Abstract
Manganese superoxide dismutase (MNSOD) is one of the major scavengers of reactive oxygen species (ROS) in mitochondria with pivotal regulatory role in ischemic disorders, inflammation and cancer. Here we report oxidative modification of MNSOD in human renal cell carcinoma (RCC) by the shotgun method using data-dependent liquid chromatography tandem mass spectrometry (LC-MS/MS). While 5816 and 5571 proteins were identified in cancer and adjacent tissues, respectively, 208 proteins were found to be up- or down-regulated (p < 0.05). Ontological category, interaction network and Western blotting suggested a close correlation between RCC-mediated proteins and oxidoreductases such as MNSOD. Markedly, oxidative modifications of MNSOD were identified at histidine (H54 and H55), tyrosine (Y58), tryptophan (W147, W149, W205 and W210) and asparagine (N206 and N209) residues additional to methionine. These oxidative insults were located at three hotspots near the hydrophobic pocket of the manganese binding site, of which the oxidation of Y58, W147 and W149 was up-regulated around three folds and the oxidation of H54 and H55 was detected in the cancer tissues only (p < 0.05). When normalized to MNSOD expression levels, relative MNSOD enzymatic activity was decreased in cancer tissues, suggesting impairment of MNSOD enzymatic activity in kidney cancer due to modifications. Thus, LC-MS/MS analysis revealed multiple oxidative modifications of MNSOD at different amino acid residues that might mediate the regulation of the superoxide radicals, mitochondrial ROS scavenging and MNSOD activity in kidney cancer.
Highlights
Superoxide dismutases (SODs) are the ubiquitous superfamily of antioxidant metalloenzymes that convert the superoxide anion (O2−) into oxygen and hydrogen peroxide (H2O2) when cells are exposed to oxidation
Double oxidation at W147 and W149 in a different MS/MS profile of the same peptide (Figure S1d) was identified by the unmodified b6,8–9,11 ions and double mass shifts of 15.99 Da in the b15–19 ions. These identified tryptophan oxidations at W147, W149, W205 and W210 were located in the hotspots that were reported to be important for the enzymatic activity of Manganese superoxide dismutase (MNSOD) [2]
We identified two asparagine oxidation sites at N206 and N209 in MNSOD, and the two oxidized asparagine residues were found in the peptide AIWN206VIN209WENVTER, which was located in the C-terminal
Summary
Superoxide dismutases (SODs) are the ubiquitous superfamily of antioxidant metalloenzymes that convert the superoxide anion (O2−) into oxygen and hydrogen peroxide (H2O2) when cells are exposed to oxidation. It was recently shown that oxidative stress-mediated MNSOD modifications compromised its enzymatic activity and led to functional deficit [1,3,18,19]. Double oxidation at W147 and W149 in a different MS/MS profile of the same peptide (Figure S1d) was identified by the unmodified b6,8–9,11 ions and double mass shifts of 15.99 Da in the b15–19 ions. These identified tryptophan oxidations at W147, W149, W205 and W210 were located in the hotspots that were reported to be important for the enzymatic activity of MNSOD [2]. Tryptophan hydroxylase (TPH), a well characterized enzyme in mammals, was shown to convert tryptophan to 5-hydroxytryptophan, the precursor for the neurotransmitter serotonin and melatonin [22], it was unclear whether TPH was involved in tryptophan oxidation in proteins
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