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.
Highlights
From the ‡Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina 29209; §Mass Spectrometry Center, Department of Chemistry & Biochemistry, University of South Carolina, Columbia, South Carolina 29205; ¶Department of Pediatrics, College of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425; ʈHoward 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
To determine if the oxidative phosphorylation (OXPHOS) deficiency was associated with fumarate accumulation and protein succination, we examined 2SC levels in several brain regions and the muscle from early (3 weeks), middle (6 weeks) and late (9 weeks) disease stage KO mice and their corresponding WT littermate controls
Increased succination in biological samples is restricted to situations where fumarate concentrations are significantly increased, for example, 2SC levels are increased in cancers derived from fumarate hydratase mutations [6, 8], and in the metabolically overwhelmed adipocyte in type 2 diabetes [4, 5, 9]
Summary
Increase up to fivefold in adipocytes grown in the presence of high (30 mM) versus normal (5 mM) glucose concentrations [2]. In the adipocyte the increase in fumarate and succinated proteins develops as a direct result of mitochondrial stress induced by nutrient excess. Excess glucose without increased ATP demand inhibits the electron transport chain resulting in an elevated NADH/NADϩ ratio. This inhibits NADϩ-dependent Krebs cycle enzymes and leads to an increase in fumarate and protein succination [9]. Considering the impact of glucotoxicity driven mitochondrial stress in the adipocyte, we predicted that deficiencies in OXPHOS associated with NADH accumulation would result in increased protein succination. Barshop’s findings support the hypothesis that MD derived from OXPHOS deficiencies may exhibit increased protein succination because of the accumulation of NADH and subsequently fumarate. In this study we report for the first time that protein succination is present in the brain in an animal model of Leigh syndrome, the Ndufs KO mouse, suggesting that this modification may be an important biochemical link between the genetic defect and the onset of neuropathology observed in Leigh syndrome
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