title: Use of E2~ubiquitin conjugates for the characterization of ubiquitin transfer by RING E3 ligases such as the inhibitor of apoptosis proteins.
abstract: Ubiquitylation of proteins is a versatile posttranslational modification that can serve to promote protein degradation, or it can have nondegradative roles, such as mediating protein-protein interactions. The Inhibitor of APoptosis (IAP) proteins are important regulators of pathways that control cell death, proliferation, and differentiation. A number of IAP family members are RING E3 ubiquitin-protein ligases, which promote direct transfer of ubiquitin from charged E2 enzymes, or E2~ubiquitin (E2~Ub) conjugates, to substrate proteins. This results in the attachment of nondegradative ubiquitin signals to other proteins, or the autoubiquitylation and degradation of IAPs. Modulating ubiquitin transfer by IAPs is the focus of a number of drug development initiatives and these studies require a detailed understanding of ubiquitylation. Here, we describe preparation of stable E2~Ub conjugates that can be used in biochemical and biophysical experiments to examine RING domain function. In the last 2 years, the availability of these conjugates has helped unveil a molecular understanding of the process of ubiquitin transfer by IAPs. The approaches described here will be suitable for studying other RING E3 ligases.
authors: Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.
authors: Adam J
authors: AJ
authors: Middleton
authors: Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.
authors: Rhesa
authors: R
authors: Budhidarmo
authors: Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand. Electronic address: catherine.day@otago.ac.nz.
authors: Catherine L
authors: CL
authors: Day
mesh_terms: D001681:Biological Assay / Q000379:methods*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D012441:Saccharomyces cerevisiae; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000235:genetics*
chemical_list: D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases
keywords: E2~ubiquitin conjugate; E3 ligase; IAP; RING; Ubiquitin