Doctoral thesis (Dissertations and theses)
Effects of protein phosphorylation on postmortem glycolysis and glycolytic enzymes in ovine muscle
Chen, Li
2019
 

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Keywords :
protein phosphorylation; glycolysis; glycolytic enzyme; glycolytic rate; phosphoproteomic; glycogen phosphorylase; phosphofructokinase; pyruvate kinase
Abstract :
[en] Glycolysis, the core metabolic pathway in muscle, is highly important to ultimate meat quality. Protein phosphorylation is one of the most frequent post-translational modifications that alter the properties of proteins by changing their structural conformation and regulating the function of these proteins in muscle. Postmortem glycolysis is a sequence of enzymatic reactions that are determined by the activities of glycolytic enzymes, while most of the glycolytic enzymes were reported as phosphoproteins in previous studies. In recent years, some proteins with different phosphorylation levels clustered in the glycolysis pathway were identified based on muscle samples of different tenderness, colour stability or water-holding capacity. The systematic research of protein phosphorylation on postmortem glycolysis was less of a concern. The objective of this research is to confirm the involvement of protein phosphorylation on postmortem glycolysis and the possible regulatory mechanism from the perspective of enzyme activity. To analyse the relationship between protein phosphorylation and postmortem glycolysis, 60 experimental sheep with the same breed, sex, environment and slaughter method were used. Based on muscle pH values at 6 h (pH6h) postmortem, 18 sheep carcasses were selected and divided into three groups (six sheep in each group): Fast glycolytic rate group (pH6h < 5.75), Moderate glycolytic rate group (5.75 < pH6h < 6.20) and Slow glycolytic rate group (pH6h > 6.20). Phosphorylation level and glycolytic related indicators such as pH value, glycogen content and lactic acid were measured and analysed. The global phosphorylation level (phosphoproteins/total proteins by electrophoresis gel with different dyes) of sarcoplasmic protein increased early postmortem and then decreased afterwards in ovine muscle. Protein bands significantly different in phosphorylation levels were identified as glycometabolism related enzymes. The phosphorylation levels of glycogen phosphorylase and pyruvate kinase may be one possible reason for the difference of glycolytic rate at 0.5 h postmortem. The glycolytic rate attributes were negatively correlated with 4 bands that were probably phosphofructokinase, enolase and adenylate kinase isoenzyme. The results revealed that the phosphorylation of sarcoplasmic proteins was related to muscle pH decline at early postmortem. One dimensional electrophoresis is not sufficient to clarify the specific phosphoproteins and phosphosites; quantitative proteomic tools (isobaric Tags for Relative and Absolute Quantitation, iTRAQ) and bioinformatics analysis were used to further understand the relationship. In total, 24 phosphoproteins clustered in glycolysis and muscle contraction were identified to be glycolytic rate related proteins; from which, 32 phosphopeptides were determined to have a significant difference among the fast, moderate and slow glycolytic rate groups. In addition, phosphorylation of pyruvate kinase at Thr157 was negatively correlated with the glycolytic rate. The current results illustrated that these glycolytic rate related phosphoproteins were related to early postmortem glycolysis, and four possible regulatory pathways were speculated: 1) Enzyme activities regulation pathway, 2) μ-calpain degradation pathway, 3) Energy metabolism pathway, 4) Protein kinase activity regulation pathway. Further, it was concluded that protein phosphorylation at early postmortem may indirectly affect the glycolysis pathway through the regulation of proteins involved in glycolysis and muscle contraction. Based on the research above, the validation of protein phosphorylation on postmortem glycolysis is necessary. In addition, the regulatory mechanism from the perspective of enzyme activity should be considered. Six sheep with the same breed, sex, environment and slaughter method were selected. Ovine muscle was treated with a kinase inhibitor, dimethyl sulfoxide, or a phosphatase inhibitor. Protein phosphorylation level revealed that the kinase inhibitor and phosphatase inhibitor were effective at modulating the level of protein phosphorylation. The pH value and lactate content revealed that a high phosphorylation level was the reason for the fast glycolysis. Protein phosphorylation was positively correlated with the activity of these rate-limiting enzymes (glycogen phosphorylase, pyruvate kinase and phosphofructokinase). Protein phosphorylation plays a role in postmortem glycolysis through the regulation of enzyme activity in ovine muscle. In conclusion, the results with different glycolytic rate groups have systematically exposed novel information of protein phosphorylation on postmortem glycolysis through the use of quantitative proteomic analysis. Protein phosphorylation is related to postmortem glycolysis and glycolytic enzymes, and the phosphorylation level of glycolytic enzymes may influence its activities, further regulating postmortem glycolysis.
Disciplines :
Food science
Author, co-author :
Chen, Li ;  Université de Liège - ULiège > Doct. sc. agro. & ingé. biol. (Paysage)
Language :
English
Title :
Effects of protein phosphorylation on postmortem glycolysis and glycolytic enzymes in ovine muscle
Defense date :
11 June 2019
Number of pages :
133
Institution :
ULiège - Université de Liège
Degree :
DOCTEUR EN SCIENCES AGRONOMIQUES ET INGENIERIE BIOLOGIQUE
Promotor :
Everaert, Nadia ;  Université de Liège - ULiège > Département GxABT
Zhang, Dequan
President :
Jacques, Philippe ;  Université de Liège - ULiège > Département GxABT > Microbial, food and biobased technologies
Jury member :
Willems, Luc  ;  Université de Liège - ULiège > Département GxABT
Clinquart, Antoine ;  Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH) > FARAH: Productions animales durables
DE SMET, Stefaan
Funders :
National Natural Science Foundation of China (31501411)
Available on ORBi :
since 05 June 2019

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