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

Yong Li, Sridhar A. Malkaram, Jie Zhou, Janos Zempleni

Research output: Contribution to journalArticle

7 Citations (Scopus)

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 one and five 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.

Original languageEnglish (US)
Pages (from-to)475-482
Number of pages8
JournalJournal of Nutritional Biochemistry
Volume25
Issue number4
DOIs
StatePublished - Jan 1 2014

Fingerprint

Biotinylation
Heat-Shock Proteins
Cell culture
Methionine
Lysine
Biotin
Reactive Oxygen Species
Cell Culture Techniques
Oxidation
HEK293 Cells
Proteins
Cell Cycle Resting Phase
G1 Phase
Proteome
Fibroblasts
Cell Cycle Checkpoints
S Phase
Mass spectrometry
Mass Spectrometry
Membrane Proteins

Keywords

  • Biotin
  • Heat shock proteins
  • Methionine oxidation
  • Reactive oxygen species

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism
  • Nutrition and Dietetics
  • Medicine(all)

Cite this

Lysine biotinylation and methionine oxidation in the heat shock protein HSP60 synergize in the elimination of reactive oxygen species in human cell cultures. / Li, Yong; Malkaram, Sridhar A.; Zhou, Jie; Zempleni, Janos.

In: Journal of Nutritional Biochemistry, Vol. 25, No. 4, 01.01.2014, p. 475-482.

Research output: Contribution to journalArticle

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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 one and five 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.",
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