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Inna N. Lavrik - One of the best experts on this subject based on the ideXlab platform.

  • Mathematical Modeling Reveals the Importance of the DED Filament Composition in the Effects of Small Molecules Targeting Caspase-8/c-FLIP_L Heterodimer
    Biochemistry (Moscow), 2020
    Co-Authors: N. V. Ivanisenko, Inna N. Lavrik
    Abstract:

    Procaspase-8 activation at the death-inducing signaling complex (DISC) triggers extrinsic apoptotic pathway. Procaspase-8 activation takes place in the death effector domain (DED) filaments and is regulated by c-FLIP proteins, in particular, by the long isoform c-FLIP_L. Recently, the first-in-class chemical probe targeting the caspase-8/c-FLIP_L heterodimer was reported. This rationally designed small molecule, FLIPin, enhances caspase-8 activity after initial heterodimer processing. Here, we used a kinetic mathematical model to gain an insight into the mechanisms of FLIPin action in a complex with DISC, in particular, to unravel the effects of FLIPin at different stoichiometry and composition of the DED filament. Analysis of this model has identified the optimal c-FLIP_L to Procaspase-8 ratios in different cellular landscapes favoring the activity of FLIPin. We predicted that the activity FLIPin is regulated via different mechanisms upon c-FLIP_L downregulation or upregulation. Our study demonstrates that a combination of mathematical modeling with system pharmacology allows development of more efficient therapeutic approaches and prediction of optimal treatment strategies.

  • Controlling Cell Death through Post-translational Modifications of DED Proteins.
    Trends in cell biology, 2020
    Co-Authors: Kamil Seyrek, Nikita V. Ivanisenko, Max Richter, Laura K. Hillert, Corinna König, Inna N. Lavrik
    Abstract:

    Apoptosis is a form of programmed cell death, deregulation of which occurs in multiple disorders, including neurodegenerative and autoimmune diseases as well as cancer. The formation of a death-inducing signaling complex (DISC) and death effector domain (DED) filaments are critical for initiation of the extrinsic apoptotic pathway. Post-translational modifications (PTMs) of DED-containing DISC components such as FADD, Procaspase-8, and c-FLIP comprise an additional level of apoptosis regulation, which is necessary to overcome the threshold for apoptosis induction. In this review we discuss the influence of PTMs of FADD, Procaspase-8, and c-FLIP on DED filament assembly and cell death induction, with a focus on the 3D organization of the DED filament.

  • Long and short isoforms of c-FLIP act as control checkpoints of DED filament assembly.
    Oncogene, 2019
    Co-Authors: Laura K. Hillert, T Kähne, Nikita V. Ivanisenko, Corinna König, Johannes Espe, Vladimir A. Ivanisenko, Inna N. Lavrik
    Abstract:

    The assembly of the death-inducing signaling complex (DISC) and death effector domain (DED) filaments at CD95/Fas initiates extrinsic apoptosis. Procaspase-8 activation at the DED filaments is controlled by short and long c-FLIP isoforms. Despite apparent progress in understanding the assembly of CD95-activated platforms and DED filaments, the detailed molecular mechanism of c-FLIP action remains elusive. Here, we further addressed the mechanisms of c-FLIP action at the DISC using biochemical assays, quantitative mass spectrometry, and structural modeling. Our data strongly indicate that c-FLIP can bind to both FADD and Procaspase-8 at the DED filament. Moreover, the constructed in silico model shows that c-FLIP proteins can lead to the formation of the DISCs comprising short DED filaments as well as serve as bridging motifs for building a cooperative DISC network, in which adjacent CD95 DISCs are connected by DED filaments. This network is based on selective interactions of FADD with both c-FLIP and Procaspase-8. Hence, c-FLIP proteins at the DISC control initiation, elongation, and composition of DED filaments, playing the role of control checkpoints. These findings provide new insights into DISC and DED filament regulation and open innovative possibilities for targeting the extrinsic apoptosis pathway.

