Merck

HyperISGylation of Old World monkey ISG15 in human cells.

PloS one (2008-06-19)
Els Pattyn, Annick Verhee, Isabel Uyttendaele, Julie Piessevaux, Evy Timmerman, Kris Gevaert, Joël Vandekerckhove, Frank Peelman, Jan Tavernier
ABSTRACT

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. 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 MoISG15, by HuUbE1, thus providing a likely explanation for OWm hyperISGylation. This study discloses the poor conjugation competence of HuISG15 compared to OWmISG15 and maps the critical determinants for efficient conjugation. HyperISGylation may greatly assist ISGylation studies and may enhance its function as positive regulator of Interferon-related immune responses or as anti-tumoral modulator.

MATERIALS
Product Number
Brand
Product Description

Millipore
抗-FLAG® M2亲和凝胶, purified immunoglobulin, buffered aqueous glycerol solution
Sigma-Aldrich
丽春红S染液(零售包装), BioReagent, suitable for electrophoresis
Sigma-Aldrich
丽春红S染液(零售包装), Dye content 75 %