Di-ubiquitin (Ub2, K63-linked) was synthesised enzymatically, using wild type ubiquitin for the proximal ubiquitin and K63R ubiquitin for the distal ubiquitin, and purified by ion exchange chromatography. Specifically linked di-ubiquitins have utility as substrates for investigating deubiquitinating enzyme (DUB) activity and polyubiquitin chain processing selectivity and in the study of ubiquitin binding and recognition in ubiquitin signalling associated processes.
K63-linked polyubiquitin molecules have been implicated in the regulation of a range of non-degradative cellular processes including trafficking, autophagy, DNA damage repair and signal transduction.
Modification of cellular proteins with polyubiquitin chains occurs in a wide range of signalling pathways and is tightly regulated in order to ensure cellular homeostasis. The function, processing and ultimate fate of polyubiquitinylated proteins is thought to be determined by the nature of the linkage between adjacent ubiquitin molecules in the polyubiquitin chain.
