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

Based on interaction interfaces of Nedd8 bound to AppBp1-Uba3 [36] , Narasimhan and colleagues predicted 7 hot spot residues on ISG15 constituting the interface with UbE1L. Three of these residues show a high degree of conservation among the different Ub(L)s (i.e. R92, E132 & R153 in ISG15), 4 other residues are presumed to confine specificity for UbE1L (R87, K90, W123 & F149) [35] . We built a similar homology model for the UbE1L, using the crystal structures of the AppBp1-Uba3-Nedd8-ATP complex. Structure superposition shows that residue 89 corresponds to L8 in Nedd8 (Figure 6a) , a well-conserved residue that is also found in Ub (Figure 1 ). Mutation of L8 in Ub significantly reduced its target conjugation ability [47] , and in yeast this residue proved to be essential for viability [48] . This suggests that residue 89 in ISG15 and L8 in Ub have a similar important role in conjugate formation. L8 in Nedd8 is part of a hydrophobic patch (L8, I44, V70) that interacts with V323 and Y331 of Uba3, embedded in the two b-sheets preceding its Cterminal domain. Mutation of these residues greatly reduced Nedd8 adenylation and consequently conjugation [36] . However, residues L8, I44 and V70 in Nedd8 correspond with the nonhydrophobic residues N89, T125 and N151 in ISG15, suggesting different types of interaction in this region. The two Uba3 bstrands correspond to the region 880-895 in the UbE1L model, but the alignment in this region is too poor to allow reliable modeling of the actual interactions. Moreover, residues 152-LRL-154, which are part of the C-terminal region of ISG15 juxtapose to N89 (Figure 4 ). This region aligns with 71-LRL-73 in Ub which was described to be of high importance for UbE1 binding affinity and for substrate specificity [49] . Our model suggests that residue 89 makes a contact with R565 in OWmUbE1L, which lies in the N-terminal domain, known to select for a specific UbL [36, 37] . In HuUbE1L, this Arg is replaced by a Trp. Strikingly, the sequence 560-GTLGTRG-566 in OWmUbE1L is very similar to the corresponding region 602-GTLGTKG-608 in HuUbE1 (see also Figure 6b ). Based upon this observation, we experimentally demonstrated that HuUbE1 could efficiently activate AgmISG15, in contrast to Hu and MoISG15. This non-redundant role for UbE1L in activating MoISG15 is also in line with the loss of MoISG15 conjugation in UbE1L knock-out mice [50] . Unlike UbE1L, which is IFN-inducible and has a tissue and cell-line specific expression profile [51] , UbE1 is ubiquitously expressed. Taken together, our findings support a role for residue 89 in ISG15 as a hot spot residue in the interface with its Activating enzyme, thus providing an explanation for the hyperISGylation seen for OWmISG15. Recently, with the discovery of UbE1L2 (Uba6) as the Activating enzyme for FAT10 and as a second Activating enzyme for Ub [7, 8] , the theory of an Activating enzyme harboring unilateral assignment for a specific Ub(L) was overthrown. Here, we describe promiscuity of UbE1 for OWmISG15 activation. This is the first report of ISG15 being activated by another Activating enzyme than UbE1L. Moreover, to the best of our knowledge, this is also the first report of an UbLother than Ubiquitin-being activated by UbE1.