Regulatory Mechanism Of Protein Acetylation On Postmortem Development Of Pork Quality

Protein acetylation is an important post-translational modification.Our previous studies confirmed that protein acetylation regulates PM mouse muscle glycolysis and p H changes.However,the regulatory effect of protein acetylation on PM pork quality has not yet been elucidated.So,this thesis analyzed the changes of acetyl protein profile in pig LD muscles within PM 24h and the effect of antemortem stress on the postmortem acetyl protein profile.By bioinformatics analysis,key acetylproteins and biological processes that may have important regulatory effects on the formation of PM pork quality were screened out.Finally,the mechanism of antemortem stress regulates PKM1 activity by acetylation was studied,and revealing a mechanism that antemortem stress regulates PM muscle glycolysis and pork quality by protein acetylation.The main results were shown as follows:（1）Protein acetylation widely involved in the conversion of muscle to meat and regulates the formation of PM meat quality through multiple channels.Samples of porcine LD muscle at PM 0,0.5,1,4,and 24h were isolated.6 samples with normal meat quality were selected and used for high-throughput acetyl proteome after measuring p H value,meat color,drip loss and other meat quality indicators.The results showed that,a total of 595 acetylation sites distributing to 163 acetylproteins were identified in pig LD muscles.Among them,460acetylation sites distributing to 110 proteins were significantly changed in acetylation level within PM 24h.These changes were closely associated with physico-chemical changes of PM muscle.All 15 glycogenolysis and glycolysis related enzymes was identified to be acetylproteins except hexokinase and most of them were down-regulated within PM 1h.11kinds of mitochondrial proteins related to ATP production were rapidly deacetylated within PM 0.5h.4 proteins that related to Ca²⁺binding or release,hemoglobin and myoglobin that related to color stability and so on were all identified have dynamic acetylation/deacetylation changes within PM 24h.Bioinformatics analysis showed that protein acetylation may regulate the maturation and quality formation of meat by multiple pathways,including regulate muscle p H by glycolysis,regulate the tenderness of meat by cell apoptosis,muscle contraction and rigor mortis,calcium signaling pathways,and protein degradation,regulate meat flavor by IMP synthesis,and even regulate meat color stability by modifying myoglobin.（2）Protein acetylation mediates the effect of antemortem stress on PM meat quality development.18 pigs were randomly assigned to 3 treatments:1Control,pigs were delivered to abattoir 12h prior to slaughter;2Transport,pigs were transported for 1h to be stressed before slaughter;3Rest,pigs were transported for 1h and then rested for 2h before slaughter.LD muscles of these 3 group pigs were collected and pork quality were determined and then acetyl proteomic analysis in muscles were conducted.The results showed that antemortem stress and resting significantly affected the PM pork quality.Meanwhile,it has a complex impact on the acetyl protein profile in porcine muscle within PM 24h.Totally 771acetylpeptides containing 681 lysine acetylation sites mapping to 176 acetylproteins were identified in PM porcine muscle.Preslaughter handling may regulate PM muscle p H changes and overall meat quality by regulating acetylation levels and enzyme activities of a variety of glycogen degrading/glycolytic enzymes;regulate rigor mortis by regulating the acetylation levels of ATP production,muscle contraction and calcium signaling-related proteins;and respond to premortem stress and cell apoptosis by regulating the acetylation level of heat shock proteins and permeability transition pore complex proteins.（3）Premortem stress regulates PKM1 activity in PM muscle by acetylation.The above analysis of the acetyl proteome shows that premortem stress regulate the acetylation level and activity of PKM1 in the LD muscle of pigs.In order to confirm its regulatory effect and explore the mechanism,firstly,mice were Intraperitoneally injected with epinephrine to simulate stress and cell acetylation/deacetylation inhibitor tests confirmed the regulation of stress on PKM1acetylation and revealed that acetylation inhibits PKM1 activity while deacetylation up-regulates its activity.Secondly,through gene cloning and site-directed mutagenesis,we screened and determined that K141 is the key acetylation site that regulates the activity of PKM1.Finally,by IP/Co IP,gene knockout and other technologies,PCAF and Sirt1 were screened and verified to be the upstream acetyltransferase and deacetylase that catalyze the acetylation and deacetylation of PKM1 and regulate its activity,thereby preliminarily revealing a mechanism that premortem stress regulates PM muscle glycolysis and meat quality by regulating the acetylation level and activity of PKM1.In summary,protein acetylation widely involved in the conversion of muscle to meat.It regulates the formation of PM pork quality by regulating glycogen degradation/glycolysis,apoptosis,calcium signal transduction,protein hydrolysis,purine metabolism,muscle contraction and rigor mortis development.Protein acetylation responds to the effects of premortem stress on PM pork quality,premortem stress regulates PM pork quality by reducing the acetylation level of PKM1,increasing its activity and muscle glycolysis.