Antagonists Induce a Conformational Change in cIAP1 That Promotes Autoubiquitination
Department of Early Discovery Biochemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. Science
(Impact Factor: 33.61).
10/2011; 334(6054):376-80. DOI: 10.1126/science.1207862
Inhibitor of apoptosis (IAP) proteins are negative regulators of cell death. IAP family members contain RING domains that impart E3 ubiquitin ligase activity. Binding of endogenous or small-molecule antagonists to select baculovirus IAP repeat (BIR) domains within cellular IAP (cIAP) proteins promotes autoubiquitination and proteasomal degradation and so releases inhibition of apoptosis mediated by cIAP. Although the molecular details of antagonist-BIR domain interactions are well understood, it is not clear how this binding event influences the activity of the RING domain. Here biochemical and structural studies reveal that the unliganded, multidomain cIAP1 sequesters the RING domain within a compact, monomeric structure that prevents RING dimerization. Antagonist binding induces conformational rearrangements that enable RING dimerization and formation of the active E3 ligase.
Available from: Andre Willasch
- "cIAP1 and cIAP2 are involved in degradation of the MAP3 kinase, NFB inducing kinase (NIK) in the NFκB pathway (23, 26–29), and contribute to activation of the classical NFκB pathway by tumor necrosis factor (TNF) stimulation (30–32). Besides preventing the XIAP interaction with caspases, SMAC mimetics induce activation of the NFκB pathway by binding to cIAP1 and cIAP2 and stimulating the E3 ubiquitin-ligase activity of the cIAP proteins (33). "
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ABSTRACT: Allogeneic hematopoietic stem cell transplantation (HSCT) is an established treatment option for high-risk hematological malignancies, and may also be offered to patients with solid malignancies refractory to conventional therapies. In case of patients' relapse, refractory tumor cells may then be targeted by cellular therapy-based combination strategies. Here, we investigated the potential of small molecule IAP (SMAC mimetic) BV6 in increasing cytokine-induced killer (CIK) cell-mediated cytotoxicity against different tumor targets. Four-hour pre-incubation with 2.5 μMol BV6 moderately enhanced CIK cell-mediated lysis of hematological (H9, THP-1, and Tanoue) and solid malignancies (RH1, RH30, and TE671). However, BV6 also increased apoptosis of non-malignant cells like peripheral blood mononuclear cells and most notably had an inhibitory effect on immune cells potentially limiting their cytotoxic potential. Hence, cytotoxicity increased in a dose-dependent manner when BV6 was removed before CIK cells were added to tumor targets. However, cytotoxic potential was not further increasable by extending BV6 pre-incubation period of target cells from 4 to 12 h. Molecular studies revealed that BV6 sensitization of target cells involved activation of caspases. Here, we provide evidence that SMAC mimetic may sensitize targets cells for CIK cell-induced cell death. However, BV6 also increased apoptosis of non-malignant cells like CIK cells and peripheral mononuclear cells. These findings may therefore be important for cell- and small molecule IAP-based combination therapies of resistant cancers after allogeneic HSCT.
Available from: Jacek Bielawski
- "The above observations that LCL85 does not alter Fas level suggests that LCL85 may target mediators of the Fas-mediated apoptosis signaling pathways. IAPs are potent inhibitors of apoptosis, including Fas-mediated apoptosis [35-37]. To determine whether IAPs play a role in metastatic human colon carcinoma apoptosis resistance, we tested the effects of IAP-specific inhibitor BV6 on metastatic human colon carcinoma cells. "
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ABSTRACT: Ceramide is a bioeffector that mediates various cellular processes, including apoptosis. However, the mechanism underlying ceramide function in apoptosis is apparently cell type-dependent and is not well-understood. We aimed at identifying molecular targets of ceramide in metastatic human colon and breast cancer cells, and determining the efficacy of ceramide analog in suppression of colon and breast cancer metastasis.
The activity of and mechanism underlying ceramide as a cytotoxic agent, and as a sensitizer for Fas-mediated apoptosis was analyzed in human cell lines established from primary or metastatic colon and breast cancers. The efficacy of ceramide analog LCL85 in suppression of metastasis was examined in preclinical mouse tumor models.
Exposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to Fas-mediated apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to Fas-mediated apoptosis. LCL85 resembles Smac mimetic BV6 in sensitization of colon carcinoma cells to Fas-mediated apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. LCL85 also decreased xIAP1 and cIAP1 protein levels and sensitized metastatic human breast cancer cells to Fas-mediated apoptosis. Silencing xIAP and cIAP with specific siRNAs significantly increased the metastatic human colon carcinoma cell sensitivity to Fas-mediated apoptosis, suggesting that IAP proteins mediates apoptosis resistance in metastatic human colon carcinoma cells and ceramide induces IAP protein degradation to sensitize the tumor cells to apoptosis induction. Consistent with its apoptosis sensitization activity, subtoxic doses of LCL85 suppressed colon carcinoma cell metastatic potential in an experimental lung metastasis mouse model, as well as breast cancer growth and spontaneous lung metastasis in a orthotopic breast cancer mouse model.
We have identified xIAP and cIAP1 as molecular targets of ceramide and determined that ceramide analog LCL85 is an effective sensitizer in overcoming resistance of human cell lines established from metastatic colon and breast cancers to apoptosis induction to suppress metastasis in vivo.
Available from: PubMed Central
- "In addition, discovery of SMAC-mimicking IAP antagonist compounds that target c-IAP protein for proteasomal degradation greatly aided the affirmation of c-IAPs as E3 ligases for NIK (31, 32). IAP antagonist treatment triggers rapid conformational change in c-IAP proteins that elevates their ubiquitin ligase activity leading to proteasomal degradation (53, 54). Some cells treated with these agents secrete a variety of NF-κB regulated inflammatory cytokines over a prolonged period even in the absence of cell death (55, 56). "
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ABSTRACT: TNF superfamily ligands and receptors are responsible for development, immunity and homeostasis of metazoan organisms. Thus, it is not surprising that signals emanating from these receptors are tightly regulated. Binding of TWEAK to its cognate receptor, FN14, triggers the assembly of receptor-associated signaling complex, which allows the activation of canonical and noncanonical NF-κB as well as MAPK signaling pathways. Ubiquitin ligases c-IAP1 and c-IAP2 and adaptor proteins TRAF2 and TRAF3 are crucial for the regulation of TWEAK signaling as they facilitate the recruitment of distal signaling components including IKK and LUBAC complexes. At the same time c-IAP1/2, together with TRAF2 and TRAF3, promote constitutive ubiquitination and proteasomal degradation of NIK – a kinase with critical role in the activation of noncanonical NF-κB signaling. While c-IAP1/2 mediated ubiquitination allows the activation of TWEAK stimulated canonical NF-κB signaling, these E3 ligases are negative regulators of noncanonical signaling. TWEAK stimulation prompts the recruitment of c-IAP1/2 as well as TRAF2 and TRAF3 to the FN14 signaling complex leading to c-IAP1/2 autoubiquitination and degradation, which stabilizes NIK and allows subsequent phosphorylation of IKKα and partial proteasomal processing of p100 to activate gene expression. Recent studies have revealed that the spatio-temporal pattern of TWEAK stimulated ubiquitination is a carefully orchestrated process involving several substrates that are modified by different ubiquitin linkages. Understanding the significance of ubiquitination for TWEAK signaling is important for the overall understanding of TWEAK biology and for the design of therapeutics that can be used in the treatment of human pathologies that are driven by TWEAK/FN14 expression and activity.
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