The mitochondrial serine protease HtrA2/Omi cleaves RIP1 during apoptosis of Ba/F3 cells induced by growth factor withdrawal

Abstract

Interleukin-3 (IL-3) deprivation of the mouse pro-B cell line Ba/F3 induces cell death that is abrogated by B-cell lymphoma 2 (Bcl-2) overexpression, but remains unaffected by the pan-caspase inhibitor carbobenzoxy-valyl-analyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD-fmk). IL-3 withdrawal causes receptor-interacting protein (RIP)1 cleavage into C-terminal fragments of 30 and 25 kDa, and only cleavage leading to the former was prevented by zVAD-fmk. siRNA experiments demonstrated that generation of the 25-kDa fragment was due to a Bcl-2-modulated release of the mitochondrial serine protease high temperature requirement protein A2 (HtrA2)/Omi. Accordingly, recombinant HtrA2/Omi efficiently cleaved mouse RIP1 in vitro, generating fragments matching those observed in IL-3-deprived Ba/F3 cells. The HtrA2/Omi cleavage site in mouse RIP1 was mapped to the intermediate domain and the corresponding N- and C-terminal fragments were impaired in their ability to activate nuclear factor-#B, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. Interestingly, knockdown of HtrA2/Omi afforded pro-tection against IL-3 withdrawal-induced death in the presence of zVAD-fmk, demonstrating a role for HtrA2/Omi in caspase-independent cell death during growth factor withdrawal by cleaving RIP1

The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

“…However, showing the limitations of this method and obviously due to a nonspecific background binding of FAM-FFCK, most of the 108 proteins turned out to be non-proteases. Nevertheless, the mitochondrial serine protease HtrA2/Omi was identified in this screen with high confidence (Figure  2), and we considered it as the most promising candidate, because it had been already associated with both caspase-dependent as well as caspase-independent PCD [25]. …”

“…ER stress, heat shock and ischemia/reperfusion) [23]. Deletion of HtrA2/Omi or mutations affecting its activity have been associated with neurodegeneration and Parkinson’s disease in mouse models [24] and patients [25]. In response to apoptotic stimuli, HtrA2/Omi is released from mitochondria into the cytoplasm, where it promotes apoptosis by binding and inhibiting IAP (inhibitor of apoptosis) proteins, thus releasing active caspases from their natural inhibitors.…”

“…In response to apoptotic stimuli, HtrA2/Omi is released from mitochondria into the cytoplasm, where it promotes apoptosis by binding and inhibiting IAP (inhibitor of apoptosis) proteins, thus releasing active caspases from their natural inhibitors. Independently, HtrA2/Omi degrades IAPs, the caspase-8 inhibitor Pea-15 and the anti-apoptotic protein HAX-1 through its serine protease activity, further promoting apoptosis [25]. …”

“…It has been reported that HtrA2/Omi can mediate caspase-independent PCD via its serine protease activity, e.g. upon interleukin-3 deprivation of the mouse pro-B cell line Ba/F3 [25], in imatinib-treated human leukemic cells [26], or in cytomegalovirus infection [27]. However, except for one study reporting cleavage and inactivation of RIPK1 by HtrA2/Omi [25], the substrates of HtrA2/Omi in necroptosis/programmed necrosis are unknown.…”

RIP1 Cleavage in the Kinase Domain Regulates TRAIL-Induced NF-κB Activation and Lymphoma Survival

“…For example, (i) many proteins are phosphorylated at a specific site only in human proteins (e.g., Ser-46 phosphorylation in human but not mouse p53), (ii) only human endothelial cells but not mouse counterparts express functional CD40, and (iii) HtrA2 cleaves mouse but not human RIP1 (41)(42)(43). In addition, human and mouse cells seem to have different susceptibility levels and responses to RIP1 kinase inhibitor Nec-1 and its inactive control, Nec1i (44).…”