Characterization Of A Novel Interactor/substrate For The Pro-apoptotic Serine Protease Omi/htra2

OmiHtrA2 is a highly conserved mammalian serine protease that belongs to the HtrA family of proteins. Omi shares homology with the bacterially expressed heat shock protease HtrA, which functions as a protease at higher temperatures and a chaperone at lower temperatures. Additionally, Omi shares sequence similarity with the mammalian homologs L56/HtrA1 and PRSP/HtrA3. Omi was first isolated as an interacting protein of Mxi2, an alternatively spliced form of the p38 stress-activated kinase, using a modified yeast two-hybrid system. Omi localizes in the mitochondria and in response to apoptotic stimuli the mature form of this protein translocates to the cytoplasm. In the cytoplasm Omi participates in both the caspase-dependent as well as caspase-independent apoptosis. Additionally, recent studies suggest that Omi may have another unique function, maintaining homeostasis within the mitochondria. In an effort to further elucidate the function of Omi, a yeast two-hybrid screening was performed to isolate novel interacting proteins. This screening identified a novel protein (HOPS), as a specific interactor of Omi. The predicted amino acid sequence of this protein does not provide any information about its potential function in mammalian cells. However, experiments show that HOPS is cleaved in vitro by Omi. Furthermore, in response to apoptotic stimuli, HOPS is also degraded in vivo. This study suggests that HOPS could be a physiological substrate of Omi that is cleaved and removed during apoptosis

Regulation of HAX-1 anti-apoptotic protein by Omi/HtrA2 protease during cell death

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