title: 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 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.
authors.affiliation: Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583-0806, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583-0806, USA.
authors.forename: Sridhar A
authors.initials: SA
authors.lastname: Malkaram
authors.affiliation: Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583-0806, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583-0806, USA. Electronic address: jzempleni2@unl.edu.
authors.forename: Janos
authors.initials: J
authors.lastname: Zempleni
mesh_terms: D000595:Amino Acid Sequence; D019921:Biotinylation; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008715:Methionine / Q000031:analogs & derivatives / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D018834:Chaperonin 60; C060293:HSPD1 protein, human; D024101:Mitochondrial Proteins; D011506:Proteins; D017382:Reactive Oxygen Species; D008715:Methionine; D008239:Lysine; C013111:methionine sulfoxide
keywords: Biotin; Heat shock proteins; Methionine oxidation; Reactive oxygen species