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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
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UbFluor: A Mechanism-Based Probe for HECT E3 Ligases.

David T Krist1, Sungjin Park2, Galyah H Boneh1

  • 1Department of Chemistry, Center for Molecular Innovation and Drug Discovery, Chemistry of Life Processes Institute, Northwestern University, Silverman Hall, 2145 Sheridan Road, Evanston, Illinois 60208.

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Researchers developed UbFluor, a novel fluorescent probe for quantifying HECT E3 ligase activity. This assay simplifies measurements, enabling high-throughput screening for drug discovery in various diseases.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Homologous to E6AP Carboxyl Terminus (HECT) E3 ubiquitin ligases are implicated in numerous diseases and are potential drug targets.
  • A significant challenge in HECT E3 research is the absence of simple assays to measure their enzymatic activity with small molecules.

Purpose of the Study:

  • To develop a simplified assay for quantifying HECT E3 ligase activity.
  • To enable high-throughput screening (HTS) of small molecules targeting HECT E3 ligases.

Main Methods:

  • Development of UbFluor, a fluorescent thioester conjugate of ubiquitin (Ub) and fluorescein-thiol (Fluor-SH).
  • Utilizing UbFluor in fluorescence polarization (FP) assays to monitor transthiolation reactions with HECT E3 ligases.
  • Assessing the probe's ability to detect functional defects in HECT E3 ligase mutants.

Main Results:

  • UbFluor directly reacts with the catalytic cysteine of HECT E3 ligases, releasing detectable fluorescence.
  • The UbFluor assay simplifies measurements by eliminating the need for ATP, E1/E2 enzymes, SDS-PAGE, and poly-ubiquitin detection.
  • The assay successfully recapitulated native ubiquitination reactions and identified defects in HECT E3 ligase mutants.

Conclusions:

  • UbFluor provides a facile and sensitive method for measuring HECT E3 ligase activity.
  • This novel probe facilitates high-throughput screening for the discovery of small molecule modulators of HECT E3 ligases.
  • UbFluor has broad applicability for studying HECT E3 ligase function and developing therapeutics for associated diseases.