Identification of a Novel Anti-apoptotic E3 Ubiquitin Ligase That Ubiquitinates Antagonists of Inhibitor of Apoptosis Proteins SMAC, HtrA2, and ARTS (original) (raw)
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Cell, 2005
members of the family promote the release of apoptogenic proteins from mitochondria. The fate of a cell un-and Xiaodong Wang* Howard Hughes Medical Institute and der apoptotic stimulation is determined by the balance between the pro-and antiapoptotic members of the Department of Biochemistry University of Texas Southwestern Medical Center Bcl-2 family. Therefore, studying the regulation of such balance will be critically important for our understand-at Dallas Dallas, Texas 75390 ing of apoptosis regulation. Among the antiapoptotic members of the Bcl-2 family proteins, Mcl-1 is unique in that it is an early-response Summary gene that can be rapidly induced and turned over (Kozopas et al., 1993; Yang et al., 1995; Yang et al., 1996). The elimination of Mcl-1, an anti-apoptotic Bcl-2 fam-This property enables Mcl-1 to function at an early step ily member, is an early and required step for DNA in a signaling cascade, consisting of Bcl-2 family prodamage-induced apoptosis. The degradation of Mcl-1 teins, and provides an acute protective function against can be blocked by proteasome inhibitors, suggesting apoptosis induced by a variety of stimuli, including a role for the ubiquitin proteasome pathway in apopto-DNA damage, adenoviral infection, growth factors' sis. Here, we demonstrate that Mcl-1 is ubiquinated at withdrawal, and treatment of cytotoxic agents (Cucofive lysines. Biochemical fractionation of cell extracts nati et al., 2003; Derouet et al., 2004; Huang et al., 2000; allowed us to identify a 482 kDa HECT-domain-contain-Le Gouill et al., 2004; Nijhawan et al., 2003; Piret et al., ing ubiquitin ligase named Mule (Mcl-1 ubiquitin ligase 2004; Zhang et al., 2002; Zhou et al., 1997). Consis-E3) that is both required and sufficient for the polytently, disappearance of Mcl-1 is associated with the ubiquitination of Mcl-1. Mule also contains a region onset of apoptosis and is achieved by the combination similar to the Bcl-2 homology region 3 (BH3) domain of synthesis blockage and continuous degradation (Cuthat allows Mule to specifically interact with Mcl-1. conati et al., 2003; Nijhawan et al., 2003). Elimination of Mule expression by RNA interference The degradation of Mcl-1 in HeLa cells can be stabilizes Mcl-1 protein, resulting in an attenuation of blocked by proteasome inhibitors, suggesting a role for the apoptosis induced by DNA-damage agents. Thus, the ubiquitin proteasome pathway in apoptosis up-Mule is a unique BH3-containing E3 ubiquitin ligase stream of Bcl-2 family of proteins (Derouet et al., 2004; apical to Bcl-2 family proteins during DNA damage-Nencioni et al., 2004; Cuconati et al., 2003; Nijhawan et induced apoptosis. al., 2003). In the current report, we demonstrate that Mcl-1 is polyubiquitinated in vivo and in vitro. Using Introduction HeLa cell extracts as a source, we established a cellfree assay for Mcl-1 ubiquitination. Biochemical frac-Apoptosis is a form of cell death orchestrated by chains tionation of HeLa cell extracts allowed us to identify of biochemical reactions. Cells undergoing apoptosis and purify a 482 kDa E3 ubiquitin ligase that mediated show characteristic morphological features such as the polyubiquitination of Mcl-1. This protein turned out condensation of cytoplasmic and nuclear contents, to be a novel homologous to E6-AP carboxyl terminus blebbing of plasma membranes, fragmentation of nu-(HECT)-domain-containing ubiquitin ligase. We named clei, and ultimately breakdown into membrane bound this protein Mule, for Mcl-1 ubiquitin ligase E3. apoptotic bodies that are rapidly phagocytized (Kerr et al., 1972). Results Mitochondria play an important role in regulating apoptosis induced by intracellular damaging signals Ubiquitination of Mcl-1 In