Analysis of ubiquitin E3 ligase activity using selective polyubiquitin binding proteins
- Resource Type
- Authors
- Craig A. Leach; James P. LaRocque; Jeffrey G. Marblestone; Michael R. Mattern
- Source
- Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1823:2094-2097
- Subject
- TRIM25
Ubiquitin binding
Ubiquitin-Protein Ligases
Muscle Proteins
Assay
Ubiquitin-conjugating enzyme
Article
Tripartite Motif Proteins
03 medical and health sciences
0302 clinical medicine
Ubiquitin
Humans
Polyubiquitin
Molecular Biology
E3 ligase
030304 developmental biology
0303 health sciences
biology
Drug discovery
Ubiquitination
Cell Biology
MuRF1
High-Throughput Screening Assays
3. Good health
Ubiquitin ligase
Biochemistry
Proteasome
Tandem Repeat Sequences
030220 oncology & carcinogenesis
Ubiquitin-Conjugating Enzymes
biology.protein
Polyubiquitin binding
Protein Binding
Transcription Factors
- Language
- ISSN
- 0167-4889
The ubiquitin proteasome pathway controls the cellular degradation of ~80–90% of the proteome in a highly regulated manner. In this pathway, E3 ligases are responsible for the conjugation of ubiquitin to protein substrates which can lead to their destruction by the 26S proteasome. Aberrant E3 ligases have been implicated in several diseases and are widely recognized as attractive targets for drug discovery. As researchers continue to characterize E3 ligases, additional associations with various disease states are being exposed. The availability of assays that allow rapid analysis of E3 ligase activity is paramount to both biochemical studies and drug discovery efforts aimed at E3 ligases. To address this need, we have developed a homogenous assay for monitoring ubiquitin chain formation using Tandem Ubiquitin Binding Entities (TUBEs). TUBEs bind selectively to polyubiquitin chains versus mono-ubiquitin thus enabling the detection of polyubiquitin chains in the presence of mono-ubiquitin. This assay reports on the proximity between the protein substrate and TUBEs as a result of polyubiquitin chain formation by an E3 ligase. This homogenous assay is a step forward in streamlining an approach for characterizing and quantitating E3 ligase activity in a rapid and cost effective manner. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.