title: System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes.
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.
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, ON M5S3E1, Canada.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Kuen-Phon
authors.initials: KP
authors.lastname: Wu
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, ON M5S3E1, Canada.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Sartori
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Hari B
authors.initials: HB
authors.lastname: Kamadurai
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Alban
authors.initials: A
authors.lastname: Ordureau
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, ON M5G 0A4, Canada.
authors.forename: Chong
authors.initials: C
authors.lastname: Jiang
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Peter Y
authors.initials: PY
authors.lastname: Mercredi
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, ON M5G 0A4, Canada.
authors.forename: Ryan
authors.initials: R
authors.lastname: Murchie
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, ON M5G1L7, Canada.
authors.forename: Jicheng
authors.initials: J
authors.lastname: Hu
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, ON M5G 0A4, Canada.
authors.forename: Avinash
authors.initials: A
authors.lastname: Persaud
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Manjeet
authors.initials: M
authors.lastname: Mukherjee
authors.affiliation: Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.
authors.forename: Nan
authors.initials: N
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Anne
authors.initials: A
authors.lastname: Doye
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, ON M5G1L7, Canada.
authors.forename: John R
authors.initials: JR
authors.lastname: Walker
authors.affiliation: Department of Biology, York University, Toronto, Ontario M3J1P3, Canada.
authors.forename: Yi
authors.initials: Y
authors.lastname: Sheng
authors.affiliation: Campbell Family Cancer Research Institute, University Health Network, Toronto, ON M5G2C1, Canada.
authors.forename: Zhenyue
authors.initials: Z
authors.lastname: Hao
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, ON M5G1L7, Canada.
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Li
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, ON M5S3E1, Canada.
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Brown
authors.affiliation: 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.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Lemichez
authors.affiliation: Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.
authors.forename: Junjie
authors.initials: J
authors.lastname: Chen
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, ON M5G1L7, Canada; Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON M5G1L7, Canada.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Tong
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: J Wade
authors.initials: JW
authors.lastname: Harper
authors.affiliation: 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.
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
authors.affiliation: Program in Cell Biology, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, ON M5G 0A4, Canada.
authors.forename: Daniela
authors.initials: D
authors.lastname: Rotin
authors.affiliation: 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.
authors.forename: Brenda A
authors.initials: BA
authors.lastname: Schulman
authors.affiliation: 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.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002465:Cell Movement; D004285:Dogs; D045325:HCT116 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008958:Models, Molecular; D009940:Organoids / Q000166:cytology / Q000378:metabolism; D019151:Peptide Library; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D019151:Peptide Library; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases