Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org). Screen for ISG15-crossreactive Deubiquitinases BACKGROUND: The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15. RESULTS: We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome. CONCLUSIONS: By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice. Posttranslational modification by ubiquitin regulates processes such as proteasomal degradation, intracellular trafficking, and transcription. Ubiquitin is attached to substrates in covalent isopeptide linkage or as an N-terminal fusion [1] [2] [3] . Ubiquitination, however, is reversible: the ubiquitin moiety can be released from substrates through the action of deubiquitinating proteases, which may rescue ubiquitinated substrates from their degradative fate [4] . In contrast, proteasome-associated DUBs enhance the rate of proteasomal degradation by removing bulky poly-ubiquitin chains from substrate proteins prior to proteolysis. Such DUBs enhance the processivity of the proteasome toward target proteins, and also recycle ubiquitin, a modifier that itself turns over slowly [5, 6] . DUBs are furthermore required to hydrolyze the ubiquitin precursor and generate the active ubiquitin monomer. Inspection of mammalian genomes shows the presence of more than 100 genes that encode putative DUBs, consistent with their specific and diverse regulatory functions. Ubiquitin-specific proteases (USPs) are the dominant family among DUBs [7] .