Regulatory Mechanism Of Heat Shock Protein 27 On The Degradation Of Myofibrils In Post-mortem Beef Muscles

Limited degradation of myofibrils by endogenous enzymes such as calpain and caspase is the main cause of tenderness improvement in postmortem muscles.Small heat shock proteins,as an important class of apoptosis regulators,were significantly associated with tenderness changes during post-mortem muscle tenderization,and may be a key regulator,but the mechanism is not clear.This thesis focused on the effect of heat shock protein 27 on the activity of caspase-3 and ?-calpain and the degradation of myofibrils during beef tenderization.Combined with proteome analysis,we detected that heat shock protein 27 may be involved in the regulation of tenderness in metabolic pathways.Explored the effect of oxidation on the regulation of myofibrils degradation by heat shock protein 27.The above research can provide theoretical basis for further improving the mechanism of postmortem muscle tenderization.The specific research contents and results are as follows:Effect of Heat Shock Protein 27 on Myofibrils Degradation during Beef TenderizationIn this part of the experiment,post-mortem beef muscles were treated mainly with exogenous heat shock protein 27,and the degradation of caspase-3,?-calpain and myofibrils were detected by SDS-PAGE and Western-blotting.Results showed that heat shock protein 27 inhibited the degradation of troponin-T and desmin in the first 3 days after post-mortem,until 5 days of treatment was basically the same as the untreated sample.Co-immunoprecipitation experiments found that heat shock protein 27 can bind with caspase-3 and ?-calpain.And exogenous heat shock protein 27 significantly reduced the degradation and activity of caspase-3 and ?-calpain.In summary,heat shock protein 27 may delay myofibrils degradation by inhibiting caspase-3 and ?-calpain activity.Effect of Heat Shock Protein 27 on the In Vitro Degradation of Myofibrils by Caspase and CalpainIn this part of the experiment,we investigated the degradation of myofibrils by caspase-3 and ?-calpain and the effect of heat shock protein 27 on this degradation in vitro.Results showed that caspase-3 can degrade tinin,nebulin,and troponin-T in vitro,whereas exogenous heat shock protein 27 had a significant inhibitory effect on this degradation;?-calpain accelerated the degradation of troponin-T and desmin,and the addition of heat shock protein 27 suppressed this degradation;exogenous heat shock protein 27 alone had no significant effect on the degradation of myofibrils.Therefore,heat shock protein 27 can retard degradation of myofibrils by caspase and calpain in vitro.Quantitative Analysis of Differential Proteome in Meat Treated with Heat Shock Protein 27 Blocking AgentIn this part of the experiment,the heat shock protein 27 blocking agent was added,and the TMT proteomics method was used to analyze the differential proteins in meat stored at different time with or without blocking agent.992 differential proteins were detected,and 597 differential proteins were associated with tenderness.The differential proteins in beef muscles are mainly concentrated in myofibril,contractile fiber,extracellular region,sarcomere,I band,Z disc,A band.The signal pathways involved were mainly metabolic pathways,oxidative phosphorylation,calcium signaling pathway.Effect of Oxidation on Myofibrils Degradation Regulated by Heat Shock Protein 27In this part of the experiment,the effects of oxidation on the degradation of myofibrils regulated by heat shock protein 27 were investigated by simulating the oxidative environment.The results showed that oxidation inhibited the degradation of troponin-T and had no significant effect on the degradation of desmin.Oxidation had no significant effect on the activity of caspase-3,but decreased the activity of ?-calpain.Immunofluorescence localization found that oxidation accelerated the translocation of heat shock protein 27 and cytochrome c from the cytoplasm to the cell membrane and release to the outside of the cell.Therefore,oxidation can accelerate the release of heat shock protein 27,reduce the activity of ?-calpain,effect the upstream activation process of caspase-3,thereby delaying the tenderization.