Regulation of HAX-1 Anti-apoptotic Protein by Omi/HtrA2 Protease during Cell Death

Abstract

Omi/HtrA2 is a nuclear-encoded mitochondrial serine protease that has a pro-apoptotic function in mammalian cells. Upon induction of apoptosis, Omi translocates to the cytoplasm and participates in caspase-dependent apoptosis by binding and degrading inhibitor of apoptosis proteins. Omi can also initiate caspase-independent apoptosis in a process that relies entirely on its ability to function as an active protease. To investigate the mechanism of Omi-induced apoptosis, we set out to isolate novel substrates that are cleaved by this protease. We identified HS1-associated protein X-1 (HAX-1), a mitochondrial anti-apoptotic protein, as a specific Omi interactor that is cleaved by Omi both in vitro and in vivo. HAX-1 degradation follows Omi activation in cells treated with various apoptotic stimuli. Using a specific inhibitor of Omi, HAX-1 degradation is prevented and cell death is reduced. Cleavage of HAX-1 was not observed in a cell line derived from motor neuron degeneration 2 mice that carry a mutated form of Omi that affects its proteolytic activity. Degradation of HAX-1 is an early event in the apoptotic process and occurs while Omi is still confined in the mitochondria. Our results suggest that Omi has a unique pro-apoptotic function in mitochondria that involves removal of the HAX-1 antiapoptotic protein. This function is distinct from its ability to activate caspase-dependent apoptosis in the cytoplasm by degrading inhibitor of apoptosis proteins.Omi/HtrA2 is a mitochondrial serine protease that is released to the cytoplasm upon induction of apoptosis (1-4). In the cytoplasm, Omi binds and cleaves IAPs 1 leading to activation of caspase-dependent apoptosis (5, 6). Omi can also induce caspase-independent apoptosis through an as yet unknown mechanism that requires its proteolytic activity (7,8). In addition to its pro-apoptotic function, Omi has another unique role in maintaining mitochondrial homeostasis, but the details of this mechanism are still unclear (9). The serine protease activity of Omi is necessary and essential for its normal function whether it acts as a pro-apoptotic protein in the cytoplasm or as a potential chaperone in the mitochondria (9). The proteolytic activity of Omi has been associated with autoprocessing to form the mature protein as well as cleavage of IAPs to activate caspase-dependent apoptosis (5,6,8). To understand the mechanism of Omi's function, it will be necessary to identify new substrates for this protease. These substrates might be mitochondrial or cytoplasmic proteins, and their degradation and removal by Omi could be part of the apoptotic process. In this report, we used the yeast two-hybrid system to isolate and characterize new Omi-interacting proteins. One of these interactors isolated from this screen was the HS1-associated protein X-1 (HAX-1) anti-apoptotic protein (10). HAX-1 interacted with Omi both in vitro and in vivo. Furthermore, HAX-1 was degraded and removed by Omi when cells were treated with various apoptotic stimuli. Using a specific inhibitor of the pro...

Omi/HtrA2 is a positive regulator of autophagy that facilitates the degradation of mutant proteins involved in neurodegenerative diseases

“…23 The cleavage of Hax-1 by Omi was confirmed, as shown in Figure 4e. In transfected HeLa cells, increased expression of Omi leads to decreased levels of Hax-1 (Figure 4f).…”

“…Degradation of Hax-1 by Omi was observed when cells were treated with various apoptotic inducers, such as cisplatin and H 2 O 2 . 23 We report here that Hax-1, just like other Bcl-2 family-related proteins, is involved in autophagy through its interaction with and inhibition of Beclin-1. It is worth noting that Hax-1 functions not only as a substrate of Omi, but also as a regulator of the mitochondrial import of Omi through presentation of Omi to another mitochondrial protease, Parl.…”

“…Hax-1 is predominantly located in mitochondria. 23 It has also been reported that Hax-1 can localize to the ER. 32 Beclin-1 is located in both the mitochondria and the ER.…”

“…The Bcl-2 family protein Bcl-xL can block Omi-induced autophagy (Figure 4a). Interestingly, Hax-1, a Bcl-2 family-related protein and a specific substrate of Omi, 23 binds to Beclin-1 and represses autophagy (Figure 4e-k). The effects of Hax-1 on autophagic inhibition can be partially rescued by overexpression of Beclin-1 (Figure 4l), and a deletion mutant of Hax that lacks the Omi binding sequence shows enhanced inhibition of Omi-induced autophagy (Figure 4l-o).…”

“…Collectively, these findings suggest that the interaction or balance between Hax-1 and Omi is crucial for various aspects of the cellular response. On the one hand, as an upstream regulator of Omi, Hax-1 ensures the proper localization of functional Omi to protect cells from stresses, on the other hand, as a specific substrate of Omi, Hax-1 can be digested by Omi to elicit autophagy in response to autophagic stimuli or to stimulate apoptosis 23 when cells are ultimately sentenced to die. It will be interesting to explore how cells can maintain the equilibrium between Hax-1 and Omi under different circumstances.…”