  • Mechanisms of Procaspase-8 Activation in the Extrinsic Programmed Cell Death Pathway
    Molekuliarnaia biologiia, 2019
    Co-Authors: N. V. Ivanisenko, Inna N. Lavrik
    Abstract:

    Caspase-8 performs initiatory functions during the induction of apoptosis through the extrinsic pathway. Apoptosis is a type of programmed cell death that plays an important role in regulating embryogenesis and maintaining homeostasis in the tissue of an adult organism, as well as differentiating and removing damaged cells. Dysregulation of the apoptosis mechanisms is associated with the pathogenesis and progression of a number of oncological and neurodegenerative diseases. Caspase-8 (also called СAP4, FLICE, MACH, MCH5) is one of two members of the death effector domain (DED)-containing caspases. Despite the fact that the role of caspase-8 in apoptosis has been well known since the mid 1990s, we are only now beginning to understand the subtle mechanisms of its activation and regulation in response to the activation of death receptors (DRs). In particular, it was demonstrated that the activation of caspase-8 requires the formation of specific oligomeric structures, which are named DED filaments. In this review, the recent data on the mechanisms of activating initiator caspase-8 in DED filaments are considered that allow us to better understand the subtle mechanisms of the initiation of the programmed cell death.

  • Model topology.
    2018
    Co-Authors: J H Buchbinder, Dennis Pischel, Kai Sundmacher, Robert J. Flassig, Inna N. Lavrik
    Abstract:

    The model of CD95 network comprises the cytosol (blue), surrounded by the cell membrane, and the nucleus (orange). The subnet of this model is CD95 DISC (gray). The CD95 DISC comprises CD95, FADD, and DED chains/filaments. Procaspase-8, c-FLIPL, and c-FLIPR+S contribute to the DED chain/filament formation and embody the source of extrinsic noise (green). In contrast intrinsic noise is introduced by stochastic gene expression (yellow arrows). The relative amounts of Procaspase-8, its cleavage products, and c-FLIP proteins were measured by western blot (thick frame), while active caspase-3 and nuclear NF-κB by imaging flow cytometry (red). Inhibitions are marked by red lines and activations by green arrows.

Ming T. Lin - One of the best experts on this subject based on the ideXlab platform.

  • Procaspase 8 and Bax Are Up-regulated by Distinct Pathways in Streptococcal Pyrogenic Exotoxin B-induced Apoptosis
    The Journal of biological chemistry, 2009
    Co-Authors: Chia Wen Chang, Wan Hua Tsai, Woei Jer Chuang, Yee Shin Lin, Ching Chuan Liu, Pei Jane Tsai, Ming T. Lin
    Abstract:

    We have previously identified integrin αvβ3 and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S, a mutant of SPE B that binds to Fas only. In the current study we found that after binding to αvβ3, SPE B stimulated the tyrosine phosphorylation of JAK2 and STAT1. STAT1 tyrosine phosphorylation was inhibited by a JAK2 inhibitor, AG490, short interfering RNA (siRNA) silencing of JAK2, and anti-αVβ3 antibody. AG490 also decreased the binding of tyrosine-phosphorylated STAT1 to the Procaspase 8 promoter, decreasing Procaspase 8 expression, suggesting that SPE B up-regulates Procaspase 8 expression via the JAK2/STAT1 pathway. Alternatively, both SPE B and G308S increased STAT1 phosphorylation at serine 727, which was inhibited by anti-Fas antibody, a p38 inhibitor, SB203580, and siRNA silencing of p38. In addition, SPE B and G308S increased binding of serine-phosphorylated STAT1 to the Bax promoter and Bax expression, which was decreased by SB203580. SPE B and G308S-stimulated Bax expression was also inhibited by anti-Fas antibody. These findings suggest that Fas mediate SPE B-induced Bax expression through p38. Silencing of JAK2 or p38 by siRNA blocked Procaspase 8 expression, whereas only p38 siRNA decreased Bax expression. Furthermore, JAK2 inhibition and p38 inhibition reduced SPE B-induced apoptosis, but only p38 inhibition blocked G308S-induced apoptosis.

