title: Proteomic identification of proteins conjugated to ISG15 in mouse and human cells.
abstract: Though the interferon-inducible protein ISG15 was one of the first ubiquitin-like modifiers to be discovered, much remains unknown about the identity of proteins conjugated to ISG15 or the biologic consequences of modification. To gain a better understanding of the cellular pathways affected by ISG15, we identified proteins targeted for ISGylation using a proteomic approach. Mass spectrometric analysis identified 76 candidate ISGylation targets in anti-ISG15 immunoprecipitates from interferon-treated mouse or human cells. Twenty-one proteins were found in both mouse and human samples, including STAT1, a known target of ISGylation. Candidates identified in both species were tested for ISGylation in a transfection system: 18 of 19 proteins tested were ISGylated in this system. Two candidates, EF-2 and VCP, were also shown to be ISGylated in an interferon-dependent manner in the absence of exogenous over-expression. Seven proteins identified from a single species, but functionally related to candidates found in both species, were also ISGylated in the over-expression system. Proteins that can be ISGylated play important roles in translation, glycolysis, stress responses, and cell motility. These data indicate that ISGylation targets proteins found in several fundamentally important cellular pathways and will contribute to understanding the physiologic role of interferon-induced ISG15 and ISG15 conjugation.
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63130, USA.
authors.forename: Nadia V
authors.initials: NV
authors.lastname: Giannakopoulos
authors.forename: Jiann-Kae
authors.initials: JK
authors.lastname: Luo
authors.forename: Vladimir
authors.initials: V
authors.lastname: Papov
authors.forename: Weiguo
authors.initials: W
authors.lastname: Zou
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Lenschow
authors.forename: Barbara S
authors.initials: BS
authors.lastname: Jacobs
authors.forename: Ernest C
authors.initials: EC
authors.lastname: Borden
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Herbert W
authors.initials: HW
authors.lastname: Virgin
authors.forename: Dong-Er
authors.initials: DE
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D016899:Interferon-beta / Q000494:pharmacology*; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D010449:Peptide Mapping / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D013045:Species Specificity; D020298:U937 Cells; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D016207:Cytokines; C489550:G1p2 protein, mouse; D020543:Proteome; D014452:Ubiquitins; C074034:ISG15 protein, human; D016899:Interferon-beta