Thompson JW, Nagel J, Hoving S, Gerrits B, Bauer A, Thomas JR, Kirschner MW, Schirle M, Luchansky SJ

Targeted degradation of proteins through the ubiquitin-proteasome system (UPS) via the activities of E3 ubiquitin ligases regulates diverse cellular processes, and misregulation of these enzymes contributes to the pathogenesis of human diseases. One of the challenges facing the UPS field is to delineate the complete cohort of substrates for a ...

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particular E3 ligase. Advances in mass spectrometry and the development of antibodies recognizing the Lys-ε-Gly-Gly (diGly) remnant from ubiquitinated proteins following trypsinolysis have provided a tool to address this question. We implemented an inducible loss of function approach in combination with quantitative diGly proteomics to find novel substrates of HUWE1, an E3 ligase implicated in cancer and intellectual disabilities. diGly proteomics results led to the identification of DDIT4 as a putative HUWE1 substrate. Cell-based assays demonstrated that HUWE1 interacts with and regulates ubiquitination and stability of DDIT4. Together these data suggest a model in which HUWE1 mediates DDIT4 proteasomal degradation. Our results demonstrate proof of concept that inducible knockdown of an E3 ligase in combination with diGly proteomics provides a potentially advantageous method for identifying novel E3 substrates, which may help to identify candidates for therapeutic modulation in the UPS.

Date: Aug. 21, 2014

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