Proteome analysis reveals ubiquitin-conjugating enzymes to be a new familyof interferon-alpha-regulated genes

Interferon (IFN)-alpha is a cytokine with antiviral, antiproliferative, and immunomodulatory properties, the functions of which are mediated via IFN-i nduced protein products. We used metabolic labeling and two-dimensional gel electrophoresis followed by MS and database searches to identify potential ly new IFN-alpha-induced proteins in human T cells. By this analysis, we sh owed that IFN-alpha induces the expression of ubiquitin cross-reactive prot ein (ISG15) and two ubiquitin-conjugating enzymes, UbcH5 and UbcH8. Norther n-blot analysis showed that IFN-alpha rapidly enhances mRNA expression of U bcH5, UbcH6 and UbcH8 in T cells. In addition, these genes were induced in macrophages in response to IFN-alpha or IFN-gamma stimulation or influenza A or Sendai virus infections. Similarly, IFNs enhanced UbcH8 mRNA expressio n in A549 lung epithelial cells, HepG2 hepatoma cells, and NK-92 cells. Cyc loheximide, a protein synthesis inhibitor, did not block IFN-induced upregu lation of UbcH8 mRNA expression, suggesting that UbcH8 is the primary targe t gene for IFN-alpha and IFN-gamma. Ubiquitin conjugation is a rate-limitin g step in antigen presentation and therefore the upregulation of UbcHs by I FNs may contribute to the enhanced antigen presentation by macrophages. Our results show that proteome analysis of cells is a suitable method for iden tifying previously unrecognized cytokine-inducible genes.