HyperISGylation of Old World Monkey ISG15 in Human Cells

Pattyn, Els; Verhee, Annick; Uyttendaele, Isabel; Piessevaux, Julie; Timmerman, Evy; Gevaert, Kris; Vandekerckhove, Joël; Peelman, Frank; Tavernier, Jan

Conjugation of Ub(L) requires the cooperative activity of at least 3 enzymes. The ubiquitination cascade is initiated by an Ub-Activating enzyme (termed Uba, Ube or E1) adenylating the Cterminus of Ub(L), thereby forming an acyl-phosphate linkage with AMP. The catalytic Cys residue in UbE1 subsequently attacks this high energy bond, forming a thiolester bond to the C-terminal Gly of Ub(L). In humans, Ub molecules are found to be activated by UbE1 (also known as A9S1) [6] or UbE1L2 (also named Uba6) [7, 8] , ISG15 by UbE1L [9] , SUMO by AOS-Uba1 [10] and Nedd8 by AppBp1-Uba3 [11] . Ub(L) molecules thiolester-linked to its Ub-Activating enzyme are transferred to a Ub-Conjugating enzyme (termed Ubc or E2), also by a thiolester linkage on a Cys residue. UbcH8 has been identified as a major Ub-Conjugating enzyme effecting ISG15 conjugation [12, 13] . Around 400 proteins are recognized as Ub-Ligases or E3s. Roughly, they can be discerned as RING (Really Interesting New Gene)-finger proteins, acting as a molecular scaffold, and HECT (Homologous to E6-AP C-Terminus)-domain proteins, which also exert a catalytic contribution. Ub-Ligases confer specificity, and place the Ub or UbL molecule in close proximity to the Lys residue of the substrate. The formation of polyubiquitin chains is a process mediated by the Ub-Conjugating enzyme together with the Ub-Ligase. Recently, the IFN-induced HERC5 has been identified as an ISG15 E3-Ligase in human cells [14] . The Estrogen-response Finger Protein (EFP), also an IFN-induced protein, functions as an E3-Ligase for ISGylation of 14-3-3d [15] . Definitely, these recently discovered ISG15-Ligases are only the onset of a more extensive list. As Ub, most UbLs are synthesized as inactive precursors, being processed by De-UBiquitinating enzymes (DUBs) exposing the mature protein with a C-terminal Gly residue. DUBs not only exert their function by protein processing, they also have a function in removal of the Ub(L) from their substrate. Ub Specific Protease 2 (USP2), USP5 (also named Isopeptidase T), USP13 (IsoT3) and USP14 have been identified as proteases with a dual specificity for Ub and ISG15 [16, 17] . USP18 (also named UBP43) specifically cleaves ISG15 [18] . Of note, USP18 also competes with Jak1 for binding to the Type I IFN receptor IFNaR2 [19] .