  • The IL-8 production by Streptococcal pyrogenic exotoxin B.
    Experimental biology and medicine (Maywood N.J.), 2009
    Co-Authors: Chia Wen Chang, Woei Jer Chuang, Yee Shin Lin, Ching Chuan Liu, Pei Jane Tsai, Ming T. Lin
    Abstract:

    We have previously identified alpha(v)beta(3) and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S, a mutant of SPE B with RSD motif, which interacts with Fas only. This study aims to evaluate how SPE B interacts with cells to induce the production of IL-8. Our results showed that following exposure to SPE B or G308S, the levels of IL-8 protein and mRNA were increased and the increase was inhibited by the addition of anti-Fas antibody, suggesting that the increased production of IL-8 by SPE B is mediated through Fas receptor. In the presence of G308S, the association of FADD and Procaspase 8, and activation of NF-kappaB were also detected. The application of siRNA of FADD and of Procaspase 8 could inhibit the NF-kappaB activity. The proteolytic activity of caspase 8 was required for the NF-kappaB activity. Further studies showed that G308S could increase the phosphorylation of ERK and the translocation of NF-kappaB into the nucleus, and the inhibition of ERK phosphorylation decreased the IL-8 production, mRNA expression and activation of NF-kappaB. In addition, siRNA of Procaspase 8 could inhibit the G308S-induced cleavage of MEKK1, binding of MEKK1 to caspase 8, activation of ERK and the NF-kappaB activity. Taken together, the production of IL-8 by SPE B in A549 cells is mediated by Fas, and followed by the activation of FADD, caspase 8, MEKK1, ERK and NF-kappaB.

Gordon C. Shore - One of the best experts on this subject based on the ideXlab platform.

  • The Procaspase-8 isoform, Procaspase-8L, recruited to the BAP31 complex at the endoplasmic reticulum
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: David G. Breckenridge, Mai Nguyen, Stephan Kuppig, Michael Reth, Gordon C. Shore
    Abstract:

    BAP31 is an integral protein of the endoplasmic reticulum membrane and a substrate of caspase-8. Here, we describe the Procaspase-8 isoform, Procaspase-8L, which is ubiquitously expressed and selectively recruited to the BAP31 complex in response to apoptotic signaling by E1A. Procaspase-8L is characterized by the N-terminal extension (Nex) domain, which extends Procaspase-8/a at the N terminus and is required for selective association of Procaspase-8L with the BAP31 complex. Gene deletion identified BAP31 and related BAP29 as required for processing of Procaspase-8L in response to E1A, by a FADD-independent mechanism that was blocked by BCL-2. Further, Bap29,31 deletion, as well as a Nex-domain dominant-negative mutant, curtailed the activation of downstream caspases (IETDase and DEVDase) and cell death in response to E1A. Preferential recruitment of Procaspase-8L by the BAP31 complex at the endoplasmic reticulum suggests an additional pathway for regulating initiator caspase-8 during apoptosis.

  • E1A-induced Processing of Procaspase-8 Can Occur Independently of FADD and Is Inhibited by Bcl-2
    The Journal of biological chemistry, 1998
    Co-Authors: Mai Nguyen, Philip E. Branton, Sophie Roy, Donald W. Nicholson, Emad S. Alnemri, Wen-chen Yeh, Tak W. Mak, Gordon C. Shore
    Abstract:

    Abstract Expression of the 243-residue form of the adenovirus E1A protein in the absence of other viral proteins triggers apoptosis by a pathway that requires p53. This pathway includes processing and activation of initiator Procaspase-8, redistribution of cytochrome c, and activation of Procaspase-3. Bcl-2 functions at or upstream of Procaspase-8 processing to inhibit all of these events and prevent cell death. This contrasts with the anti-apoptotic influence of Bcl-2 family proteins in the cell death pathway induced by Fas ligand or tumor necrosis factor (TNF), in which Bcl-2 typically acts downstream of Fas/TNFR1-mediated activation of caspase-8. Moreover, E1A induces Procaspase-8 processing and cell death in cells deleted of FADD, an adaptor protein critical for Fas/TNFR1 activation of caspase-8. The results indicate that E1A is capable of activating caspase-8 by a Bcl-2-inhibitable pathway that does not involve autocrine stimulation of FADD-dependent death receptor pathways.

