skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

Abstract

Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

Authors:
 [1];  [2];  [1];  [2];  [1];  [3];  [1];  [4];  [1];  [5];  [1]
  1. Univ. of Toronto, ON (Canada). Terrence Donnelly Center for Cellular and Biomolecular Research. Banting and Best Dept. of Medical Research. Dept. of Molecular Genetics
  2. Mount Sinai Hospital, Toronto, ON (Canada). Lunenfeld-Tanenbaum Research Inst.
  3. Cornell Univ., Argonne, IL (United States). Dept. of Chemistry and Chemical Biology
  4. Mount Sinai Hospital, Toronto, ON (Canada). Lunenfeld-Tanenbaum Research Inst.; Univ. of Montreal, QC (Canada). Inst. of Immunology and Cancer Research
  5. Mount Sinai Hospital, Toronto, ON (Canada). Lunenfeld-Tanenbaum Research Inst.; Univ. of Toronto, ON (Canada). Dept. of Molecular Genetics
Publication Date:
Research Org.:
Univ. of Toronto, ON (Canada)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); National Inst. of Health (NIH) (United States); Canadian Inst. of Health Research (CIHR) (Canada); Canadian Cancer Society Research Inst. (Canada)
Contributing Org.:
Mount Sinai Hospital, Toronto, ON (Canada); Cornell Univ., Argonne, IL (United States); Univ. of Montreal, QC (Canada)
OSTI Identifier:
1247362
Grant/Contract Number:  
AC02-06CH11357; GM103403; MOP-136956; MOP-126129
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 113; Journal Issue: 13; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
ENGLISH
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; Cul1 affinity; SCF inhibitors; Fbxw7; Fbxw11; β-Trcp

Citation Formats

Gorelik, Maryna, Orlicky, Stephen, Sartori, Maria A., Tang, Xiaojing, Marcon, Edyta, Kurinov, Igor, Greenblatt, Jack F., Tyers, Mike, Moffat, Jason, Sicheri, Frank, and Sidhu, Sachdev S. Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface. United States: N. p., 2016. Web. doi:10.1073/pnas.1519389113.
Gorelik, Maryna, Orlicky, Stephen, Sartori, Maria A., Tang, Xiaojing, Marcon, Edyta, Kurinov, Igor, Greenblatt, Jack F., Tyers, Mike, Moffat, Jason, Sicheri, Frank, & Sidhu, Sachdev S. Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface. United States. https://doi.org/10.1073/pnas.1519389113
Gorelik, Maryna, Orlicky, Stephen, Sartori, Maria A., Tang, Xiaojing, Marcon, Edyta, Kurinov, Igor, Greenblatt, Jack F., Tyers, Mike, Moffat, Jason, Sicheri, Frank, and Sidhu, Sachdev S. 2016. "Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface". United States. https://doi.org/10.1073/pnas.1519389113. https://www.osti.gov/servlets/purl/1247362.
@article{osti_1247362,
title = {Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface},
author = {Gorelik, Maryna and Orlicky, Stephen and Sartori, Maria A. and Tang, Xiaojing and Marcon, Edyta and Kurinov, Igor and Greenblatt, Jack F. and Tyers, Mike and Moffat, Jason and Sicheri, Frank and Sidhu, Sachdev S.},
abstractNote = {Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.},
doi = {10.1073/pnas.1519389113},
url = {https://www.osti.gov/biblio/1247362}, journal = {Proceedings of the National Academy of Sciences of the United States of America},
issn = {0027-8424},
number = 13,
volume = 113,
place = {United States},
year = {Mon Mar 14 00:00:00 EDT 2016},
month = {Mon Mar 14 00:00:00 EDT 2016}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 48 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Control of Meiotic and Mitotic Progression by the F Box Protein β-Trcp1 In Vivo
journal, June 2003


Ablation of Fbxw7 Eliminates Leukemia-Initiating Cells by Preventing Quiescence
journal, March 2013


Cand1 Promotes Assembly of New SCF Complexes through Dynamic Exchange of F Box Proteins
journal, March 2013


The ubiquitin-specific protease USP47 is a novel β-TRCP interactor regulating cell survival
journal, December 2009


A Strategy for Modulation of Enzymes in the Ubiquitin System
journal, January 2013


PHENIX: a comprehensive Python-based system for macromolecular structure solution.
text, January 2010


A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants
journal, November 1993


Roles of F-box proteins in cancer
journal, March 2014


Insights into SCF ubiquitin ligases from the structure of the Skp1–Skp2 complex
journal, November 2000


Emerging therapies targeting the ubiquitin proteasome system in cancer
journal, January 2014


Coot model-building tools for molecular graphics
journal, November 2004


High-affinity Human Antibodies from Phage-displayed Synthetic Fab Libraries with a Single Framework Scaffold
journal, July 2004


Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex
journal, April 2002


Fbw7 dimerization determines the specificity and robustness of substrate degradation
journal, December 2013


Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation
journal, February 2021


An allosteric inhibitor of substrate recognition by the SCFCdc4 ubiquitin ligase
journal, June 2010


Tumor Suppression by the Fbw7 Ubiquitin Ligase: Mechanisms and Opportunities
journal, October 2014


WD40 Repeat Propellers Define a Ubiquitin-Binding Domain that Regulates Turnover of F Box Proteins
journal, November 2010


Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway
journal, May 2013


PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010


Homodimer of Two F-box Proteins βTrCP1 or βTrCP2 Binds to IκBα for Signal-dependent Ubiquitination
journal, January 2000


Fbxw7α- and GSK3-mediated degradation of p100 is a pro-survival mechanism in multiple myeloma
journal, March 2012


NMRPipe: A multidimensional spectral processing system based on UNIX pipes
journal, November 1995


The Fbw7 and BetaTRCP E3 ubiquitin ligases and their roles in tumorigenesis
journal, January 2012


Multi-functionality of a tryptophan residue conserved in substrate-binding groove of GH19 chitinases
journal, January 2021


Systematic analysis and nomenclature of mammalian F-box proteins
journal, November 2004


Phaser crystallographic software
journal, July 2007


[20] Processing of X-ray diffraction data collected in oscillation mode
book, January 1997


Works referencing / citing this record:

Humanization and directed evolution of the selenium-containing scFv phage abzyme
journal, January 2018


Identification and Characterization of Mutations in Ubiquitin Required for Non-covalent Dimer Formation
journal, September 2019


UBB pseudogene 4 encodes functional ubiquitin variants
journal, March 2020


Proteomic identification of the oncoprotein STAT3 as a target of a novel Skp1 inhibitor
journal, November 2016


Specific targeting of the deubiquitinase and E3 ligase families with engineered ubiquitin variants: Gorelik and Sidhu
journal, November 2016


A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants
journal, October 2017


Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21
journal, July 2016


Protein Engineering in the Ubiquitin System: Tools for Discovery and Beyond
journal, February 2020


Structure and Function of Viral Deubiquitinating Enzymes
journal, November 2017


Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation
journal, August 2017


Peptides meet ubiquitin: Simple interactions regulating complex cell signaling
journal, September 2018


Emerging drug development technologies targeting ubiquitination for cancer therapeutics
journal, July 2019


Peptide design by optimization on a data-parameterized protein interaction landscape
journal, October 2018