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Assaying Proteasomal Degradation in a Cell-free System in Plants
Published on: March 27, 2014
Cellular and viral Fos proteins are degraded by different proteolytic systems
C Acquaviva1, C Salvat, F Brockly
1Institut de Génétique Moléculaire/UMR5535 /IFR24, CNRS, 1919, route de Mende, 34293-Montpellier Cedex 05-France.
Abstract:
c-Fos proto-oncoprotein is a short-lived transcription factor degraded by the proteasome in vivo. Its mutated forms expressed by the mouse osteosarcomatogenic retroviruses, FBJ-MSV and FBR-MSV, are stabilized two- and threefold, respectively. To elucidate the mechanisms underlying v-Fos(FBJ) and v-Fos(FBR) protein stabilization, we conducted a genetic analysis in which the half-lives and the sensitivities to various cell-permeable protease inhibitors of a variety of cellular and viral protein mutants were measured. Our data showed that the decreased degradation of v-Fos(FBJ) and v-Fos(FBR) is not simply explained by the deletion of a c-Fos destabilizing C-terminal domain. Rather, it involves a complex balance between opposing destabilizing and stabilizing mutations which are distinct and which include virally-introduced peptide motifs in both cases. The mutations in viral Fos proteins conferred both total insensitivity to proteasomal degradation and sensitivity to another proteolytic system not naturally operating on c-Fos, explaining the limited stabilization of the two proteins. This observation is consistent with the idea that FBR-MSV and FBJ-MSV expression machineries have evolved to ensure controlled protein levels. Importantly, our data illustrate that the degradation of unstable proteins does not necessarily involve the proteasome and provide support to the notion that highly related proteins can be broken down by different proteolytic systems in living cells.
Insights
Viral Fos proteins (v-Fos) are stabilized due to mutations that confer proteasome insensitivity and activate alternative degradation pathways. This reveals distinct protein breakdown mechanisms in cells.
Area of Science:
- Molecular Biology
- Cellular Biology
- Virology
Background:
- c-Fos proto-oncoprotein is a transient transcription factor normally degraded by the proteasome.
- Mutated viral Fos proteins (v-Fos) from FBJ-MSV and FBR-MSV exhibit increased stability.
- Understanding the stabilization mechanisms of viral Fos proteins is crucial for comprehending viral oncogenesis and protein degradation pathways.
Purpose of the Study:
- To investigate the molecular mechanisms responsible for the stabilization of viral Fos proteins (v-Fos(FBJ) and v-Fos(FBR)).
- To determine if the stabilization is solely due to the absence of a destabilizing C-terminal domain or involves other factors.
- To explore the proteolytic systems involved in the degradation of these viral oncoproteins.
Main Methods:
- Genetic analysis of cellular and viral protein mutants.
- Measurement of protein half-lives.
- Assessment of sensitivity to various cell-permeable protease inhibitors.
Main Results:
- Decreased degradation of v-Fos(FBJ) and v-Fos(FBR) is not solely due to the deletion of a c-Fos destabilizing domain.
- Stabilization involves a complex interplay of distinct destabilizing and stabilizing mutations, including virally-introduced peptide motifs.
- Mutations confer complete proteasomal degradation insensitivity and sensitivity to an alternative proteolytic system, limiting overall stabilization.
Conclusions:
- Viral Fos protein stabilization results from a balance of mutations affecting distinct proteolytic pathways.
- Protein degradation is not exclusively mediated by the proteasome, even for unstable proteins.
- Related proteins can be degraded by different proteolytic systems within the same cell.
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