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Updated: Jul 23, 2026
Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations
Published on: November 7, 2019
Analysis of ubiquitination in vivo using a transgenic mouse model
M Tsirigotis1, S Thurig, M Dubé
1Ottawa Regional Cancer Centre, Ottawa, ON, Canada.
Abstract:
The primary pathway for the proteolytic destruction of cellular proteins is through ubiquitin-mediated targeting to the proteasome. This pathway is pivotal not only in the elimination of damaged or misfolded proteins but also in the temporal, developmental, or signal-mediated destruction of normal cellular substrates. The list of known substrates of the ubiquitin/proteasome pathway is long, but most substrates have been identified in yeast or, more recently, in cultured mammalian cells. It is likely that many mammalian substrates with developmental or disease relevance have yet to be identified because their ubiquitination occurs in tissue or organ systems that cannot be adequately modeled in vitro. We have developed a transgenic mouse model that will allow the isolation and identification of these substrates. The human UbC promoter was used to drive expression of a hexahistidine-tagged version of human ubiquitin in a variety of mouse tissues from early embryonic stages, as assessed by a green fluorescent protein marker. Cleavage of the fusion protein by endogenous enzymes produced epitope-tagged ubiquitin that was detected both in monomeric form and conjugated to cellular proteins. This mouse model should facilitate in the analysis of normal and disease-related ubiquitination events in vivo.
Insights
Researchers developed a transgenic mouse model to identify cellular protein substrates targeted for destruction by the ubiquitin-proteasome system. This model aids in studying ubiquitination in vivo, crucial for understanding development and disease.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The ubiquitin-proteasome system is the primary pathway for cellular protein degradation.
- This system eliminates damaged proteins and regulates normal protein turnover during development and signaling.
- Current methods primarily identify substrates in yeast or cell cultures, potentially missing tissue-specific targets.
Purpose of the Study:
- To develop a novel transgenic mouse model for identifying ubiquitin-proteasome system substrates in vivo.
- To enable the isolation and characterization of ubiquitinated proteins in various mouse tissues.
- To facilitate the study of ubiquitination in physiological and pathological contexts.
Main Methods:
- Generation of transgenic mice expressing a hexahistidine-tagged human ubiquitin under the UbC promoter.
- Utilized a green fluorescent protein marker to assess expression from early embryonic stages.
- Detected epitope-tagged ubiquitin (monomeric and conjugated) after cleavage by endogenous enzymes.
Main Results:
- Successfully generated a transgenic mouse model expressing tagged ubiquitin across multiple tissues.
- Demonstrated the presence of both free and conjugated tagged ubiquitin in vivo.
- Confirmed the model's capability to reflect ubiquitination events in various cellular compartments.
Conclusions:
- The developed transgenic mouse model is a valuable tool for identifying novel ubiquitin-proteasome substrates.
- This model facilitates the in vivo analysis of ubiquitination in diverse tissues and developmental stages.
- It holds significant potential for advancing research in developmental biology and disease mechanisms involving protein degradation.
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