High‐Throughput Screening of HECT E3 Ubiquitin Ligases Using UbFluor

Peter K. Foote1, David T. Krist1, Alexander V. Statsyuk2

1 Department of Chemistry, Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois, 2 Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.24
Online Posting Date:  September, 2017
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Abstract

HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin thioesters and a complex mixture of polyubiquitin products provide further opportunities for off‐target inhibition and increase the complexity of the assay. UbFluor is a novel ubiquitin thioester that bypasses the E1 and E2 enzymes and undergoes direct transthiolation with HECT E3 ligases. The release of fluorophore upon transthiolation allows fluorescence polarization detection of HECT E3 activity. In the presence of inhibitors, HECT E3 activity is ablated, and thus no reaction and no change in FP are observed. This assay has been adapted for high‐throughput screening of small molecules against HECT E3 ligases, and its utility has been proven in the discovery of HECT E3 ligase inhibitors. © 2017 by John Wiley & Sons, Inc.

Keywords: ubiquitin; HECT E3 ligase; high‐throughput screen; fluorescence polarization; thioester

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Determination of Ligase and UbFluor Conditions for High‐Throughput Screening
  • Basic Protocol 2: High‐Throughput Screening Against HECT E3 Ligase Using UbFluor
  • Basic Protocol 3: Dose‐Response Validation of HIT Compounds Against HECT E3 Ligase with UbFluor
  • Basic Protocol 4: Single‐Point Validation of HIT Compounds Against HECT E3 Ligase in the Native Ubiquitination Cascade
  • Basic Protocol 5: Dose‐Response Analysis of HIT Compounds Against HECT E3 Ligase in the Native Ubiquitination Cascade
  • Basic Protocol 6: Exclusion of E1/E2 Transthiolation by HIT Compounds
  • Basic Protocol 7: Determination of Transthiolation or Isopeptide Ligation Inhibition by HIT Compounds
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determination of Ligase and UbFluor Conditions for High‐Throughput Screening

  Materials
  • Dimethyl sulfoxide (DMSO)
  • 100 mM iodoacetamide (IAA) in DMSO (freshly prepared from solid)
  • HECT ligase stock solution: catalytic HECT domain, HECT domain plus WW domains, or full‐length enzyme; commercially available (e.g., LifeSensors, Boston Biochem) or from E. coli expression by previously published procedures (Kathman et al., ; Krist et al., )
  • 10× UbFluor assay buffer (see recipe)
  • Tween‐20
  • UbFluor stock solution (synthesis available in Krist et al., )
  • Liquid handler (e.g., TTP Labtech Mosquito or Labcyte Echo 550)
  • Reagent dispenser (e.g., Integra VIAFILL rapid reagent dispenser; optional)
  • 384‐well plate (low volume, low binding; Corning, cat. no. 3820)
  • Centrifuge for 384‐well plates (e.g., Eppendorf Centrifuge 5810)
  • Platform plate shaker (e.g., Corning LSE Digital Microplate Shaker)
  • Plate reader (e.g., Tecan Infinite M1000 Pro)

Basic Protocol 2: High‐Throughput Screening Against HECT E3 Ligase Using UbFluor

  Materials
  • Dimethyl sulfoxide (DMSO)
  • 100 mM iodoacetamide (IAA) in DMSO (freshly prepared from solid)
  • Stock solutions of screening compounds in DMSO
  • HECT ligase stock solution: catalytic HECT domain, HECT domain plus WW domains, or full‐length enzyme; commercially available (e.g., LifeSensors, Boston Biochem) or obtained from E. coli expression by previously published procedures (Kathman et al., ; Krist et al., )
  • 10× UbFluor assay buffer (see recipe)
  • Tween‐20
  • UbFluor stock solution (synthesis available in Krist et al., )
  • Liquid handler (e.g., TTP Labtech Mosquito or Labcyte Echo 550)
  • Reagent dispenser (e.g., Integra VIAFILL rapid reagent dispenser; optional)
  • 384‐well plate (low volume, low binding; Corning, cat. no. 3820)
  • Centrifuge for 384‐well plates (e.g., Eppendorf Centrifuge 5810)
  • Platform plate shaker (e.g., Corning)
  • Plate reader with automated plate stacker (e.g., Tecan Infinite M1000 Pro)

Basic Protocol 3: Dose‐Response Validation of HIT Compounds Against HECT E3 Ligase with UbFluor

  Materials
  • Dimethyl sulfoxide (DMSO)
  • 100 mM iodoacetamide (IAA) in DMSO (freshly prepared from solid)
  • Stock solutions of hit compounds in DMSO (from protocol 2)
  • HECT ligase stock solution: catalytic HECT domain, HECT domain plus WW domains, or full‐length enzyme; commercially available (e.g., LifeSensors, Boston Biochem) or obtained from E. coli expression by previously published procedures (Kathman et al., ; Krist et al., )
  • 10× UbFluor assay buffer (see recipe)
  • Tween‐20
  • UbFluor stock solution (synthesis available in Krist et al., )
  • Liquid handler (e.g., TTP Labtech Mosquito or Labcyte Echo 550)
  • Reagent dispenser (e.g., Integra VIAFILL rapid reagent dispenser; optional)
  • 384‐well plate (low volume, low binding; Corning, cat. no. 3820)
  • Centrifuge for 384‐well plates (e.g., Eppendorf Centrifuge 5810)
  • Platform plate shaker (e.g., Corning)
  • Plate reader with automated plate stacker (e.g., Tecan Infinite M1000 Pro)

