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

The interaction interfaces of Nedd8 bound to its Activating Enzyme, AppBp1-Uba3, [36] and SUMO bound to Sae1-Sae2 [37] have recently been mapped and show a remarkable high degree of similarity. We built a similar homology model for the UbE1L-ISG15 complex (Figure 6a ), based on the crystal structure of the AppBp1-Uba3-Nedd8-ATP complex [38] . This superposition brings the critical residue 89 in ISG15 in the interaction interface with UbE1L. Therefore, we compared the sequences of Hu and OWmUbE1L in their predicted interfaces with ISG15 and found an interaction region displaying significant substitutions (i.e. the sequence 563-GTSGTWG-569 in HuUbE1L corresponding to 560-GTLGTRG-566 in OWmUbE1L). Residue W568 in HuUbE1L juxtaposes to ISG15 N89 (Figure 6a ). This residue is substituted with an Arg residue in OWmUbE1L. Strikingly, the corresponding sequence in OWmUbE1L shows high similarity with HuUbE1 ( Figure 6b ). This observation prompted us to test whether AgmISG15 could be activated by HuUbE1. We performed an in vitro assay analyzing the loading of either HuISG15 or AgmISG15 with HuUbE1 or HuUbE1L. Thiolester formation of both ISG15 orthologues with HuUbE1L was observed, as expected. Importantly, as anticipated from the sequence similarity, HuUbE1 was also found to be able to form thiolester bonds with AgmISG15, whilst no loading of HuISG15 could be seen (Figure 7a ). The thiolester nature of the bond was verified by addition of b-ME. Western blot analysis evaluating the total ISGylation pattern further confirmed this alternative activation pathway for AgmISG15 ( Figure 7b for the experiment in HekT cells, Figure 7c in COS cells). The cells were transfected with combinations of an empty vector or vectors encoding HuUbE1L, HuUbE1 and UbcH8 together with FLAGtagged HuISG15, AgmISG15 or MoISG15. Visualising the ISGylation pattern on the different samples only observed significantly enhanced ISGylation by Hu and MoISG15 upon ectopic expression of UbE1L. A beneficial effect on Hu/ MoISGylation efficiency upon ectopic expression of UbE1 was minimal. By contrast, even with the higher basal ISGylation level of AgmISG15, overexpression of both UbE1L and UbE1 were able to further markedly enhance its conjugation capacity.