Proteomic identification of proteins conjugated to ISG15 in mouse and human cells

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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.

Section snippets

Materials and methods

Cell culture and antibodies. Ubp43−/− MEFs [30] were cultured in DMEM (Cellgro, Mediatech, Herndon, VA) supplemented with 10% low-endotoxin FBS (HyClone, Logan, UT), 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM Hepes, 1 mM sodium pyruvate, and 2 mM l-glutamine (Biosource, Camarillo, CA). U937 cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 2 mM l-glutamine (Invitrogen). BMMs were prepared [31] from groups of 2 to 3 C57/BL6 (wild-type) or Ifnar1−/− mice [32]

Identification of candidate ISGylated targets by mass spectrometry

As a first step towards understanding the biological role of ISGylation in mediating the pleiotropic effects of IFN, it is important to elucidate the cellular components targeted by this protein modification system. To identify ISGylation targets by mass spectrometry, we enriched conjugates by ISG15 immunoaffinity purification. Due to the sensitivity of mass spectrometry, trace contaminants could potentially be identified along with true ISGylation targets, making it difficult to distinguish

Discussion

Using immunoprecipitation and mass spectrometry, we identified 76 potential targets of ISGylation. In order to minimize false positives, we chose to purify and identify ISGylated proteins from both mouse and human IFN-treated cells. By using different monoclonal antibodies and washing six of eight samples with 3–4 M NaCl, we believed that we could enrich candidate ISGylated proteins and reduce background contaminants. Supplementary Table 1 contains a listing of all proteins identified in either

Acknowledgments

We thank Dr. Reid Townsend at the Siteman Cancer Center Proteomics Core (DDRCC Grant 52574, Washington University School of Medicine) and Dr. Mark Crankshaw at Protein and Nucleic Acid Chemistry Laboratories (PNACL; Washington University School of Medicine) for their invaluable expertise and assistance. We thank Dr. Keun-Il Kim at the Sookmyung Women’s University for his expert advice and suggestions throughout the course of this work. Ifnar1−/− mice back-crossed to C57/BL6 were kindly provided

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    These two authors contributed equally to this work.

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