Vivo and In Vitro such as DNA damage in mammalian cells (Danial and Proteins targeted for proteasome degradation are usu-Korsmeyer, 2004). Apoptotic stimuli exert their effects ally modified by a polyubiquitin chain (Hershko and on mitochondria to cause the release of proapoptotic Ciechanover, 1998). To test whether Mcl-1 is ubiqfactors like cytochrome c and Smac/Diablo. These facuitinated, we treated HeLa cells expressing Flag-Mcl-1 tors either directly activate caspases, a group of intrawith a proteasome inhibitor MG132 to block the procellular cysteine proteases that execute apoptosis by teasome activity and performed immunoprecipitation cleaving their substrates or releasing caspase inhibition analysis for Flag-Mcl-1 to test for the accumulation of imposed by the inhibitor-of-apoptosis proteins (IAPs; higher-molecular-weight forms of Mcl-1 that could be Du et al., 2000; Liu et al., 1996; Verhagen et al., 2000). ubiquitin modified. As shown in Figure 1A, higher-Mitochondrial response to apoptotic stimuli is regumolecular-weight protein bands recognized by antilated by the pro-and antiapoptotic Bcl-2 family of pro-Mcl-1 antibody were indeed accumulated upon MG132 teins (Gross et al., 1999; Martinou and Green, 2001). treatment (Figure 1A, lanes 3 and 4). To rule out a possi-Antiapoptotic proteins such as Bcl-2, Bcl-xL, and Mcl-1 bility that these higher-molecular-weight bands were protect mitochondrial integrity, while the proapoptotic from Mcl-1-associated protein rather than Mcl-1 itself, the protein mixture was first heated in the presence of 1% SDS and 5 mM DTT to disassociate protein-protein
PloS one, 2011
Here we identified an evolutionarily highly conserved and ubiquitously expressed protein (C9orf82) that shows structural similarities to the death effector domain of apoptosis-related proteins. RNAi knockdown of C9orf82 induced apoptosis in A-549 and MCF7/casp3-10b lung and breast carcinoma cells, respectively, but not in cells lacking caspase-3, caspase-10 or both. Apoptosis was associated with activated caspases-3, -8, -9 and -10, and inactivation of caspases 10 or 3 was sufficient to block apoptosis in this pathway. Apoptosis upon knockdown of C9orf82 was associated with increased caspase-10 expression and activation, which was required for the generation of an 11 kDa tBid fragment and activation of Caspase-9. These data suggest that C9orf82 functions as an anti-apoptotic protein that modulates a caspase-10 dependent mitochondrial caspase-3/9 feedback amplification loop. We designate this ubiquitously expressed and evolutionarily conserved anti-apoptotic protein Conserved Anti-Ap...
… International Journal of …, 2011
ARTS (Sept4 i2) is a mitochondrial pro-apoptotic tumor suppressor protein. In response to apoptotic signals, ARTS translocates to the cytosol where it promotes caspase activation through caspase de-repression and proteasome mediated degradation of X-linked Inhibitor of Apoptosis Protein (XIAP). Here we show that XIAP regulates the levels of ARTS by serving as its ubiquitin ligase, thereby providing a potential feedback mechanism to protect against unwanted apoptosis. Using both in vitro and in vivo ubiquitination assays we found that ARTS is directly ubiquitinated by XIAP. Moreover, we found that XIAP-induced ubiquitination and degradation is prevented by removal of the first four amino acids in the N-terminus of ARTS, which contains a single lysine residue at position 3. Thus, this lysine at position 3 is a likely target for ubiquitination by XIAP. Importantly, although the stabilized ARTS lacking its first 4 residues binds XIAP as well as the full length ARTS, it is more potent in promoting apoptosis than the full length ARTS. This suggests that increased stability of ARTS has a significant effect on its ability to induce apoptosis. Collectively, our data reveal a mutual regulatory mechanism by which ARTS and XIAP control each other's levels through the ubiquitin proteasome system.
