Identification Of A Novel Anti-oxidative Protein In Inner Mitochondrial Membrane

Oxidative stress leads to various diseases including cancer,neurodegenerative diseases,and cardiovascular dysfunctions by damaging cellular functions.However,proper level of reactive oxygen species（ROS）is essential for cell signaling.Thus,it is crucial for cells to maintain redox homeostasis by multiple antioxidants.Mitochondria are a major site of ROS production,that makes the mitochondrial proteins face a high risk of oxidative damage.Particularly,the inner mitochondrial membrane（IMM）is not only enriched with plenty of proteins essential for mitochondrial functions,but also is a key site for mitochondrial ROS production.However,although a few antioxidant proteins in matrix and intermembrane space have been identified,whether there exists specific antioxidant proteins in IMM remains unknown.Here our research identifies a novel mitochondrial inner membrane delimited anti-oxidative protein,ROMO1（reactive oxygen species modulator 1）,which contains four cysteines in its 79 amino acids and has a markedly low redox potential（-408m V）.Quantitative redox-proteomics profiling reveals that ROMO1 overexpression leads to a dramatic reductive shift with 68%cysteines of mitochondrial proteins in reductive state,whereas ROMO1 knockout shows the converse effect with 66%cysteines of mitochondrial proteins in peroxidized state.Interestingly,these ROMO1-regulated cysteines are specifically enriched in mitochondrial inner membrane proteins and oxidation-reduction process.These results indicate that ROMO1 acts as an essential antioxidant in IMM.Mechanistically,our research finds that ROMO1 interacts with various proteins in IMM and its Cys15 and Cys42 are crucial for its function.Specifically,our research studied the regulatory roles of ROMO1 on mitochondrial respiratory chain,mitochondrial Ca²⁺uniporter（MCU）,mitochondrial inner membrane fusion protein OPA1,and mitochondrial permeability transition pore（m PTP）.Our research finds that ROMO1 enhances mitochondrial respiratory efficiency and reduces ROS production via interacting with multiple subunits of respiratory chain and protecting the Cys residues in reductive state.ROMO1 also interacts with MCU and enhances mitochondrial Ca²⁺uptake through reductive protection of MCU Cys96 and Cys190.Meanwhile,ROMO1 promotes cristae formation through reductive protection of OPA1 Cys786/853/856 and prevents m PTP opening through Cys reductive protection of several potential m PTP components.Importantly,our research finds that while ROMO1 is critical for cardiac functions,its overexpression effectively protects the heart against various oxidative stress-associated cardiomyopathies,including ischemia-reperfusion injury and transverse aortic constriction induced heart failure.Taken together,our research identifies ROMO1 as a novel IMM anti-oxidative protein,which plays essential roles in mitochondrial redox homeostasis.These findings not only reveal a new mechanism of mitochondrial redox homeostasis,but also provides a potential target for treating oxidative stress-associated diseases.