  • BCL-XL COOPERATIVELY ASSOCIATES WITH THE BAP31 COMPLEX IN THE ENDOPLASMIC RETICULUM, DEPENDENT ON Procaspase-8 AND CED-4 ADAPTOR
    The Journal of biological chemistry, 1998
    Co-Authors: Gordon C. Shore
    Abstract:

    Abstract Bap31 is a polytopic integral membrane protein of the endoplasmic reticulum and forms a complex with Bcl-2/Bcl-XL and Procaspase-8 (Ng, F. W. H., Nguyen, M., Kwan, T., Branton, P. E., Nicholson, W. D., Cromlish, J. A., and Shore, G. C. (1997) J. Cell Biol.139, 327–338). In co-transfected human cells, Procaspase-8 is capable of interacting with Ced-4, an important adaptor molecule inCaenorhabditis elegans that binds to and activates theC. elegans Procaspase, proCed-3. Here, we show that the predicted death effector homology domain within the cytosolic region of Bap31 interacts with Ced-4 and contributes to recruitment of Procaspase-8. Bcl-XL, which binds directly but weakly to the polytopic transmembrane region of Bap31, indirectly and cooperatively associates with the Bap31 cytosolic domain, dependent on the presence of Procaspase-8 and Ced-4. Ced-4Δc does not interact with Bcl-XL but rather displaces it from Bap31, suggesting that an endogenous Ced-4-like adaptor is a normal constituent of the Bap31 complex and is required for stable association of Bcl-XL with Bap31 in vivo. These findings indicate that Bap31 is capable of recruiting essential components of a core death regulatory machinery.

  • p28 bap31 a bcl 2 bcl xl and Procaspase 8 associated protein in the endoplasmic reticulum
    Journal of Cell Biology, 1997
    Co-Authors: Mai Nguyen, Philip E. Branton, Donald W. Nicholson, Tony Kwan, James A Cromlish, Gordon C. Shore
    Abstract:

    We have identified a human Bcl-2–interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and Procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like Procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of Procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.

Peter H Krammer - One of the best experts on this subject based on the ideXlab platform.

  • Molecular architecture of the DED chains at the DISC: regulation of Procaspase-8 activation by short DED proteins c-FLIP and Procaspase-8 prodomain
    Cell death and differentiation, 2015
    Co-Authors: Kolja Schleich, Selcen Ozturk, Peter H Krammer, Uwe Warnken, Martina Schnölzer, J H Buchbinder, S Pietkiewicz, T Kähne, M Naumann, Inna N. Lavrik
    Abstract:

    The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, Procaspase-8, Procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that Procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating Procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of Procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and Procaspase-10 that are present in 10 times lower amounts compared with Procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with Procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor Procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of Procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that Procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in Procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

  • stoichiometry of the cd95 death inducing signaling complex experimental and modeling evidence for a death effector domain chain model
    Molecular Cell, 2012
    Co-Authors: Kolja Schleich, Nicolai Fricker, Selcen Ozturk, Kerstin Kammerer, Petra Richter, Peter H Krammer, Uwe Warnken, Martina Schnölzer, Inna N. Lavrik
    Abstract:

    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.

