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.
Catic
André
A
Program in Immunology, Harvard Medical School, Boston, Massachusetts, United States of America; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
Fiebiger
Edda
E
Korbel
Gregory A
GA
Blom
Daniël
D
Galardy
Paul J
PJ
Ploegh
Hidde L
HL
D000818:Animals; D003227:Conjugation, Genetic; D003429:Cross Reactions; D016207:Cytokines / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D007700:Kinetics; D051379:Mice; D010802:Phylogeny; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014452:Ubiquitins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D016207:Cytokines; C489550:G1p2 protein, mouse; D014452:Ubiquitins; C074034:ISG15 protein, human; D046988:Proteasome Endopeptidase Complex