down-regulated the expression of XIAP and c-IAP1. Small hairpin RNAs directed against CKI revealed that the CKIα isoform contributed significantly to the inhibition of TRAIL-induced apoptosis. These findings suggest that CKIα plays an antiapoptotic role through the generation of phosphorylated sites at the level of the Death-Inducing Signaling Complex, thereby conferring resistance to caspase cleavage mediated by TRAIL.
