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Mol Cell. 2016 Apr 7;62(1):121-36. doi: 10.1016/j.molcel.2016.02.005. Epub 2016 Mar 3.

System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes.

Author information

1
Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, ON M5S3E1, Canada.
2
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
3
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
4
Program in Cell Biology, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, ON M5G 0A4, Canada.
5
Structural Genomics Consortium, University of Toronto, Toronto, ON M5G1L7, Canada.
6
Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.
7
Inserm U1065, Centre Méditerranéen de Médecine Moléculaire, C3M, Equipe Labellisée La Ligue Contre Le Cancer, Université de Nice-Sophia Antipolis, 151 Route St Antoine de Ginestière, BP 2 3194, 06204 Nice Cedex, France.
8
Department of Biology, York University, Toronto, Ontario M3J1P3, Canada.
9
Campbell Family Cancer Research Institute, University Health Network, Toronto, ON M5G2C1, Canada.
10
Structural Genomics Consortium, University of Toronto, Toronto, ON M5G1L7, Canada; Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON M5G1L7, Canada.
11
Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, ON M5S3E1, Canada; Department of Molecular Genetics, University of Toronto, 1 King's College Cir, Toronto, ON M5S1A8, Canada.
12
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA; Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address: brenda.schulman@stjude.org.
13
Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, ON M5S3E1, Canada; Department of Molecular Genetics, University of Toronto, 1 King's College Cir, Toronto, ON M5S1A8, Canada. Electronic address: sachdev.sidhu@utoronto.ca.

Abstract

HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.

PMID:
26949039
PMCID:
PMC4988125
DOI:
10.1016/j.molcel.2016.02.005
[Indexed for MEDLINE]
Free PMC Article

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