ArticleMethods in molecular biology (Clifton, N.J.)2012
Measuring rates of ubiquitin chain formation as a functional readout of ligase activity.
Article in Methods in molecular biology (Clifton, N.J.), 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed.
- A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates.Science advances · 2024Article
- Crosstalk between ubiquitination and translation in neurodevelopmental disorders.Frontiers in molecular neuroscience · 2024Review
- Redefining the catalytic HECT domain boundaries for the HECT E3 ubiquitin ligase family.Bioscience reports · 2022Article
- ISG15 and ISGylation in Human Diseases.Cells · 2022Review
- Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways.eLife · 2019Article
- Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly.The Journal of biological chemistry · 2018Article
- Real-time detection of N-end rule-mediated ubiquitination via fluorescently labeled substrate probes.The New phytologist · 2018Article
- The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly.The Journal of biological chemistry · 2017Article
- Article
- UbFluor: A Fluorescent Thioester to Monitor HECT E3 Ligase Catalysis.Current protocols in chemical biology · 2017Article
- Hepatitis B Virus-Induced Parkin-Dependent Recruitment of Linear Ubiquitin Assembly Complex (LUBAC) to Mitochondria and Attenuation of Innate Immunity.PLoS pathogens · 2016Article
- Convergent evolution in the assembly of polyubiquitin degradation signals by the Shigella flexneri IpaH9.8 ligase.The Journal of biological chemistry · 2014Article
- The active form of E6-associated protein (E6AP)/UBE3A ubiquitin ligase is an oligomer.The Journal of biological chemistry · 2014Article
- Measuring activity in the ubiquitin-proteasome system: from large scale discoveries to single cells analysis.Cell biochemistry and biophysics · 2013Review
- E6AP/UBE3A ubiquitin ligase harbors two E2~ubiquitin binding sites.The Journal of biological chemistry · 2013Article
- Tripartite motif ligases catalyze polyubiquitin chain formation through a cooperative allosteric mechanism.The Journal of biological chemistry · 2013Article
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Authors and funding
2 authors.
Funding
Abstract
Specificity within the pathways of ubiquitin conjugation are defined by protein-binding affinities among the components. Enzyme kinetics provides a facile high-resolution experimental approach for quantitating such protein-binding affinities and yields additional mechanistic insights into the transition state of the enzyme-catalyzed reaction. Most ubiquitin ligases form free polyubiquitin chains at a slow rate in the absence of their cognate target protein as a normal step in their overall catalytic cycle. Rates of polyubiquitin chain formation can, therefore, be used as a reporter function kinetically to characterize binding interactions within the ligation pathway. We describe experimental approaches for: (1) precisely quantitating functional E1 and E2 concentrations by their stoichiometric formation of (125)I-ubiquitin thiolester; (2) semiquantitative screens to define the cognate E2(s) for ubiquitin ligases based on their ability to support polyubiquitin chain formation; (3) initial rate studies to quantify K (m) and k (cat) as a measure of the ability of specific E2-ubiquitin thiolester substrates to support ligase-catalyzed polyubiquitin chain formation; and (4) an isopeptidase T-based technique for distinguishing between free and conjugated polyubiquitin chains formed in the functional assays. These kinetic methods provide mechanistic insights that are otherwise inaccessible by other experimental approaches and yield a precision in characterizing protein interactions that exceeds that of other techniques.
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