New Developments in Ubiquitomics: Discovery of poly-ubiquitylated proteins by novel chain-selective TUBEs

By July 10, 2018 October 28th, 2019 No Comments

LifeSensors Scientists Apurva Chaturvedi, Peter Foote, Li Liang, Dominic Vasturia, and Rajesh Singh recently presented research at FASEB in SnowMass, Colorado. A downloadable PDF of this poster can be found here.



The work presented here focuses on developing easy-to-use tools to distinguish between specific ubiquitin chain linkages, which are involved in a multitude of cellular functions. We believe that the next wave of novel drugs will come from the ubiquitin (Ub) proteasome pathway. However, the field is hampered by a lack of tools to analyze ubiquitin ligase/DUB drug targets and their substrates. Traditional methods for mass spectrometry-based proteomics are insensitive, not reproducible, costly and time consuming. LifeSensors has developed novel chain-selective TUBEs (Tandem-repeated Ub-Binding Entities). These affinity matrices bind to their respective poly-ubiquitin chain type with ~10-20 nM affinity. TUBEs have revolutionized the Ub field by allowing poly-ubiquitylated proteins to be selectively enriched from cellular extracts. A structure-based approach to engineer unique high affinity Ub binding domains (UBDs) for Ub-linkages has been implemented to design highly selective affinity matrices. Development of TUBEs that selectively bind M1-, K48-, and K63-linked poly-ubiquitylated proteins has helped understand the role of modified proteins in cell physiology. Biochemical and biophysical experiments demonstrate highly selective interactions of the matrices to distinct lysine-linked poly-Ub chains. The power of these selective affinity matrices has made it possible to perform linkage-specific Ub proteomics. Applications of these affinity matrices in far-Western detection, pull-down and proteomics will be described here. To discover selective drugs, it is important to establish rank order of potency of compounds in vitro biochemical and in cellular/animal studies. Application of TUBEs-based biomarker methodology such as UbiTest, which helps to identify ubiquitylation of biomarkers, will be described. Development of chainselective TUBE affinity matrices that selectively bind and isolate poly-ubiquitylated proteins will dramatically accelerate the pace of discovery in this important area of biology.

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