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

In this study we also present the Ub-Conjugating enzymes UbcH10, H16 and H17 as new ISGylation targets. UbcH10 (UbE2C) acts as an UbcH for the Anaphase Promoting Complex (APC), promoting cyclinA degradation and mitotic exit. During the G1 phase, UbcH10 is auto-ubiquitinated allowing reaccumulation of cyclinA and entry in the S phase [53] . UbcH10 is often referred to as the cancer-related UbcH as it is markedly overexpressed in the majority of cancerous cell lines [54] . Recently, also UbcH16 (E2-25K or HIP2) has been identified as an APC-dependent UbcH promoting Ub K48-linked chain extensions on pre-attached Ubs [55] . UbcH17 (UbE2T) has recently been identified as the UbcH essential to ubiquinate FANCD2, which needs to be ubiquitinated in order to bind BRCA2 and take part in the DNA repair process. UbcH17depleted cells have abnormal chromosomes, characteristic for In vitro assay revealing the capability of AgmISG15 to form thiolester bonds with UbE1, contrary to HuISG15. 2.5 mM UbcH8 was incubated with 200 nM of either UbE1 or UbE1L, and 7.5 mM of either mature Hu-or AgmISG15 under conditions as described in Materials and Methods. The samples were divided in two aliquots; and were subjected to SDS-PAGE under non-reducing or reducing conditions. The proteins were stained with IRDye Blue protein stain and visualized using the Odyssey infrared imaging system. The extra band representing thiolester formation between UbE1 and AgmISG15 is flanked by two asterisks. These thiolester bonds are disrupted by the addition of reducing agent (here b-ME). (B) UbE1 can efficiently activate AgmISG15, but not Hu or MoISG15. HekT cells were transfected with an empty vector, or combinations of vectors encoding HuUbE1L, HuUbE1 and UbcH8 together with HuISG15, AgmISG15 or MoISG15 as indicated. The total ISGylation patterns were evaluated by Western blot analysis using the FLAG tag of the ISG15 proteins. Fanconi anemia [56, 57] . These findings may help explain the role for ISG15 in anti-tumor defense.