Abstract
Cardiac-specific overexpression of SOD2 (mitochondrial manganese superoxide dismutase, mouse model of SOD2-tg) induces supernormal and physiologically hypertrophic phenotypes in the mouse heart. However, the reductive stress imposed by SOD2 overexpression also results in protein aggregation of SOD2 pentamer and differential acetylation of SOD2 in the compartments of mitochondrial matrix, mitochondrial inner membrane, and cytosol. We studied SOD2 acetylation in the matrix, submitochondrial particle (SMP), and cytosol from the SOD2-tg mouse heart. LC/MS/MS analysis revealed that K68, K75, K89, K114, K122, K130, K132, K134, and K154 residues of the cytosolic SOD2 were acetylated. LC/MS/MS analysis further indicated that K68, K122, and K130 of SOD2 in SMP and K68, K122, K 130, K202 of matrix SOD2 were acetylated. LC/MS/MS also detected a signaling peptide, 12QLAPVLGYLGSR23, in the SMP SOD2. The results implicated that mitochondrial localization of SOD2 resulted in specific deacetylation of K75, K89, K114, K132, K134, K154 residues, and site-specific acetylation of K202 residue in the matrix. In vitro acetylation of matrix SOD2 with acetic anhydride de-aggregated pentameric SOD2, restored the profile of hyperacetylation observed in cytosolic SOD2, and decreased the enzymatic activity of matrix SOD2. As indicated by the 3D structure of SOD2, specific acetylation of K89, K134, and K154 detected in cytosolic SOD2 induces unfolding tertiary structure and breaking salt bridges for stabilizing the quaternary structure. Stable isotope labeling of both wild type and SOD2-tg matrix proteins followed by LC/MS analysis indicated significant up-regulation of stress-induced mitochondrial hsp70 (GRP75) and down-regulation of presequence protease (PREP), thus supporting increased reductive stress, compromised mitochondrial localization, and weakening correct folding of the nascent SOD2 in the SOD2-tg murine heart. In conclusion, hyperacetylation maintains the unfolded cytosolic SOD2 and mediates importation of SOD2 across the mitochondrial membrane. Activation of the matrix SOD2 is subsequently initiated by deacetylation that further induces SOD2 misfolding for pentameric aggregation.
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