Identifying the mechanisms behind non-apoptotic functions of mitochondrial matrix-localized MCL-1 - PROJECT SUMMARY Aberrant regulation of apoptosis is a hallmark of human pathologies, including neurodegeneration, autoimmunity, and cancer. Intrinsic apoptosis is regulated by the BCL-2 family which is composed of anti-apoptotic proteins (e.g., BCL-2 and MCL-1) which sequester pro-apoptotic BH3-only proteins (e.g., BIM and BAD) or directly inhibit pro-apoptotic effectors, BAX and BAK, preventing their oligomerization. Many cancer cells overexpress anti- apoptotic proteins to promote aberrant survival. MCL-1 is unique among anti-apoptotic proteins because it is essential in early embryonic development and for the survival of many cell lineages (e.g., hematopoietic stem cells, lymphocytes, neutrophils, neurons, and cardiomyocytes). Our lab previously reported that MCL-1 has two isoforms –– one that localizes to the outer mitochondrial membrane (OMM) and one that localizes to the mitochondrial matrix. Although OMM MCL-1’s canonical anti-apoptotic function has been well characterized, the roles of matrix MCL-1 are still largely unknown. Evidence suggests that matrix MCL-1 serves to maintain normal mitochondrial fission/fusion, oxidative phosphorylation, and cristae ultrastructure. The basis for these physiologic roles of MCL-1 remains unknown. Metabolomic investigation of Mcl1–deficient (Mcl1–/–) murine embryonic fibroblasts (MEFs) and MEFs lacking matrix-localized Mcl1 revealed that they are highly sensitive to glutamine deprivation as compared to wild-type MEFs. Additionally, MCL-1 protein levels decrease after 24 hours of glutamine withdrawal in wild-type MEFs. These data suggest a link between MCL-1 and glutamine metabolism that could be connected to the mitochondrial defects that are observed upon Mcl1 deletion. The goal of this proposal is to determine the functions of matrix MCL-1 and gain a mechanistic understanding of these functions. I hypothesize that matrix-localized MCL-1 plays an essential, non-apoptotic role in maintaining mitochondrial morphology and bioenergetics. To address this hypothesis, I will use a novel mutant mouse that endogenously expresses a truncated MCL-1 protein (MCL-1OM) which blocks apoptosis but cannot be imported into the mitochondrial matrix. First, I will ectopically express Mcl1 mutants back into Mcl1–/– MEFs to determine which version(s) of Mcl1 can rescue the death triggered by glutamine withdrawal. I will also perform mRNA-Seq on Mcl1–/–, Mcl1+/+, Mcl1–/+, and Mcl1–/OM MEFs to interrogate the metabolic rewiring induced by loss of matrix-localized MCL-1. Second, I will assess mitochondrial function (e.g., Seahorse XF Mito Stress Test and TMRE staining), determine the proteins interacting with matrix-localized MCL-1, ectopically express Opa1 and Drp1 mutants in Mcl1–/– MEFs, and perform confocal imaging on these cells to determine the functions of matrix- localized MCL-1 in mitochondrial morphology. Finally, I will perform in vivo experiments on Mcl1-/OM mice to analyze T cell activation, effector function, and memory T cell generation. These findings will provide new, mechanistic insights into the non-apoptotic roles of matrix-localized MCL-1 and could shed light on the potential consequences of using MCL-1 inhibitors for clinical applications.