Journal of Biological Chemistry, 2001
To identify human proteins that bind to the Smac and caspase-9 binding pocket on the baculoviral inhibitor of apoptosis protein (IAP) repeat 3 (BIR3) domain of human XIAP, we used BIR3 as an affinity reagent, followed by elution with the BIR3 binding peptide AVPIA, microsequencing, and mass spectrometry. The mature serine protease Omi (also known as HtrA2) was identified as a mitochondrial direct BIR3-binding protein and a caspase activator. Like mature Smac (also known as Diablo), mature Omi contains a conserved IAP-binding motif (AVPS) at its N terminus, which is exposed after processing of its N-terminal mitochondrial targeting sequence upon import into the mitochondria. Mature Omi is released together with mature Smac from the mitochondria into the cytosol upon disruption of the outer mitochondrial membrane during apoptosis. Finally, mature Omi can induce apoptosis in human cells in a caspase-independent manner through its protease activity and in a caspase-dependent manner via its ability to disrupt caspase-IAP interaction. Our results provide clear evidence for the involvement of a mitochondrial serine protease in the apoptotic pathway, emphasizing the critical role of the mitochondria in cell death.
Regulation of the Proapoptotic ARTS Protein by Ubiquitin-mediated Degradation
Journal of Biological Chemistry, 2005
ARTS is a mitochondrial protein that promotes apoptosis induced by a variety of proapoptotic stimulators. ARTS induces apoptosis, at least in part, through binding to and antagonizing IAPs (inhibitors of apoptosis proteins). As a result of ARTS binding to IAPs, caspase inhibition is removed and apoptosis can be executed. Here we show that high cellular levels of ARTS protein sensitize cells toward apoptosis. Accordingly, in healthy cells ARTS levels are kept low through constant ubiquitin-mediated degradation. Upon proapoptotic stimuli, the ubiquitination process is inhibited, resulting in increased levels of ARTS. Increased ARTS in turn leads to a decrease of Bcl-2 and Bcl-xL protein levels, cytochrome c release from mitochondria and apoptosis.
Regulation of Apoptosis by Inhibitors of Apoptosis (IAPs)
2013
Inhibitors of Apoptosis (IAPs) are a family of proteins with various biological functions including regulation of innate immunity and inflammation, cell proliferation, cell migration and apoptosis. They are characterized by the presence of at least one N-terminal baculoviral IAP repeat (BIR) domain involved in protein-protein interaction. Most of them also contain a C-terminal RING domain conferring an E3-ubiquitin ligase activity. In drosophila, IAPs are essential to ensure cell survival, preventing the uncontrolled activation of the apoptotic protease caspases. In mammals, IAPs can also regulate apoptosis through controlling caspase activity and caspase-activating platform formation. Mammalian IAPs, mainly X-linked IAP (XIAP) and cellular IAPs (cIAPs) appeared to be important determinants of the response of cells to endogenous or exogenous cellular injuries, able to convert the survival signal into a cell death-inducing signal. This review highlights the role of IAP in regulating apoptosis in Drosophila and Mammals.
Regulation of apoptosis by XIAP ubiquitin-ligase activity
Genes & Development, 2008
Because IAPs are frequently overexpressed in human tumors, they have become major pharmacological targets for developing new cancer therapeutics. However, the precise physiological function of individual mammalian IAPs and their role as E3 ubiquitin-ligases in situ remain largely obscure. Here, we investigated the function of XIAP ubiquitin-ligase activity by inactivating the RING motif via gene targeting in the mouse. Removing the RING stabilized XIAP in apoptotic thymocytes, demonstrating that XIAP ubiquitin-ligase activity is a major determinant of XIAP protein stability. Surprisingly, the increased amounts of "XIAP-BIR-only" protein did not lead to attenuated but rather increased caspase activity and apoptosis. ⌬RING embryonic stem cells and fibroblasts had elevated caspase-3 enzyme activity, and XIAP ⌬RING embryonic fibroblasts were strongly sensitized to TNF-␣-induced apoptosis. Similar results were obtained with XIAP deficient mice. Furthermore, deletion of the RING also improved the survival of mice in the Eµ-Myc lymphoma model. This demonstrates a physiological requirement of XIAP ubiquitin-ligase activity for the inhibition of caspases and for tumor suppression in vivo.