  • Regulation of CD95/Fas signaling at the DISC
    Cell Death & Differentiation, 2011
    Co-Authors: Inna N. Lavrik, Peter H Krammer
    Abstract:

    CD95 (APO-1/Fas) is a member of the death receptor (DR) family. Stimulation of CD95 leads to induction of apoptotic and non-apoptotic signaling pathways. The formation of the CD95 death-inducing signaling complex (DISC) is the initial step of CD95 signaling. Activation of Procaspase-8 at the DISC leads to the induction of DR-mediated apoptosis. The activation of Procaspase-8 is blocked by cellular FLICE-inhibitory proteins (c-FLIP). This review is focused on the role in the CD95-mediated signaling of the death effector domain-containing proteins Procaspase-8 and c-FLIP. We discuss how dynamic cross-talk between Procaspase-8 and c-FLIP at the DISC regulates life/death decisions at CD95.

  • A New C-Terminal Cleavage Product of Procaspase-8, p30, Defines an Alternative Pathway of Procaspase-8 Activation†
    Molecular and cellular biology, 2009
    Co-Authors: Julia C. Hoffmann, Peter H Krammer, Alexander Pappa, Inna N. Lavrik
    Abstract:

    Apoptosis can be triggered by a number of factors, including UV or γ-irradiation, chemotherapeutic drugs, and signaling from death receptors (11, 12). CD95 (APO-1/Fas) is a member of the death receptor family, a subfamily of the tumor necrosis factor receptor (TNF-R) superfamily (1, 30). Eight members of the death receptor subfamily have been characterized so far: TNF-R1 (DR1, CD120a, p55, p60), CD95 (DR2, APO-1, Fas), DR3 (APO-3, LARD, TRAMP, WSL1), TRAIL-R1 (APO-2, DR4), TRAIL-R2 (DR5, KILLER, TRICK2), DR6, EDA-R, and NGF-R (13). Cross-linking of CD95 by its natural ligand, CD95L (CD178) (29), or by agonistic antibodies induces apoptosis in sensitive cells (31, 36). The death-inducing signaling complex (DISC) is formed within seconds after CD95 stimulation (9). The DISC consists of oligomerized, probably trimerized CD95 receptors, the adaptor molecule FADD, two isoforms of Procaspase-8 (Procaspase-8a and -8b), Procaspase-10, and c-FLIPL/S/R (6, 19, 21, 25, 27). The interactions between molecules at the DISC are based on homotypic contacts. The death domain of the receptor interacts with the death domain of FADD, while the death effector domain (DED) of FADD interacts with the N-terminal tandem DEDs of Procaspase-8 and -10 and c-FLIPL/S/R. Two isoforms of Procaspase-8 (Procaspase-8a and Procaspase-8b) were reported to be bound to the DISC (24). Both isoforms possess two tandem DEDs, as well as the catalytic subunits p18 and p10 (see Fig. ​Fig.1A).1A). Procaspase-8a contains an additional 2-kDa (15-amino-acid [aa]) fragment, which results from the translation of exon 9. This small fragment is located between the second DED and the large catalytic subunit, resulting in different lengths of Procaspase-8a and -8b (p55 and p53 kDa), respectively. FIG. 1. A new 30-kDa protein is detected by the anti-caspase-8 MAb C15. (A) Scheme of Procaspase-8 and its cleavage products. The binding sites of the anti-caspase-8 MAbs C5 and C15 are indicated. (B) The B-lymphoblastoid cell lines SKW6.4, Raji, and BJAB and ... Activation of Procaspase-8 is believed to follow an “induced-proximity” model in which high local concentrations and a favorable mutual orientation of Procaspase-8 molecules at the DISC lead to their autoproteolytic processing (2, 3, 20). There is strong evidence from several in vitro studies that autoproteolytic activation of Procaspase-8 occurs after oligomerization at the receptor complex (20). Furthermore, it has been shown that homodimers of Procaspase-8 have proteolytic activity and that proteolytic processing of Procaspase-8 occurs between precursor homodimers (3). Procaspase-8a/b (p55/p53) processing at the DISC has been described to involve two sequential cleavage steps (see Fig. ​Fig.1A).1A). This process is referred to as the “two-step model” (3, 17). The first cleavage step occurs between the two protease domains, and the second cleavage step takes place between the prodomain and the large protease subunit (see Fig. ​Fig.1A)1A) (15). During the first cleavage step, the cleavage at Asp374 generates the two subunits p43/p41 and p12. Both cleavage products remain bound to the DISC: p43/p41 by DED interactions and p12 by interactions with the large protease domain of p43/p41. The second cleavage step takes place at Asp216 and Asp384, producing the active enzyme subunits p18, p10, and the prodomain p26/p24. As a result of Procaspase-8 processing, the active caspase-8 heterotetramer p182-p102 is formed at the DISC. This heterotetramer is subsequently released into the cytosol, starting the apoptotic signaling cascade (14). Recent studies have shown that processing of Procaspase-8 at the DISC is more complicated and can involve additional steps like the generation of a prolonged prodomain of Procaspase-8, termed CAP3 (p27), that is quickly converted to p26 (see Fig. ​Fig.1A)1A) (7). In addition to its central role in death receptor-induced apoptosis, caspase-8 was reported to be required for proliferation of lymphocytes (12, 23). Recently caspase-8 was shown to be an important factor for NF-κB activation following T-cell receptor stimulation (28). The mechanism underlying the dual role of caspase-8 activity and its regulation is largely unknown. In the present study, we show that upon death receptor stimulation, p30 is formed by cleavage at Asp210, a yet-unknown cleavage product of Procaspase-8, which comprises the C terminus of Procaspase-8. p30 turned out to be a key intermediate product in the course of Procaspase-8 processing. Furthermore, we suggest that the p30-mediated activation of Procaspase-8 plays an important role in the amplification of the death signal. Taken together, our findings provide a new mechanism of Procaspase-8 activation and extend the current two-step cleavage model by an alternative activation pathway.

