Ubiquitin Ligase

A ubiquitin ligase is an enzyme that helps regulate protein fate by attaching the small protein ubiquitin to specific cellular targets. Working with ubiquitin-activating E1 and conjugating E2 enzymes, it recognizes a substrate and promotes transfer of ubiquitin to a lysine residue; repeated additions can form a polyubiquitin chain that directs the protein to the proteasome for degradation, while other ubiquitin signals alter localization or activity. Ubiquitin ligases therefore control processes such as cell-cycle progression, DNA repair, transcription, and immune responses. Studying their substrate specificity and dysfunction provides insight into disease mechanisms and supports efforts to develop targeted therapies.

Ubiquitin Ligase - Related Videos

Research

JoVE Journal - Biology

In Vitro Analysis of E3 Ubiquitin Ligase Function

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Cited by 8 •

2021

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

Research

JoVE Journal - Biochemistry
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Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

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Cited by 5 •

2019

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

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Cited by 2 •

2021

Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

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Cited by 2 •

2022

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs

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Cited by 14 •

2019

This work describes the synthesis and characterization of a pomalidomide-based, bifunctional homo-PROTAC as a novel approach to induce ubiquitination and degradation of the E3 ubiquitin ligase cereblon (CRBN), the target of thalidomide analogs.

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