Dataset Information

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.

SUBMITTER: Piroli GG

PROVIDER: S-EPMC4739666 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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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 Gerardo G GG Manuel Allison M AM Clapper Anna C AC Walla Michael D MD Baatz John E JE Palmiter Richard D RD Quintana Albert A Frizzell Norma N

Molecular & cellular proteomics : MCP 20151008 2

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 ...[more]

PMID: 26450614

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Project description:BACKGROUND: NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), containing one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), is one of the core nuclear-encoded subunits existing in human mitochondrial complex I. Defects in this subunit have been associated with Parkinson's disease, Alzheimer's disease, Bipolar disorder, and Schizophrenia. The aim of this study is to examine the mitochondrial targeting of NDUFV2 and dissect the pathogenetic mechanism of one human deletion mutation present in patients with early-onset hypertrophic cardiomyopathy and encephalopathy. METHODS: A series of deletion and point-mutated constructs with the c-myc epitope tag were generated to identify the location and sequence features of mitochondrial targeting sequence for NDUFV2 in human cells using the confocal microscopy. In addition, various lengths of the NDUFV2 N-terminal and C-terminal fragments were fused with enhanced green fluorescent protein to investigate the minimal region required for correct mitochondrial import. Finally, a deletion construct that mimicked the IVS2+5_+8delGTAA mutation in NDUFV2 gene and would eventually produce a shortened NDUFV2 lacking 19-40 residues was generated to explore the connection between human gene mutation and disease. RESULTS: We identified that the cleavage site of NDUFV2 was located around amino acid 32 of the precursor protein, and the first 22 residues of NDUFV2 were enough to function as an efficient mitochondrial targeting sequence to carry the passenger protein into mitochondria. A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein. The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability. CONCLUSIONS: The mitochondrial targeting sequence of NDUFV2 is located at the N-terminus of the precursor protein. Maintaining a net positive charge and an amphiphilic structure with the overall balance and distribution of basic and hydrophobic amino acids in the N-terminus of NDUFV2 is important for mitochondrial targeting. The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

Dataset Information

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.

Publications

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.

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