Lysine biotinylation and methionine oxidation in the heat shock protein HSP60 synergize in the elimination of reactive oxygen species in human cell cultures

Abstract

Previous studies suggest that the number of proteins containing covalently bound biotin is larger than previously thought. Here, we report the identity of some of these proteins. Using mass spectrometry we discovered 108 novel biotinylation sites in the human embryonic kidney HEK293 cell proteome; members of the heat shock protein (HSP) superfamily were overrepresented among the novel biotinylated proteins. About half of the biotinylated proteins also displayed various degrees of methionine oxidation, which is known to play an important role in the defense against reactive oxygen species; for biotinylated HSPs, the percent of methionine sulfoxidation approached 100%. Protein structure analysis suggests that methionine sulfoxides localize in close physical proximity to the biotinylated lysines on the protein surface. Mass spectrometric analysis revealed that between 1 and 5 of the methionine residues in the C-terminal KEEKDPGMGAMGGMGGGMGGGMF motif are oxidized in HSP60. The likelihood of methionine sulfoxidation is higher if one of the adjacent lysine residues is biotinylated. Knockdown of HSP60 caused a 60% increase in the level of reactive oxygen species in fibroblasts cultured in biotin-sufficient medium. When HEK293 cells were transferred from biotin-sufficient medium to biotin-free medium, the level of reactive oxygen species increased by >9 times compared with baseline controls and a time-response relationship was evident. High levels of methionine sulfoxidation coincided with cell cycle arrest in the G0/G1 and S phases in biotin-depleted cells. We conclude that biotinylation of lysines synergizes with sulfoxidation of methionines in heat-shock proteins such as HSP60 in the defense against reactive oxygen species.

High‐dose biotin restores redox balance, energy and lipid homeostasis, and axonal health in a model of adrenoleukodystrophy

“…To the best of our knowledge, this is the first time that biotin or high-dose biotin was shown to induce the endogenous antioxidant response of NRF-2. A different mechanism of action of the antioxidant role of biotin dependent on the biotinylation of HSP60 at its lysines, a mitochondrial heat-shock protein that ameliorates oxidative stress in cells, was previously reported (36). .…”

Novel roles of holocarboxylase synthetase in gene regulation and intermediary metabolism

“…A second study of HSP focused on HSP60 because of its pivotal role in ameliorating oxidative stress . HSP is unique in that the protein is dually modified by methione sulfoxidation and lysine biotinylation (underlined) in the C‐terminal KEEKDPGMGAMGGMGGGMGGGMF motif . The levels of reactive oxygen species are about 10 times higher in biotin‐depleted HEK293 cells compared with biotin‐sufficient controls, and the biotin‐depleted cells arrest in the G0/G1 and S phases of the cell cycle.…”

Resveratrol compounds inhibit human holocarboxylase synthetase and cause a lean phenotype in Drosophila melanogaster

“…MCC converts 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA in leucine metabolism in cytoplasm. Recently, 108 new biotinylated protein were discovered by mass spectrometry analysis [2]. The biological function of their biotinylation is unknown, despite some evidence that biotinylation plays a role in the functionality of heat shock proteins [2,3].…”