When the cell responds to stress, it will stop the translation process and selectively turn on the expression of some stress response. In addition, other biological pathways in the cell will also be shut down or disrupted, including nuclear and cytoplasmic transport, RNA splicing, and All cell cycle related activities will be affected. Inhibition of translation and subsequent decomposition of multiple polymers will lead to an increase in the concentration of non-ribosome-bound mRNA in the cytoplasm, which ultimately leads to the formation of cell aggregates called Stress granules. The stress-induced cell changes prompt the cells to gain adaptability in a short time, but after the stress is eliminated, the need for stress particles is reversed, thereby restoring the normal progress of intracellular activities and rebuilding the homeostatic environment of the cells.
Therefore, after the stress is removed, the stress particles gradually degrade, translation, and other biological pathways return to normal, but so far, how the depolymerization process of the stress particles is regulated after the stress is removed is still unknown.
On June 24th, the Howard Hughes Institute’s St Jude Children’s Research Hospital J. Paul Taylor’s research group published a paper in Science entitled“Ubiquitination is essential for the recovery of cellular activities after heat shock”， reveal the specific mechanism of ubiquitination in restoring cell activity and mediating depolymerization of stress granules under stress condition was revealed.
The reason why the authors focus on Ubiquitylation is that related studies on the dynamic changes of stress granules indicate that ubiquitin-like modification of SUMO may be very important for stress granules. In addition, studies have shown that the level of protein ubiquitination contained in stress particles is extremely low, indicating that the coupling of ubiquitination is completely unnecessary for the dynamic changes of stress particles. Therefore, in many disputes, the authors believe that ubiquitination may unlock the secret key to the depolymerization of stress particles.
In order to uncover the answer to this question, the authors hope to test the response of the Human embryonic kidney in response to different forms of stress. The authors exposed the cells to heat stress (42°C) and oxidative stress (arsenous acid). Potassium), osmotic stress (sorbitol), ultraviolet stress (UV) and protease inhibitor (bortezomib) stress, and unstressed cells were used as controls. After treatment, a Tandem ubiquitin-binding entity was performed to capture pan The changes in the chemical omics are analyzed using the large-scale proteomics method of unlabeled Liquid chromatography with tandem mass spectrometry. (Proteomics services at AxisPharm)
Through a large-scale analysis of ubiquitinomics in cells after different stress treatments, the authors found that the proteomic changes of ubiquitination modification depend on different stress treatments. For example, the ubiquitination modification of certain proteins only occurs under heat shock treatment or UV exposure. So what are the characteristics of the ubiquitinated modified proteome during different stresses? Taking heat shock stress treatment as an example, the authors examined the dynamic characteristics of ubiquitination modification during heat shock treatment and recovery. The authors used different polyubiquitination antibodies or polyubiquitination modified antibodies at different modification sites (K48 or K63) and found no significant site or chain type specificity for the ubiquitination modification after heat shock and recovery.
In addition, the authors also analyzed the TUBE ubiquitinomics changes after different cell heat shock treatments. For example, in neurons derived from induced pluripotent stem cells or mouse cortical neurons, the authors found that heat shock treatment and There is not much difference in ubiquitination omics changes during the recovery process. Therefore, this result shows that the ubiquitinomics changes caused by stress treatment do not have much cell specificity.
The authors classified and analyzed the functions of proteins affected by ubiquitination omics changes during heat shock stress, and found that they were consistent with the previous results: under the regulation of heat shock stress, factors related to nucleoplasmic transport were recruited into stress particles and affected the nucleoplasmic transport process. In addition, through gene ontology analysis, the authors found that changes in ubiquitinomics also appear in some proteins related to translation and metabolic processes. However, compared with arsenite-induced oxidative stress, different types of stress still lead to different patterns of ubiquitination modification.
It is worth mentioning that a large class of changes in heat shock ubiquitin chemistry is mRNA binding proteins. This result indicates that heat shock-dependent ubiquitination modifications may appear in some messenger ribonucleoprotein complex proteins. Ubiquitination occurs on the fully assembled complex rather than on one of the individual RNA binding proteins.
The authors want to know whether the ubiquitination changes caused by heat shock stress have a certain effect on the depolymerization of stress particles. Through immunostaining, the authors found that polyubiquitinated antibodies quickly co-localized with the stress particles produced after heat shock treatment. In addition, Taylor’s laboratory published another Science paper at the same time. The study found that the ubiquitination of G3BP1, a key regulator of stress granules, is critical for the depolymerization of cell stress granules after recovery from heat shock treatment. The authors found that exposure to a certain concentration of ubiquitination inhibitors on cells treated with heat shock can cause problems with the depolymerization of stress particles after heat shock treatment is restored. Therefore, this result shows that ubiquitination is critical to the restoration of the normal biological activity of cells after heat shock treatment and to promote the depolymerization of stress particles.
In order to further prove the role of ubiquitination in the restoration of normal biological activity of cells after the stress is relieved, the authors hope to detect the effects of ubiquitination modification on the process of nuclear and cytoplasmic transport. To this end, the authors constructed a Shuttle-tdTomato nucleocytoplasmic reporter assay. This system fuses the fluorescent protein with the Nuclear localization signal and the Nuclear export signal. The authors found that the ability of cells to transport nucleoplasm after treatment with ubiquitination inhibitors is still impaired after the stress is relieved. Therefore, the level of ubiquitination is critical to the recovery of nucleoplasm transport function after heat shock stress.
In summary, this work found that heat stress induces targeted ubiquitination of a series of specific proteins, and this ubiquitination modification is necessary for cells to recover from a variety of heat stress-induced cellular stresses, including Reversal of mRNP remodeling, depolymerization of stress granules, nucleocytoplasmic transport function and restoration of protein synthesis. This work established a novel identification method of ubiquitination chemistry and uncovered the large-scale proteomics changes of ubiquitination modification in the process of cell response to different stresses.
Reference
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4. Keiten-Schmitz, J. et al. The Nuclear SUMO-Targeted Ubiquitin Quality Control Network Regulates the Dynamics of Cytoplasmic Stress Granules. Molecular cell 79, 54-67.e57, doi:10.1016/j.molcel.2020.05.017 (2020).
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