title: Holocarboxylase synthetase catalyzes biotinylation of heat shock protein 72, thereby inducing RANTES expression in HEK-293 cells.
abstract: In a recent mass spectrometry screen, we identified 108 new proteins that were modified endogenously by covalent binding of biotin; members of the heat shock superfamily of proteins, including heat shock protein 72 (HSP72), were overrepresented among the biotinylated proteins. Mammals respond to infections by secreting extracellular HSP72 (eHSP72), which elicits an immune response. Here, using mass spectrometry and site-directed mutagenesis, we identified five biotinylation sites in HSP72. We used coimmunoprecipitation, mass spectrometry, and limited proteolysis assays to demonstrate that HSP72 interacts physically with the protein biotin ligase holocarboxylase synthetase (HLCS), leading to biotinylation of residues K112, K128 K348, K361, K415, and, probably, additional lysines. Finally, we demonstrated that HLCS-dependent biotinylation of eHSP72 increases expression of the chemokine regulated on activation normal T-expressed and presumably secreted (RANTES) by human embryonic kidney (HEK-293) cells. In conclusion, we report a novel endogenous modification of HSP72 and demonstrated that binding of biotin to eHSP72 prepares cells for a strong immune response.
authors.affiliation: Department of Nutrition and Health Sciences, University of Nebraska at Lincoln, Lincoln, Nebraska.
authors.forename: Jing
authors.initials: J
authors.lastname: Xue
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.forename: Janos
authors.initials: J
authors.lastname: Zempleni
mesh_terms: D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D018946:Chemokine CCL5 / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D057809:HEK293 Cells; D050884:HSP72 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans
chemical_list: C483024:CCL5 protein, human; D018946:Chemokine CCL5; D050884:HSP72 Heat-Shock Proteins; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases
keywords: RANTES; biotin; heat shock protein 72; holocarboxylase synthetase; posttranslational modification