Quantitative Lys-ϵ-Gly-Gly (diGly) proteomics coupled with inducible RNAi reveals ubiquitin-mediated proteolysis of DNA damage-inducible transcript 4 (DDIT4) by the E3 ligase HUWE1.
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 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 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase), an E3 ligase implicated in cancer and intellectual disabilities. diGly proteomics results led to the identification of DNA damage-inducible transcript 4 (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 that may help to identify candidates for therapeutic modulation in the UPS.
['Thompson|Joel W|JW|', 'Nagel|Jane|J|', 'Hoving|Sjouke|S|', 'Gerrits|Bertran|B|', 'Bauer|Andreas|A|', 'Thomas|Jason R|JR|', 'Kirschner|Marc W|MW|', 'Schirle|Markus|M|', 'Luchansky|Sarah J|SJ|']
D015972:Gene Expression Regulation, Neoplastic D057809:HEK293 Cells D006367:HeLa Cells D006801:Humans D008239:Lysine D013058:Mass Spectrometry D009369:Neoplasms D009842:Oligopeptides D046988:Proteasome Endopeptidase Complex D059748:Proteolysis D040901:Proteomics D034622:RNA Interference D014157:Transcription Factors D025521:Tumor Suppressor Proteins D025801:Ubiquitin D043222:Ubiquitin Thiolesterase D044767:Ubiquitin-Protein Ligases D000076084:Ubiquitin-Specific Peptidase 7
Proteomics DDIT4 HUWE1 Ubiquitin-Proteasome System Ubiquitin Ubiquitin Ligase Quantitative diGly Proteomics Protein Degradation Stress Response