The yeast two-hybrid system was used to isolate and characterize protein interactors of the N-terminal domain of the serine protease Omi/HtrA2 (high temperature requirement protein A2) encompassing amino acids 31-133. This large domain of Omi/HtrA2 is usually cleaved and removed through autoproteolysis to produce the mature form of the protein. Whether the N-terminal domain has any function after its removal is unknown. Omi/HtrA2 is involved in a variety of diseases including cancers, neurodegenerative disorders, and metabolic disorders, but thus far, it is assumed that its normal function is the degradation of specific substrates. To characterize any potential function of Omi/HtrA2’s unique amino terminus, specific interactors were isolated. One such interactor was the small GTPase Rab2A protein. We discuss the implications of this interaction and its biological significance.

While mitochondria are traditionally associated with energy production, recent studies identified its function in controlling the onset of apoptosis. Mitochondrial apoptotic control results from sequestering pro-apoptotic proteins, which are secreted following cellular stress. HtrA2/OMI is secreted from mitochondria to the cytosol following apoptotic induction, binds and degrades the inhibitor of apoptosis protein, XIAP in mammals, activating the caspase cascade. This study characterizes the expression of Drosophila HtrA2/OMI, a mitochondrial protein, its processing by Rhomboid-7 and demonstrates its pro-apoptotic function. Following exposure to apoptotic stress, dOMI is secreted from mitochondria and its expression profile displays an increase in a cleaved form consistent with Rhomboid-7 processing. dOMI expression resulted in sensitization of cells to apoptotic stress, observed through an increase in caspase activity. These data further validate the use of Drosophila in the study of mitochondrial driven apoptosis while implicating a potential role for Rhomboid-7 in apoptosis through proteolytic cleavage of dOMI.

While mitochondria are traditionally associated with energy production, recent studies identified its function in controlling the onset of apoptosis. Mitochondrial apoptotic control results from sequestering pro-apoptotic proteins, which are secreted following cellular stress. HtrA2/OMI is secreted from mitochondria to the cytosol following apoptotic induction, binds and degrades the inhibitor of apoptosis protein, XIAP in mammals, activating the caspase cascade. This study characterizes the expression of Drosophila HtrA2/OMI, a mitochondrial protein, its processing by Rhomboid-7 and demonstrates its pro-apoptotic function. Following exposure to apoptotic stress, dOMI is secreted from mitochondria and its expression profile displays an increase in a cleaved form consistent with Rhomboid-7 processing. dOMI expression resulted in sensitization of cells to apoptotic stress, observed through an increase in caspase activity. These data further validate the use of Drosophila in the study of mitochondrial driven apoptosis while implicating a potential role for Rhomboid-7 in apoptosis through proteolytic cleavage of dOMI.

Omi/HtrA2 is a mitochondrial serine protease with high homology to the bacterial HtrA proteins. Omi promotes caspase-dependent apoptosis by binding and degrading IAPs-inhibitor of apoptosis proteins. Omi can also induce caspase-independent apoptosis but the actual mechanism is still unknown. IAP's are not the only substrates cleaved by Omi. There are at least two more known substrates of Omi, the HAX-1 and the ped/pea-15 proteins. HS1-associated protein X-1 (HAX-1) is a mitochondrial protein, degraded by Omi after induction of caspase-dependent apoptosis. Ped/pea-15 is also an anti-apoptotic protein and is cleaved by Omi after induction of caspase-independent apoptosis. The proteolytic activity of Omi is necessary and essential for its pro-apoptotic function. Recent studies suggest the proteolytic activity of Omi is regulated by specific protein-protein interactions. Presenilin was identified to be such a regulator of Omi. It binds to the PDZ domain of Omi via its carboxy-terminus and this interaction significantly increases the proteolytic activity of the enzyme. My project was aimed to investigate the normal function of Omi in cell death and the mechanism of its regulation by isolating and characterizing novel Omi interactors. I screened a human melanocyte cDNA library using the yeast-two-hybrid system and Omi as the "bait" protein. Human Rad21 protein was isolated as a specific novel interactor of Omi. Human Rad21 interacted with the PDZ domain of Omi, the part of the protein known to be involved in protein-protein interactions. Human Rad21 is a nuclear protein that plays a role in DNA double-strand break repair and sister chromatid cohesion during metaphase. Several reports suggest hRad21 has also a role in apoptosis; it is cleaved by caspase-3 and part of the protein becomes cytoplasmic. Human Rad21 was not cleaved by Omi in vitro and therefore it is unlikely to be a substrate. When tested in a proteolytic assay Rad21 was able to increase the proteolytic activity of Omi. My work suggests a new mechanism whereby Omi and hRad21 can co-operate to induce cell death. This mechanism necessitates direct interaction of hRad21 with the PDZ domain of Omi resulting in increased proteolytic activity of the enzyme.

Human High Temperature requirement A2 (HtrA2) also known as Omi, is a serine protease located in the mitochondria with an important function in both cell survival and death. My results show the proteolytic activity of Omi/HtrA2 varies under different conditions. I characterized the optimal condition for Omi/HtrA2 protease activity using an in vitro assay system. Additionally, I identified a new allosteric regulation of Omi/HtrA2 through interaction with a specific substrate, the MUL1 protein. MUL1 is a multifunctional E3 ubiquitin ligase anchored in the outer mitochondrial membrane with domains both inside mitochondria and in the cytoplasm. The data shown here strongly supports the hypothesis that Omi/HtrA2 activity is modulated by a number of different mechanisms. Some of these conditions, such as pH or substrate denaturation might reflect the state of mitochondria under stress. It has been known that Omi/HtrA2 is a stress activated protease, but the mechanism of its regulation has not been fully elucidated. Furthermore, the allosteric regulation of Omi/HtrA2 by specific substrates, can be another mechanism that provides a feedback loop to increase the activity of the enzyme. The findings from this project contribute new information on the mechanisms of activation of Omi/HtrA2 protease. They support the hypothesis that mitochondrial stress might be involved in the regulation of Omi/HtrA2 protease.

Human HtrA2 is part of the HtrA family of ATP-independent serine proteases that are conserved in both prokaryotes and eukaryotes and localizes to the inter-membrane space of the mitochondria. Several recent reports have suggested that HtrA2 is important for maintaining proper mitochondrial homeostasis and may play a role in several neurodegenerative disorders. One disorder HtrA2 is implicated in is Alzheimer's disease (AD). AD is characterized by the presence of oligomers and fibrils of the amyloid 13 (AI3) peptide that is generated from cleavage of the amyloid precursor protein (APP) by 13- and y-secretases. HtrA2 degrades APP at the mitochondria, and binds the neurotoxic Al3 (1-42) peptide. In this report, the ability of HtrA2 to prevent the aggregation of a model suqstrate CS and the toxic Al3 (1-42) peptide were investigated. Using CS aggregation assays, HtrA2 was seen to have a moderate ability to delay and prevent the aggregation of CS, and this activity was significantly increased following removal of the PDZ domain. Additionally, using EM and lD-WG NMR analyses HtrA2 was seen to significantly delay the aggregation of the Al3 (1-42) peptide via a dual proteolytic and chaperone-like function. These results show a novel chaperone-like activity for HtrA2 and a model emerges from this work in which HtrA2 monitors the inter-membrane space of the mitochondria using a dual proteolytic and chaperone-like function to turnover stress-damaged proteins. Furthermore HtrA2, along with other quality control factors, may be involved in the metabolism of regular as well as aberrant levels of intramitochondrial Al3 (1-42) peptide, which is known to lead to oxidative stress and mitochondrial dysfunction. / Thesis / Master of Science (MSc)

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.