Ubiquitin code: Accelerating the discovery of poly-ubiquitylated proteins by novel chain-selective affinity matrices

Ubiquitin (Ub) is attached via isopeptide bonds to lysine residues in the target protein or to another Ub to form poly-Ub chains. 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. 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 enriched/purified from cellular extracts. Development of TUBEs that selectively bind K48-, K63-, and M1-linked poly-ubiquitylated proteins has helped understand the role of modified proteins in cell physiology. However, the roles of other Ub-linkages remain obscured mainly due to the lack of tools that specifically recognize them. Structure-based approach to engineer unique high affinity Ub binding domains(UBDs) for rare Ub-linkages has been implemented to design highly selective affinity matrices. Biochemical and biophysical experiments demonstrate highly selective interactions of the matrices to distinct lysine-linked poly-Ub chains. Power of these highly selective affinity matrices has made it possible to perform Ub proteomics bypassing SILAC mass spectrometry. Application of these affinity matrices in Far Western detection, pull-down and imaging will be described in the poster. To conclude, we have identified unique binding partners for K6-, K11-, K27-, K29-, K33- and K48-linked poly-Ub chains. Development of chain selective affinity matrices (TUBEs) that selectively bind to poly-ubiquitylated proteins will dramatically accelerate the pace of discovery in this important area of biology. Citation Format: Apurva Chaturvedi, Peter Foote, Sarah Julius, Essam Abed, Rajesh K. Singh. Ubiquitin code: Accelerating the discovery of poly-ubiquitylated proteins by novel chain-selective affinity matrices [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3540.