title: Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome.
abstract: Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys(77) and Cys(48) were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.
authors.affiliation: From the ‡Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina 29209;
authors.forename: Gerardo G
authors.initials: GG
authors.lastname: Piroli
authors.affiliation: From the ‡Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina 29209;
authors.forename: Allison M
authors.initials: AM
authors.lastname: Manuel
authors.affiliation: From the ‡Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina 29209;
authors.forename: Anna C
authors.initials: AC
authors.lastname: Clapper
authors.affiliation: §Mass Spectrometry Center, Department of Chemistry & Biochemistry, University of South Carolina, Columbia, South Carolina 29205;
authors.forename: Michael D
authors.initials: MD
authors.lastname: Walla
authors.affiliation: ¶Department of Pediatrics, College of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425;
authors.forename: John E
authors.initials: JE
authors.lastname: Baatz
authors.affiliation: ‖Howard Hughes Medical Institute and Department of Biochemistry, University of Washington, Seattle, Washington 98195;
authors.forename: Richard D
authors.initials: RD
authors.lastname: Palmiter
authors.affiliation: ‖Howard Hughes Medical Institute and Department of Biochemistry, University of Washington, Seattle, Washington 98195; **Center for Integrative Brain Research and Center for Developmental Therapeutics, Seattle Children's Research Institute, Seattle, Washington 98101.
authors.forename: Albert
authors.initials: A
authors.lastname: Quintana
authors.affiliation: From the ‡Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina 29209; norma.frizzell@uscmed.sc.edu.
authors.forename: Norma
authors.initials: N
authors.lastname: Frizzell
mesh_terms: D000818:Animals; D001933:Brain Stem / Q000378:metabolism / Q000473:pathology; D002952:Citric Acid Cycle; D003545:Cysteine / Q000378:metabolism; D004195:Disease Models, Animal; D042967:Electron Transport Complex I / Q000235:genetics* / Q000378:metabolism; D005650:Fumarates / Q000378:metabolism; D006801:Humans; D007888:Leigh Disease / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D011499:Protein Processing, Post-Translational / Q000235:genetics; D040901:Proteomics; D013386:Succinates / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D005650:Fumarates; C534449:Ndufs4 protein, mouse; D013386:Succinates; D042967:Electron Transport Complex I; D003545:Cysteine