hyperisgylation of old world monkey isg15 in human cellshyperisgylation旧世界猴isg15在人类细胞.pdfVIP

HyperISGylation of Old World Monkey ISG15 in Human Cells Els Pattyn1,2, Annick Verhee1,2, Isabel Uyttendaele1,2, Julie Piessevaux1,2, Evy Timmerman1,2, Kris 1,2 ¨ 1,2 1,2 1,2 Gevaert , Joel Vandekerckhove , Frank Peelman , Jan Tavernier * 1 Department of Medical Protein Research, VIB, Ghent, Belgium, 2 Department of Biochemistry, Ghent University, Ghent, Belgium Abstract Background: ISG15 is an Ubiquitin-like protein, highly induced by Type I Interferons. Upon the cooperative activity of specific Ubiquitinating enzymes, ISG15 can be conjugated to its substrates. Increasing evidence points to a role for protein ISGylation in anti-viral and anti-tumoral defense. Principal Findings: We identified ISG15 from Old World Monkeys (OWm) as a hyper-efficient protein modifier. Western blot analysis visualized more efficient conjugation of OWmISG15 relative to HuISG15 in human (Hu), monkey and mouse (Mo) cell-lines. Moreover, the substrates of OWmISG15 identified upon Tandem Affinity Purification followed by LC-MS/MS identification largely outnumbered these of HuISG15 itself. Several Ubiquitin-Conjugating enzymes were identified as novel ISGylated substrates. Introduction of a N89D mutation in HuISG15 improved its ISGylation capacity, and additional Q31K/ T33A/D133N mutations yielded a HuISG15 variant with an ISGylation efficiency comparable to OWmISG15. Homology modeling and structural superposition situate N89 in the interaction interface with the Activating enzyme. Analysis of the UbE1L residues in this interface revealed a striking homology between OWmUbE1L and HuUbE1, the Activating enzyme of Ubiquitin. In line with this observation, we found efficient activation of AgmISG15, but not HuISG15 or M