Basic Protocol 4: Single‐Point Validation of HIT Compounds Against HECT E3 Ligase in the Native Ubiquitination Cascade

  Materials
  • 10× UbFluor assay buffer (see recipe)
  • HECT E3 ligase stock solution: catalytic HECT domain, HECT domain plus WW domains, or full‐length enzyme; commercially available (e.g., LifeSensors, Boston Biochem) or obtained from E. coli expression by previously published procedures (Kathman et al., ; Krist et al., )
  • Stock solutions of hit compounds in DMSO (following initial validation in protocol 3)
  • Dimethyl sulfoxide (DMSO)
  • 100 mM iodoacetamide (IAA) in DMSO (freshly prepared from solid)
  • E2 ubiquitin‐conjugating enzyme: commercially available (e.g., Life Sensors, Boston Biochem) or from E. coli expression (Krist et al., )
  • UBA1 E1 enzyme: commercially available (e.g., LifeSensors, Boston Biochem) or from E. coli expression (Carvalho et al., )
  • Ubiquitin (Sigma‐Aldrich)
  • Adenosine triphosphate (ATP; Sigma‐Aldrich)
  • 40 mM MgCl 2
  • 6× Laemmli loading buffer (see recipe)
  • 7.5% SDS‐polyacrylamide gel
  • SDS‐PAGE running buffer (see recipe)
  • Western blot transfer buffer (see recipe)
  • Nitrocellulose blotting membrane (e.g., Bio‐Rad)
  • Absorbent filter paper (e.g., Bio‐Rad)
  • Blocking buffer: 5% blotting‐grade blocker in TBS‐T
  • TBS‐T (see recipe)
  • Anti‐ubiquitin antibody (rabbit)
  • Goat anti‐rabbit secondary antibody with HRP conjugate
  • ECL substrate (e.g., Bio‐Rad Clarity Western ECL Substrate)
  • 1.5‐ml microcentrifuge tubes
  • Heating block (e.g., VWR Advanced Mini Block Heater)
  • Electrophoresis and blot transfer apparatus (e.g., Bio‐Rad Mini‐PROTEAN Tetra Cell SDS‐PAGE system with protein blotting modules)
  • Rocking platform
  • Imaging system (e.g., Bio‐Rad ChemiDoc XRS+ Molecular Imager)

Basic Protocol 5: Dose‐Response Analysis of HIT Compounds Against HECT E3 Ligase in the Native Ubiquitination Cascade

  Materials
  • 10× UbFluor assay buffer (see recipe)
  • Stock solutions of hit compounds in DMSO (following validation in Basic Protocols 3‐5)
  • Dimethyl sulfoxide (DMSO)
  • 100 mM iodoacetamide (IAA) in DMSO (freshly prepared from solid)
  • E2 ubiquitin‐conjugating enzyme: commercially available (e.g., LifeSensors, Boston Biochem) or from E. coli expression (Krist et al., )
  • UBA1 E1 enzyme: commercially available (e.g., LifeSensors, Boston Biochem) or from E. coli expression (Carvalho et al., )
  • Fluorescein‐labeled ubiquitin (FUb; LifeSensors)
  • Adenosine triphosphate (ATP; Sigma‐Aldrich)
  • 40 mM MgCl 2
  • 6× non‐reducing Laemmli loading buffer (see recipe)
  • 15% SDS‐polyacrylamide gel
  • SDS‐PAGE running buffer (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Electrophoresis apparatus (e.g., Bio‐Rad Mini‐PROTEAN Tetra Cell SDS‐PAGE system)
  • Imaging system (e.g., GE Healthcare Typhoon 9400)

Basic Protocol 6: Exclusion of E1/E2 Transthiolation by HIT Compounds

  Materials
  • 10× UbFluor assay buffer (see recipe)
  • Stock solutions of hit compounds in DMSO (following validation in Basic Protocols 3‐6)
  • Dimethyl sulfoxide (DMSO)
  • 100 mM iodoacetamide (IAA) in DMSO (freshly prepared from solid)
  • HECT E3 ligase stock solution: catalytic HECT domain, HECT domain plus WW domains, or full‐length enzyme; commercially available (e.g., LifeSensors, Boston Biochem) or obtained from E. coli expression by previously published procedures (Kathman et al., ; Krist et al., )
  • E2 ubiquitin‐conjugating enzyme: commercially available (e.g., LifeSensors, Boston Biochem) or from E. coli expression (Krist et al., )
  • UBA1 E1 enzyme: commercially available (e.g., LifeSensors, Boston Biochem) or from E. coli expression (Carvalho et al., )
  • Fluorescein‐labeled ubiquitin (FUb; LifeSensors)
  • Adenosine triphosphate (ATP; Sigma‐Aldrich)
  • 40 mM MgCl 2
  • Discharge assay buffer (see recipe)
  • 6× non‐reducing Laemmli loading buffer (see recipe)
  • 15% SDS‐polyacrylamide gel
  • SDS‐PAGE running buffer (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • 0.5‐ml Zeba Spin Desalting Columns (Thermo Fisher Scientific)
  • Electrophoresis apparatus (e.g., Bio‐Rad Mini‐PROTEAN Tetra Cell SDS‐PAGE system)
  • Imaging system (e.g., GE Healthcare Typhoon 9400)
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Literature Cited

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