title: 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.
abstract: 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.
authors.affiliation: From the Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139.
authors.forename: Joel W
authors.initials: JW
authors.lastname: Thompson
authors.affiliation: From the Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139.
authors.forename: Jane
authors.initials: J
authors.lastname: Nagel
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, CH-4056 Basel, Switzerland, and.
authors.forename: Sjouke
authors.initials: S
authors.lastname: Hoving
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, CH-4056 Basel, Switzerland, and.
authors.forename: Bertran
authors.initials: B
authors.lastname: Gerrits
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, CH-4056 Basel, Switzerland, and.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bauer
authors.affiliation: From the Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Thomas
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Marc W
authors.initials: MW
authors.lastname: Kirschner
authors.affiliation: From the Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139.
authors.forename: Markus
authors.initials: M
authors.lastname: Schirle
authors.affiliation: From the Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139, sarah.luchansky@novartis.com.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Luchansky
mesh_terms: D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D009369:Neoplasms / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics; D034622:RNA Interference; D014157:Transcription Factors / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin / Q000737:chemistry; D043222:Ubiquitin Thiolesterase / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D000076084:Ubiquitin-Specific Peptidase 7
chemical_list: C469991:DDIT4 protein, human; D009842:Oligopeptides; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; C502830:HUWE1 protein, human; D044767:Ubiquitin-Protein Ligases; C464569:USP7 protein, human; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7; D046988:Proteasome Endopeptidase Complex; D008239:Lysine
keywords: DDIT4; HUWE1; Protein Degradation; Proteomics; Quantitative diGly Proteomics; Stress Response; Ubiquitin; Ubiquitin Ligase; Ubiquitin-Proteasome System