Ubiquitylation in apoptosis: a post-translational modification at the edge of life and death
Nature Reviews Molecular Cell Biology, 2011
Apoptosis is mediated by the assembly of signalling complexes that culminates in the activation of a cell death programme. It is evident that these complexes are subject to substantial post-translational regulation through modification by the 76-amino-acid protein ubiquitin. The ubiquitylation of constituent components in the apoptotic pathway often destabilizes them by targeting them for proteasomal degradation. However, just as importantly, the ubiquitin chain-mediate d assembl y of apoptotic signalling complexes illuminates how ubiquityl ation can have non-degradative functions. Recent progress provides insight into how these seemingly disparate outcomes of ubiquitylatio n in apoptosis are mediated. This Review addresses the intersection of these two exciting fields: apoptosis and the ubiquitin-proteasom e system (UPS). We first describe the biochemistry of ubiquitylation and the various forms of ubiquitin modification, ranging from monoubiquitylation to linkage-specific polyubiquitin chain formation. This is followed by an introduction to the apoptotic pathways. We then discuss how apoptotic pathways are regulated by various components of the UPS, including ubiquitin E3 ligases and deubiquitinases (DUBs), before describing how the deregulation of ubiquitylation, and subsequently of apoptosis, can result in human diseases, such as cancer.
Targeting Inhibitors of Apoptosis Proteins (IAPs) For New Breast Cancer Therapeutics
Journal of Mammary Gland Biology and Neoplasia, 2012
Apoptosis resistance is a hallmark of human cancer. Research in the last two decades has identified key regulators of apoptosis, including inhibitor of apoptosis proteins (IAPs). These critical apoptosis regulators have been targeted for the development of new cancer therapeutics. In this article, we will discuss three members of IAP proteins, namely XIAP, cIAP1 and cIAP2, as cancer therapeutic targets and the progress made in developing new cancer therapeutic agents to target these IAP proteins. Apoptosis and human cancer Apoptosis, or programmed cell-death, is a fundamental cellular process to remove damaged or unwanted cells in multiple cellular organisms. Improper regulation of apoptosis is therefore linked in many human diseases, including cancer, autoimmune diseases, inflammation and neurological diseases. 1-3 In fact, defective regulation of apoptosis is a hallmark of human cancer. 4 Basic apoptosis pathways Apoptosis is a tightly regulated process. Several major apoptosis pathways have been identified and characterized in the last two decades, although these pathways often have extensive cross-talks. The intrinsic and extrinsic apoptosis pathways are two of the best studied (Figure 1). 5 The intrinsic, or mitochondria, apoptotic pathway integrates a variety of cell stress signals and is initiated by permeabilization of the outer membrane of mitochondria and loss of mitochondrial potential. On the molecular level, the intrinsic pathway involves the translocation and oligomerization of Bax or Bak, members of the Bcl-2 family proteins, which forms a pore in the outer member of mitochondria and leads to the release of proapoptotic molecules such as cytochrome C. Upon its release from mitochondria into cytosol, cytochrome C, together with dATP, Apaf-1 and procaspase-9, forms the apoptosome, which processes the procaspase-9 zymogen into the active form of caspase-9. Caspase-9 then cleaves and activates caspase-3,-6 and-7, which leads to further processing of downstream cell-death substrates, and ultimately apoptosis. The extrinsic, or death-receptor, apoptotic pathway, is initiated by the binding of death ligands such as Fas/Apo-1, TNF-alpha, Apo2L/TRAIL, and Apo3L ligands to their cognate cell-surface receptors, FasR, TNFR1, DR4/DR5 and DR3, respectively. The binding of these Phone: