Novel Approach for Characterizing Ubiquitin E3 Ligase Function
Abstract
The ubiquitin-proteasome system (UPS) is central to the regulation of numerous cellular events, and its dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases function in concert with E1 and E2 enzymes to recruit specific substrates, coordinating their ubiquitylation and subsequent proteasomal degradation or modulation of cellular activity. E3 ligases have been implicated in a wide range of pathologies, making them attractive targets for drug discovery. However, the lack of high-throughput screening (HTS)-compliant E3 ligase assays has hindered the discovery of E3 inhibitors. Here, we describe a novel HTS-compliant E3 ligase assay platform that utilizes a ubiquitin binding domain’s inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been successfully used with both RING and HECT E3 ligase families, demonstrating broad utility. The platform’s utility is further exemplified by the identification of inhibitors of the E3 ligase CARP2. As more E3 ligases are associated with disease states, the ability to rapidly quantify their activity becomes increasingly important in drug discovery.
Keywords: ubiquitylation, assay, drug discovery, high-throughput screening, CARP2
Introduction
Posttranslational modifications, such as protein ubiquitylation, are crucial for proper cell function and are essential in the maintenance of protein homeostasis. The UPS is central to controlled proteolysis, which manages numerous cellular events; dysregulation can lead to disease. Cellular protein levels are determined by both synthesis and degradation rates, with many proteins regulated via ubiquitin tagging for proteasomal degradation.
Ubiquitin, a 76-amino acid polypeptide, is covalently attached to proteins through an enzymatic cascade involving E1 (activating), E2 (conjugating), and E3 (ligase) enzymes. Polyubiquitin chains, particularly those linked via lysine 48 (K48), target proteins for degradation by the proteasome. However, not all ubiquitin attachments result in degradation; different linkages (e.g., K6, K11, K27, K29, K33, K63) can modulate protein activity or localization.
E3 ligases, a family of approximately 600 proteins, are the final enzymes in the ubiquitin cascade and confer substrate specificity. They represent attractive drug targets due to their limited number of substrates and selective regulatory pathways. Despite this, only one E3 inhibitor has entered clinical trials to date, partly due to the lack of robust, HTS-compliant assays for E3 activity.
Polyubiquitin binding domains are structural motifs that recognize ubiquitin moieties conjugated to substrates. These domains, found in proteins such as the proteasome subunits S5a and Rpn13, can bind polyubiquitin chains with high affinity, especially when multiple ubiquitin moieties are present.
Assay Platform Overview
We developed a novel E3 ligase assay platform that leverages the inherent affinity of a ubiquitin binding domain (UBA2 from Rad23) for polyubiquitin chains. This approach allows for the capture and isolation of polyubiquitylated proteins from in vitro ubiquitylation reactions. The relative amounts of polyubiquitylated proteins are then measured using an enzyme-linked immunosorbent assay (ELISA) format, enabling quantification of E3 ligase activity. The assay is compatible with both RING and HECT E3 ligase families and can be configured for high-throughput screening.
Materials and Methods
Protein Expression and Purification
Plasmids encoding various E3 ligases and related proteins were obtained from commercial sources or collaborators.Proteins such as SUMO-UBA2, K48-only ubiquitin, 6His-Ubel (E1), 6His-UbcH5b/c/H7 (E2s), SUMO-CARP2, SUMO-MuRF1, 6His-E6AP, and SUMO-Hrd1 were expressed in BL21(DE3) bacteria and purified using standard molecular biology techniques.All constructs were sequence-verified.
UBA Assay for E3 Ligase Autoubiquitylation
MicroFluor 96-well plates were coated with purified SUMO-UBA2 and blocked with 3% BSA.E3 ligase reactions were set up with varying concentrations of E3, 5 nM Ubel (E1), 100 nM E2, and 1.1 μM K48 ubiquitin in reaction buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl₂, 0.2 mM ATP, 1 mM β-mercaptoethanol).After incubation, plates were washed, and autoubiquitylation was detected by sequential incubation with rabbit anti-ubiquitin and anti-rabbit HRP or FITC antibodies.Luminescence was measured using a plate reader.
Ubiquitin Chain Recognition Assay
Monomeric ubiquitin or purified ubiquitin chains were incubated in UBA2-coated plates, washed, and detected as above.
Gel-Based In Vitro Autoubiquitylation Assay
Reactions were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-ubiquitin antibodies.
Apparent Km Determination
Serial dilutions of E1 or E2 enzymes were added to reaction mixtures in UBA2-coated plates to determine saturating concentrations and apparent Km values using the Michaelis-Menten equation.
High-Throughput Screening (HTS)
Test compounds were screened at 50 μM in UBA2-coated plates with CARP2, E1, E2, and ubiquitin.Reactions were terminated by washing, and ubiquitylated products were detected as above.Percent inhibition was calculated relative to controls, and hits were identified as compounds inhibiting CARP2 autoubiquitylation by more than 4 standard deviations from the mean.
Results
UBA2-Coated Plates Preferentially Bind Polyubiquitin
SUMO-UBA2-coated plates bound K48-linked polyubiquitin chains with high affinity, while monoubiquitin was not retained after washing.Both K48- and K63-linked chains showed dose-dependent binding, demonstrating the assay’s utility for different polyubiquitin linkages.
Detection of E3 Ligase Autoubiquitylation Activity
The platform detected CARP2 autoubiquitylation only when all components (E1, E2, E3, ubiquitin) were present.A CARP2 mutant lacking a functional RING domain (H333A) showed no activity, confirming assay specificity.Optimal E1 and E2 concentrations were determined for robust CARP2 activity.
Sensitivity Compared to Gel-Based Assays
The ELISA format was significantly more sensitive than gel-based analysis for detecting E3 autoubiquitylation.
Broad Utility for RING and HECT E3 Ligases
The assay successfully measured dose-dependent autoubiquitylation for RING E3 ligases (MuRF1, Hrd1) and the HECT E3 ligase E6AP.Apparent Km measurements identified UbcH7 as the optimal E2 for E6AP, consistent with published data.
High-Throughput Screening Capability
The assay was adapted for HTS, screening CARP2 at 8 nM, with DMSO tolerance up to 5%.The assay demonstrated linear kinetics and robust Z’ values, supporting its suitability for HTS.Hits were confirmed by rescreening.
Discussion
This novel E3 ligase assay platform provides a robust, sensitive, and HTS-compatible method for quantifying E3 ligase activity. By utilizing the high affinity of ubiquitin binding domains for polyubiquitin chains, the assay enables the detection of E3-dependent ubiquitylation events in a simple ELISA format. The platform is broadly applicable to various E3 ligase families and can facilitate the discovery of E3 modulators,SPOP-i-6lc a critical need in drug discovery given the central role of E3 ligases in disease.