CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis

Abstract

We have identified and characterized CLARP, a caspase-like apoptosis-regulatory protein. Sequence analysis revealed that human CLARP contains two amino-terminal death effector domains fused to a carboxylterminal caspase-like domain. The structure and amino acid sequence of CLARP resemble those of caspase-8, caspase-10, and DCP2, a Drosophila melanogaster protein identified in this study. Unlike caspase-8, caspase-10, and DCP2, however, two important residues predicted to be involved in catalysis were lost in the caspase-like domain of CLARP. Analysis with f luorogenic substrates for caspase activity confirmed that CLARP is catalytically inactive. CLARP was found to interact with caspase-8 but not with FADD͞MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathway. Expression of CLARP induced apoptosis, which was blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-f luoromethylketone (zVAD-fmk). Moreover, CLARP augmented the killing ability of caspase-8 and FADD͞MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis.Apoptosis, a morphologically distinguished form of programmed cell death, is critical not only during development and tissue homeostasis but also in the pathogenesis of a variety of diseases, including cancer, autoimmune disease, viral infection, and neurodegenerative disorders (1-2). Whereas numerous genes that control apoptosis have been identified and partially characterized, the precise mechanisms by which these genes interact to execute the apoptotic program remains poorly understood. In mammals, a family of cysteine proteases (designated caspases) related to the Caenorhabditis elegans gene ced-3 appears to represent the effector arm of the apoptotic program (3-4). The first member of the family identified was interleukin 1␤-converting enzyme (5). To date, more than 10 caspases have been identified and partially characterized (3). Each caspase contains characteristic conserved sequences important for proteolytic activity and induction of apoptosis (3-4). Caspase activation is induced by a wide array of death signals and leads to precipitous cleavage of protein substrates and execution of the apoptotic program (1, 2).Engagement of the surface receptors, Fas͞APO-1͞CD95 and tumor necrosis factor receptor 1 (TNFR-1), leads to the activation of several caspases and apoptosis (6). Both CD95 and TNFR-1 use the adapter protein FADD͞MORT-1 to link cytoplasmic receptor sequences to the upstream FLICE͞ MACH͞Mch5 protease (caspase-8) and Mch4 (caspase-10) (7-14). Both FADD͞MORT-1 and caspase-8 interact through conserved death effector domains (DED) located in the N-terminal region of FADD͞MORT-1 and pro-domain of caspase-8 (7, 8, 11-13). Upon binding of the corresponding ligands, the CD95 re...

c-FLICE inhibitory protein expression inhibits T-cell activation

“…Second, c-FLIP has been shown to exhibit proapoptotic effect. 4,6,8,9,37,38 Consistent with these reports, we found an increase in the death of thymocytes from transgenic mice with c-FLIP overexpression (Figure 4), likely contributing to the reduction in total thymocyte number in c-FLIP-transgenic mice. It may be noted that apoptosis induced by c-FLIP overexpression was independent of the developmental stage of thymocytes; increased cell death was found in immature as well as in mature thymocyte populations (Figure 4), which may explain why the number of CD4 À CD8 À thymocyte was also reduced in c-FLIP-transgenic mice ( Table 1).…”

“…1,2 Cellular FLICE-inhibitory protein (c-FLIP) specifically regulates the DR apoptotic process. [3][4][5][6][7][8][9] c-FLIP is expressed in both a long-form (c-FLIP L ) and a short-form (c-FLIP S ) due to alternative splicing. The DEDs of c-FLIP L and c-FLIP S interact with the DEDs of FADD and procaspasse-8.…”

Activation of the NF-κB pathway by Caspase 8 and its homologs

“…The anti-apoptotic property of MRIT has been previously explained on the basis of its ability to compete with Caspases 8 or 10 to bind to the DED of FADD (Goltsev et al, 1997;Hu et al, 1997;Inohara et al, 1997;Irmler et al, 1997;Shu et al, 1997;Srinivasula et al, 1997). Our results demonstrate the ability of this protein to activate the anti-apoptotic NF-kB pathway and thus provide an alternative/ additional mechanism for its ability to protect against cell death.…”

“…The prodomain of Caspase 8 consists of two homologous DEDs and serves to keep Caspase 8 in an inactive form (Chinnaiyan and Dixit, 1997). DEDscontaining prodomains are also found in two additional cellular proteins; Caspase 10 (Mch4, FLICE2), a proteolytically active Caspase 8 homolog (FernandesAlnemri et al, 1996;Vincenz and Dixit, 1997), and MRIT (Casper, c-FLIP, I-FLICE, FLAME, CASH, CLARP), a Caspase 8 homolog which is devoid of protease activity (Goltsev et al, 1997;Han et al, 1997;Hu et al, 1997;Inohara et al, 1997;Irmler et al, 1997;Shu et al, 1997;Srinivasula et al, 1997).…”

Modulation of the NF-κB pathway by virally encoded Death Effector Domains-containing proteins

“…Caspase 8 is the apical caspase of the caspase cascade and possesses an N-terminal prodomain consisting of two homologous Death E ector Domains (DEDs) Fernandes-Alnemri et al, 1996;Muzio et al, 1996). DEDs-containing prodomains are also found in two additional cellular proteins; Caspase 10 (Mch4, FLICE2) (FernandesAlnemri et al, 1996;Vincenz and Dixit, 1997), and MRIT (c-FLIP, Casper, I-FLICE, FLAME, CASH, CLARP) (Goltsev et al, 1997;Han et al, 1997;Hu et al, 1997b;Inohara et al, 1997;Irmler et al, 1997;Shu et al, 1997;Srinivasula et al, 1997). Recently, several viruses were also found to encode proteins with homology to the DEDs (Bertin et al, 1997;Hu et al, 1997a;Thome et al, 1997).…”