Effect Of Heat Shock Protein 90 On 20S Proteasome In Oxidative Stress

BackgroudOxidative stress is ubiquitous in the biosphere, which due to the ROS (reactive oxygen species) imbalances between generation and elimination, will lead to human diseases and cell damage. ROS is the formation of a class of oxygen, the chemical nature of active oxygen than the ground state of the oxygen metabolites, including oxygen free radicals and non-radical substances. Some exogenous chemicals, drugs, cytokines or other environmental factors such as ultraviolet radiation, ionizing radiation can also promote the generation of ROS. Oxidative stress occur when absence of antioxidants or O2-·,H2O2, and OH·excessive accumulation. In many diseases and environmental damage, the imbalance of ROS generation and elimination can cause aging, tumorigenes, cardiovascular disease, neurodegenerative diseases, rheumatoid arthritis,lung inflammation lesions and other diseases. ROS generation and accumulation may cause serious cell damage and result in physiological dysfunction and cell death.The basic mechanism of injury is oxygen free radicals damage cells, causing damage to the lipids and membrane, also damage to proteins, enzymes, nucleic acids chromosome, ultimately leading to cell necrosis and apoptosis.The mechanism of oxidative stress injury has been more in-depth research in many studies, but the research about oxidative damage to cells under stress and the mechanism of protein quality control is not enough. However, this part is critical to maintain the normal function and survival of cells under oxidative stress. The protein quality control system is composed with molecular chaperone and proteolytic enzymes. First, the molecular chaperone identify the damaged or misfolded proteins result from oxidative stress and assist their recovery correct conformation; Secondly, the molecular chaperone protein will transfer the damaged proteins to specific ATP dependent proteolytic enzymes and degrade them. In the protein quality control system, the molecular chaperone Hsp90 and the 20S Proteasome which is the core catalytic hydrolysis unit of the ubiquitin-26S Proteasome system play an important role.Under the oxidative stress,Hsp90 is essential for maintaining the function of 20S Proteasome system and synergistic the protein quality control.However, how Hsp90 affects the 20S Proteasome?This mechanism needs to be further clarified. To explore the protein quality control mechanism in the oxidative stress condition will provide important theoretical basis for the relevant mechanisms of disease, disease treatment, disease prevention and drug development.Heat shock protein 90 (heat shock protein, Hsp90) is a protective molecules closely related with the stress, which is interact with the substrate proteins (mostly signal transduction pathway signaling molecules)with the assistance of co-chaperones, by adjusting its folding and aggregation to protect its biological function, and thus prevent or promote cell growth, apoptosis/death. Proteasome is a ubiquitous giant protein complexes.In eukaryotes, the Proteasome locate in the nucleus and cytoplasm.The main role of Proteasome is degradate the unwanted or damaged protein in the cell, which is used to control the special protein and remove the misfolding protein. Proteasome degradation pathway is essential for many cellular processes, including cell cycle, gene expression, oxidative stress, and at the same time,it is also a target for oxidative stress.The vitro studies have shown that the normal function of the Proteasome, in addition to depend the target protein ubiquitination and ubiquitin protein identification, but also closely related with the role of molecular chaperone.Therefore, to explore the relationship between Hsp90αand 20S Proteasome will help clarify the stress protective mechanism of Hsp90αas a protective molecular.Objectives1. Established model of oxidative stress in HepG2 cells;.2. Under the conditions of oxidative stress, the expression of Hsp90αand the 20S Proteasome in HepG2 cells, the co-localization as well as the interaction between the two proteins;.3. To establish a cell line stably inhibiting the Hsp90αexpression by siRNA interference.4. On the basis of the previous step, detect the changes of Proteasome activity after lower expression of Hsp90α, and to further investigate the molecular mechanism of the protective function of heat shock proteins in cells under oxidative stress.Methods1. Established model of oxidative stress in HepG2 cells:The cells were divided into control group, group of oxidative stress 2h,4h,6h,8h,12h,24h group.Oxidative stress factors:500μmol/L H2O2 to stimulate oxidative stress for the appropriate time.Use the ROS probe to observe the distribution and content of ROS in cells under different conditions, and use MTT to observe cell survival.2. Detect the intracellular synthesis,intracellular co-localization and cell interactions of Hsp90αand 20S Proteasome respectively by Western-blotting,immunofluorescence and coimmunoprecipitation. 3. The recombinant plasmid pSilencerHsp90αcontaining the 21nt small interfering RNA of human Hsp90αwas subcloned, purified and identified by DNA sequence analysis. Then the recombinant plasmid pSilencerHsp90a was introduced into HepG2 cell by electroporation. After G418 selection, positive clones were isolated and cultured to form a cell line stably inhibiting Hsp90αexpression. Use this cell line as the siRNA interference group and use the non-transfected HepG2 cells as the control group. The positive clones were identified by quantitative RT-PCR and Western blotting.4. Detecting the Proteasome chymotrypsin activity after lower the expression of Hsp90α.Results1. ROS probe results:ROS is significantly increased in the HepG2 cells after oxidative stress and the distribution of ROS transfer from cytoplasm to the nucleus;.2. The result of MTT colorimetric assay cell viability:Under 500μM H2O2 concentration,the inhibition of cell proliferation was time dependent. With the time prolongation, the inhibition gradually increased.(Control group> group of oxidative stress in 4h,6h group,8h group,12h group,24h group, P<0.05);.3. The results of western-blotting detecting the expression of Hsp90αand the 20S Proteasome:oxidative stress led to increased expression of intracellular Hsp90α, but the expression of 20S Proteasome was significantly reduced;.4. The result of immunofluorescence:after oxidative stress, Hsp90αand 20S Proteasome presented same dispersed in the cytoplasm, and the point-like aggregates of 20S Proteasome in the cytoplasm are significantly increased; after 24h of oxidative stress,20S Proteasome aggregated in the cytoplasm showed a more significant increase, and massive aggregates appeared;. 5. The results of immunoprecipitation detecting the interaction between Hsp90αand 20S Proteasome:the results_displayed that the interaction between Hsp90αand 20S Proteasome significantly increased after oxidative stress, either detected by the Anti-Hsp90αco-immunoprecipitation or the Anti-20S Proteasome immunoprecipitation;.6.The results of siRNA interference:The sequence of specific siRNA was correct by sequence analysis. The level of Hsp90αmRNA and protein expression were reduced in siRNA interference group.7.Proteasome Activity Assay Results:compared with the control group, Proteasome activity was not significantly decreased (P>0.05) after H2O2 stimulating HepG2 cells 6h; However, Proteasome activity decreased significantly (P<0.05) after H2O2 stimulated HepG2 cells till 24h; Proteasome activity had a more significant decline (P<0.01) in the group of siRNA interference. However, compared with the group of application Proteasome inhibitors, Proteasome activity had no significant change (P>0.05).Conclusions1. HepG2 cells was stimulated with H2O2 in different times, cells produced different degrees of damage. Within 24h, the longer expoed to H2O2,the more of ROS gereration and the more obvious inhibition of cell proliferation were observed.2. Oxidative stress can affect the expression of Hsp90αand 20S Proteasome and the changes of distribution and interaction between the two protein, suggesting that in this process, Hsp90αplays a protective role, reduces the direct damage caused by stress, and maintains the stability of Proteasome function.3. The cell lines stably low expressing Hsp90a is successfully established, and this new cell line model establish a basis for subsequent experiments.4. After reduced the expression of Hsp90αand use the Proteasome inhibition, Proteasome activity showed a downward trend.It is a further evidence that Hsp90a has a protective role for Proteasome.