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J Biol Chem. 2014 Oct 17;289(42):28942-55. doi: 10.1074/jbc.M114.573352. Epub 2014 Aug 21.

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.

Author information

  • 1From the Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139.
  • 2Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, CH-4056 Basel, Switzerland, and.
  • 3Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.
  • 4From the Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139, sarah.luchansky@novartis.com.

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.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

KEYWORDS:

DDIT4; HUWE1; Protein Degradation; Proteomics; Quantitative diGly Proteomics; Stress Response; Ubiquitin; Ubiquitin Ligase; Ubiquitin-Proteasome System

PMID:
25147182
[PubMed - indexed for MEDLINE]
PMCID:
PMC4200252
Free PMC Article
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