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
Piroli, G. G. (Department of Pharmacology. Physiology and Neuroscience. University of South Carolina)
Manuel, A. M. (Department of Pharmacology. Physiology and Neuroscience. University of South Carolina)
Clapper, A. C. (Department of Pharmacology. Physiology and Neuroscience. University of South Carolina)
Walla, M. D. (Mass Spectrometry Center. Department of Chemistry and Biochemistry. University of South Carolina)
Baatz, J. E. (Department of Pharmacology. Physiology and Neuroscience. University of South Carolina)
Palmiter, Richard D (Howard Hughes Medical Institute and Department of Biochemistry. University of Washington)
Quintana Puebla, Alberto (Seattle Children's Research Institute)
Frizzell, N. (Department of Pharmacology. Physiology and Neuroscience. University of South Carolina)

Data:	2016
Resum:	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 voltagedependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys77 and Cys48 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.
Nota:	We thank Ms. Jennifer Bethard, Medical University of South Carolina for the mass spectrometric analysis of the 2D gel spots. We would like to thank Professor John Baynes, USC for helpful discussion. We thank Dr. Boris Kantor, USC Viral Vector Core for production of the lentiviral vectors. This work was supported by grants from the National Institutes of Health (R03 HD077187, R01 NS092938, R37 DK19971 and P20 GM109091), the American Diabetes Association (1-11-JF-13), and a University of South Carolina Research Foundation ASPIRE-I award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Drets:	Tots els drets reservats.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Animals ; Brain Stem ; Citric Acid Cycle ; Cysteine ; Disease Models, Animal ; Electron Transport Complex I ; Fumarates ; Humans ; Leigh Disease ; Mice ; Mice, Knockout ; Mitochondria ; Protein Processing, Post-Translational ; Proteomics ; Succinates ; Tandem Mass Spectrometry
Publicat a:	Molecular and Cellular Proteomics, Vol. 15 Núm. 2 (february 2016) , p. 445-461, ISSN 1535-9484

DOI: 10.1074/mcp.M115.051516
PMID: 26450614

17 p, 2.6 MB

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