  • The role of CAP3 in CD95 signaling: new insights into the mechanism of Procaspase-8 activation.
    Cell death and differentiation, 2005
    Co-Authors: Alexander Golks, Peter H Krammer, Dirk Brenner, Ingo Schmitz, Carsten Watzl, Andreas Krueger, Inna N. Lavrik
    Abstract:

    Formation of the CD95 (APO-1/Fas) death inducing signaling complex (DISC) plays a central role in CD95 signaling. Previously, CD95 DISC composition was analyzed by two-dimensional gel electrophoresis and four major cytotoxicity-associated proteins (CAP1-4) were found. CAP1 and CAP2 were defined to be unmodified and phosphorylated FADD, respectively. CAP4 was identified as Procaspase-8a. CAP3, however, has remained elusive. In this study, we demonstrate that CAP3 is an intermediate of Procaspase-8 processing. CAP3 is generated within seconds of DISC formation and subsequently processed to the prodomain of Procaspase-8a that is known as p26 (CAP5). These findings lead to new insights into the mechanism of Procaspase-8 processing and apoptosis initiation.

Dean A. Fennell - One of the best experts on this subject based on the ideXlab platform.

  • Prognostic and therapeutic relevance of FLIP and Procaspase-8 overexpression in non-small cell lung cancer
    Cell death & disease, 2013
    Co-Authors: Joel S. Riley, Caitriona Holohan, Nyree Crawford, Ryan Hutchinson, Darragh G. Mcart, Ian Paul, S. Van Schaeybroeck, Manuel Salto-tellez, Patrick G. Johnston, Dean A. Fennell
    Abstract:

    Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and Procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and Procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and Procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and Procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1–3 selective inhibitors such as entinostat to exploit high Procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.

  • Procaspase 8 overexpression in non-small-cell lung cancer promotes apoptosis induced by FLIP silencing
    Cell death and differentiation, 2009
    Co-Authors: Timothy R. Wilson, Caitriona Holohan, Nyree Crawford, Dean A. Fennell, Kathy Gately, Kelly M. Redmond, Kirsty Mclaughlin, Kenneth J. O'byrne, C. Le-clorrenec, Patrick B. Johnston
    Abstract:

    We found that Procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of Procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent Procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.