Novel Mitochondrial Substrates of Omi Indicate a New Regulatory Role in Neurodegenerative Disorders

Abstract

The mitochondrial protease OMI (also known as HtrA2) has been implicated in Parkinson's Disease (PD) and deletion or protease domain point mutations have shown profound neuropathologies in mice. A beneficial role by OMI, in preserving cell viability, is assumed to occur via the avoidance of dysfunctional protein turnover. However relatively few substrates for mitochondrial Omi are known. Here we report our identification of three novel mitochondrial substrates that impact metabolism and ATP production. Using a dual proteomic approach we have identified three interactors based upon ability to bind to OMI, and/or to persist in the proteome after OMI activity has been selectively inhibited. One candidate, the chaperone HSPA8, was common to each independent study. Two others (PDHB subunit and IDH3A subunit) did not appear to bind to OMI, however persisted in the mito-proteome when OMI was inhibited. Pyruvate dehydrogenase (PDH) and isocitrate dehydrogenase (IDH) are two key Kreb's cycle enzymes that catalyse oxidative decarboxylation control points in mitochondrial respiration. We verified both PDHB and IDH3A co-immunoprecipitate with HSPA8 and after elution, were degraded by recombinant HtrA2 in vitro. Additionally our gene expression studies, using rotenone (an inhibitor of Complex I) showed Omi expression was silenced when pdhb and idh3a were increased when a sub-lethal dose was applied. However higher dose treatment caused increased Omi expression and decreased levels of pdhb and idh3a transcripts. This implicates mitochondrial OMI in a novel mechanism relating to metabolism.

Nonproteasomal Targets of the Proteasome Inhibitors Bortezomib and Carfilzomib: a Link to Clinical Adverse Events

“…Mitochondrial HtrA2/Omi has been shown to play a protective role in both in vitro and in vivo models of neurodegeneration and lymphocyte survival (29,(38)(39)(40). Following exposure to either bortezomib or carfilzomib, HtrA2/Omi expression increased in SH-SY5Y cells to levels similar to that reported for MG-132 (35), which may be the result of an unfolded protein response leading to oxidative stress. In these cells, processing of 2 substrates of HtrA2/ Omi, eIF4G1, a translation factor, and pyruvate dehydrogenase, a Kreb cycle regulatory enzyme involved in generating oxidative species, are inhibited by bortezomib but not carfilzomib.…”

“…HtrA2/Omi is expressed as mitochondrial and endoplasmic reticulum forms, each with distinct sets of substrates (35)(36)(37). Mitochondrial HtrA2/Omi has been shown to play a protective role in both in vitro and in vivo models of neurodegeneration and lymphocyte survival (29,(38)(39)(40).…”

“…In these cells, processing of 2 substrates of HtrA2/ Omi, eIF4G1, a translation factor, and pyruvate dehydrogenase, a Kreb cycle regulatory enzyme involved in generating oxidative species, are inhibited by bortezomib but not carfilzomib. Because both of these substrates have been implicated in neurodegenerative diseases (35,41), inhibition of their processing may represent downstream mechanisms by which bortezomib alters neuronal cell function. Our data are consistent with a model in which bortezomib reduces neurite length by dual inhibition of the proteasome (resulting in oxidative and proteotoxic stress) and the neuronal prosurvival protease HtrA2/Omi.…”

Allosteric Regulation of Serine Protease HtrA2 through Novel Non-Canonical Substrate Binding Pocket

“…HtrA2 is involved in both caspase dependent as well as caspase independent apoptotic pathways [5], [6], [7]. Literature suggests it might have chaperoning functions as well and recently has been found to be associated with several neurodegenerative disorders [8], [9], [10]. Based on information from literature [4], [11], this multitasking ability of HtrA2 can be attributed to its serine protease activity which is intricately coordinated by its unique substrate binding process, complex trimeric structure, interdomain networking and conformational plasticity.…”

HtrA2 Peptidase

“…Several mitochondrial proteins have been proposed as substrates for HtrA2, including Hax1 [29], beta-amyloid precursor protein (APP) [8], Pyruvate dehydrogenase E1 component beta subunit (PDHB) [30] and isocitrate dehydrogenase [NAD] subunit alpha (IDH3A) [30].…”