Abstract 13974: Mitochondrial Chaperonin CLPB Determines MICU1 Protein Turnover and Mitochondrial Calcium Overload in Ischemia-Reperfusion Injury

Abstract

Background: Mitochondrial calcium uptake 1 protein (MICU1) fine-tunes mitochondrial calcium ( m Ca 2+ ) uptake across the inner mitochondrial membrane (IMM) by regulating the open probability of the mitochondrial calcium uniporter channel complex (mtCU). The loss of MICU1 is associated with numerous chronic disease states and contributes to mitochondrial calcium overload. Here we report that in response to hypoxia, MICU1 protein turnover is higher compared to all other mtCU components, suggesting a dynamically regulated post-translational system controls MICU1 expression. Methods: Biotin-based proximity labeling and liquid chromatography- mass spectrometry proteomics, protein biochemistry, live-cell Ca 2+ imaging, confocal and super-resolution imaging, CRISPR/Cas9 based gene editing, AAV9 based gene delivery, and mouse left coronary artery ligation for myocardial ischemia-reperfusion (IR) injury were utilized to discern the role of CLPB in MICU1 turnover, m Ca 2+ overload, and IR injury. Results and Conclusions: To identify the components regulating MICU1 protein turnover, we performed an unbiased proteomics screen utilizing MICU1 fused to BioID2, a biotinylation proximity labeling strategy combined with avidin-based isolation and mass spectrometry. We identified caseinolytic peptidase B protein homolog (CLPB), a mitochondrial chaperonin/disaggregase, as a potential determinant of the MICU1 protein turnover. We confirmed that CLPB lacks proteolytic activity using in vitro protein degradation assays. Next, to identify the CLPB partner protease, we performed a subsequent BIOID2 screen of the CLPB interactome. We identified that in response to hypoxia, the intermembrane space (IMS) localized protease YME1L was necessary for MICU1 protein turnover. Subsequent experiments suggest that CLPB regulated MICU1 protein turnover is a critical molecular event in the onset of cell death in the context of cardiac injury. As knockdown of CLPB is sufficient to decrease infarct size and protect against cardiac ischemia reperfusion injury in mice. In conclusion, we detail a previously unknown protein-quality control system regulating MICU1 and influencing mitochondrial calcium uptake and downstream cellular events.