Interactome Mapping of the Mitochondrial Intermembrane Space Proteases Identifies a Novel Function of HTRA2

Mitochondrial proteases are enzymes responsible for the breakdown of proteins within the mitochondria. A number of these unique proteases localize to specific sub-compartments of the mitochondria, but the functions of these enzymes are poorly defined. To better characterize these proteases, I used proximity-dependent biotinylation (BioID) to map the interactomes of seven proteases localized to the mitochondrial intermembrane space (IMS). In total, I identified 802 high confidence proximity interactions with 342 unique proteins. While all seven proteases co-localized with the IMS markers OPA1 and CLPB, 230 of the interacting partners were unique to just one or two protease bait proteins, highlighting the ability of BioID to differentiate unique interactomes within the confined space of the IMS. Notably, high temperature requirement peptidase 2 (HTRA2) interacted with eight of 13 components of the mitochondrial intermembrane space bridging (MIB) complex, a multiprotein assembly essential for the maintenance of mitochondrial cristae structure. Knockdown of HTRA2 disrupted cristae in HEK 293 and OCI-AML2 cells and led to increased intracellular levels of the MIB subunit IMMT. Using a cell-free assay, we demonstrated that HTRA2 could degrade recombinant IMMT, while two additional core MIB complex subunits, SAMM50 and CHCHD3 remained undegraded. Based on these findings, I further investigated the role of HTRA2 in acute myeloid leukemia (AML). Knockdown of HTRA2 in OCI-AML2 cells showed a decrease in cell growth and an increase in cell differentiation. Additionally, mice injected with HTRA2 knockdown AML cells displayed reduced engraftment, highlighting the role for HTRA2 in tumour cell growth in vivo. Lastly, I collaborated in the development of additional mitochondrial BioID datasets to help characterize the potential functions of these mitochondrial proteins. Overall, my characterization of the IMS protease interactome provides important biological context to an understudied class of proteins and represents a rich dataset that can be further mined to uncover novel IMS protease biology.Ph.D