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Identification of NDUFAF1 in mediating K-Ras induced mitochondrial dysfunction by a proteomic screening approach.

ABSTRACT: Increase in aerobic glycolysis and mitochondrial dysfunction are important biochemical features observed in human cancers. Recent studies suggest oncogenic K-Ras can cause suppression of mitochondrial respiration and up-regulation of glycolytic activity through a yet unknown mechanism. Here we employed proteomic approach and used a K-RasG12V inducible cell system to investigate the impact of oncogenic K-Ras on mitochondria and cell metabolism. Mitochondria isolated from cells before and after K-Ras induction were subjected to protein analysis using stable isotope labeling with amino acids (SILAC) and liquid chromatography coupled with mass spectrometry (LC-MS). 70 mitochondrial proteins with significant expression alteration after K-Ras induction were identified. A majority of these proteins were involved in energy metabolism. Five proteins with significant decrease belong to mitochondrial respiratory chain complex I. NADH dehydrogenase 1 alpha subcomplex assembly factor 1 (NDUFAF1) showed most significant decrease by 50%. Such decrease was validated in primary human pancreatic cancer tissues. Knockdown of NDUFAF1 by siRNA caused mitochondrial respiration deficiency, accumulation of NADH and subsequent increase of glycolytic activity. Our study revealed that oncogenic K-Ras is able to induce significant alterations in mitochondrial protein expression, and identified NDUFAF1 as an important molecule whose low expression contributes to mitochondrial dysfunction induced by K-Ras.

SUBMITTER: Wang P

PROVIDER: S-EPMC4414165 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Identification of NDUFAF1 in mediating K-Ras induced mitochondrial dysfunction by a proteomic screening approach.

Wang Peng P Song Ming M Zeng Zhao-lei ZL Zhu Chao-feng CF Lu Wen-hua WH Yang Jing J Ma Ming-zhe MZ Huang A-min AM Hu Yumin Y Huang Peng P

Oncotarget 20150201 6

Increase in aerobic glycolysis and mitochondrial dysfunction are important biochemical features observed in human cancers. Recent studies suggest oncogenic K-Ras can cause suppression of mitochondrial respiration and up-regulation of glycolytic activity through a yet unknown mechanism. Here we employed proteomic approach and used a K-RasG12V inducible cell system to investigate the impact of oncogenic K-Ras on mitochondria and cell metabolism. Mitochondria isolated from cells before and after K- ...[more]

PMID: 25714130

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Project description:Endothelial cell dysfunction is associated with bioavailable nitric oxide deficiency and an excessive generation of reactive oxygen species. We modeled this condition by chronically inhibiting nitric oxide generation with subpressor doses of N(G)-monomethyl-L-arginine (L-NMMA) in C57B6 and Tie-2/green fluorescent protein mouse strains. L-NMMA-treated mice exhibited a slight reduction in vasorelaxation ability, as well as detectable abnormalities in soluble adhesion molecules (soluble intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1, and matrix metalloproteinase 9), which represent surrogate indicators of endothelial dysfunction. Proteomic analysis of the isolated microvasculature using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy revealed abnormal expression of a cluster of mitochondrial enzymes, which was confirmed using immunodetection. Aconitase-2 and enoyl-CoA-hydratase-1 expression levels were decreased in L-NMMA-treated animals; this phenotype was absent in nitric oxide synthase-1 and -3 knockout mice. Depletion of aconitase-2 and enoyl-CoA-hydratase-1 resulted in the inhibition of the Krebs cycle and enhanced pyruvate shunting toward the glycolytic pathway. To assess mitochondrial mass in vivo, co-localization of green fluorescent protein and MitoTracker fluorescence was detected by intravital microscopy. Quantitative analysis of fluorescence intensity showed that L-NMMA-treated animals exhibited lower fluorescence of MitoTracker in microvascular endothelia as a result of reduced mitochondrial mass. These findings provide conclusive and unbiased evidence that mitochondriopathy represents an early manifestation of endothelial dysfunction, shifting cell metabolism toward "metabolic hypoxia" through the selective depletion of both aconitase-2 and enoyl-CoA-hydratase-1. These findings may contribute to an early preclinical diagnosis of endothelial dysfunction.

Dataset Information

Identification of NDUFAF1 in mediating K-Ras induced mitochondrial dysfunction by a proteomic screening approach.

Publications

Identification of NDUFAF1 in mediating K-Ras induced mitochondrial dysfunction by a proteomic screening approach.

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