Abstract B070: Deciphering the ubiquitin code with polyubiquitin chain selective affinity matrices

Post-translational modification of proteins by ubiquitin (Ub) is a versatile process, highly dynamic, and involved in nearly all aspects of biologic functions in eukaryotes. Dysregulation of ubiquitylation has been implicated in a plethora of pathologic conditions ranging from developmental abnormalities and autoimmune to neurodegenerative diseases and a variety of cancers. Ub is attached, via isopeptide bonds, to lysine residues in the target protein through a cascade of enzymatic reactions. These Ub-moieties can then serve as substrates for the conjugation of additional Ubs, forming poly-ubiquitin chains, again through the formation of isopeptide bonds between the C-terminus of one Ub and any of seven lysine, or N-terminal methionine in the target Ub. The reversibility, heterogeneity, and diversity of these modifications combined with the lack of suitable tools have made it difficult to properly isolate and characterize poly-ubiquitylated cellular proteins. This chain complexity known as the “Ub code” requires diverse technical approaches to unravel its complexity. Recent publications have demonstrated that antibodies against Ub display linkage bias or are of poor quality for experiments requiring precise measurements. However, in 2009 a novel technology called TUBEs (Tandem-repeated Ub-Binding Entities) was developed. TUBEs have revolutionized the Ub field by allowing poly-ubiquitylated proteins to be purified from cellular extracts. Development of TUBEs that selectively bind K48, K63 poly-ubiquitylated proteins has helped understand the role of modified proteins in cell physiology. In addition, derivatives of chain selective TUBEs have been used to develop novel assays for DUBs and ubiquitin ligases. This presentation will describe variants of TUBEs and their applications, as well as the new iterations of TUBEs aimed at identifying Ub-linkage specific functions and their biologic relevance. Citation Format: Rajesh Singh, Don Bosco Than, Sarah Julius, Xiaolong Lu, Peter Foote, Chengcheng Song. Deciphering the ubiquitin code with polyubiquitin chain selective affinity matrices [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B070.