title: Lysine Acetylation Activates Mitochondrial Aconitase in the Heart.
abstract: High-throughput proteomics studies have identified several thousand acetylation sites on more than 1000 proteins. Mitochondrial aconitase, the Krebs cycle enzyme that converts citrate to isocitrate, has been identified in many of these reports. Acetylated mitochondrial aconitase has also been identified as a target for sirtuin 3 (SIRT3)-catalyzed deacetylation. However, the functional significance of mitochondrial aconitase acetylation has not been determined. Using in vitro strategies, mass spectrometric analyses, and an in vivo mouse model of obesity, we found a significant acetylation-dependent activation of aconitase. Isolated heart mitochondria subjected to in vitro chemical acetylation with either acetic anhydride or acetyl-coenzyme A resulted in increased aconitase activity that was reversed with SIRT3 treatment. Quantitative mass spectrometry was used to measure acetylation at 21 lysine residues and revealed significant increases with both in vitro treatments. A high-fat diet (60% of kilocalories from fat) was used as an in vivo model and also showed significantly increased mitochondrial aconitase activity without changes in protein level. The high-fat diet also produced an increased level of aconitase acetylation at multiple sites as measured by the quantitative mass spectrometry assays. Treatment of isolated mitochondria from these mice with SIRT3 abolished the high-fat diet-induced activation of aconitase and reduced acetylation. Finally, kinetic analyses found that the increase in activity was a result of increased maximal velocity, and molecular modeling suggests the potential for acetylation at K144 to perturb the tertiary structure of the enzyme. The results of this study reveal a novel activation of mitochondrial aconitase by acetylation.
authors.affiliation: †Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, United States.|‡Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Jolyn
authors.initials: J
authors.lastname: Fernandes
authors.affiliation: †Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Alexis
authors.initials: A
authors.lastname: Weddle
authors.affiliation: †Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Caroline S
authors.initials: CS
authors.lastname: Kinter
authors.affiliation: †Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, United States.|‡Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.|∥Reynolds Oklahoma Center on Aging, Donald W. Reynolds Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Humphries
authors.affiliation: ‡Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.|§Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Timothy
authors.initials: T
authors.lastname: Mather
authors.affiliation: †Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, United States.|‡Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.|∥Reynolds Oklahoma Center on Aging, Donald W. Reynolds Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Luke I
authors.initials: LI
authors.lastname: Szweda
authors.affiliation: †Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, United States.|∥Reynolds Oklahoma Center on Aging, Donald W. Reynolds Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Kinter
mesh_terms: D000107:Acetylation; D000154:Aconitate Hydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology*; D009206:Myocardium / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D056566:Sirtuin 3 / Q000235:genetics / Q000378:metabolism
chemical_list: D056566:Sirtuin 3; D000154:Aconitate Hydratase; D008239:Lysine