Mechanisms In The Regulation Of Protein Synthesis Of Transcription Factor Junb In The Myoblasts

Skeletal muscle is the body’s largest tissue, which constitutes about 40-50% of the body’s mass. At birth, the number of muscle fibers have been identified, therefore postnatal growth of porcine skeletal muscle was dependent on the positive balance of muscle protein, when protein synthesis is greater than protein degradation, skeletal muscle growth. Research shows that anabolic and catabolic processes are mechanistically strongly interdependent in skeletal muscle.Therefore and on a practical point of view, any manipulation of muscle mass in animal wasting diseases should be more efficient if we can target the components of signalling pathways that are at the crossroads of protein synthesis and breakdown. Studies have shown that the transcription factor Jun B as a crossroads of protein synthesis and breakdown can promote protein synthesis and inhibit protein degradation. The mechanisms that Jun B inhibits protein degradation have been elucidated, but Jun B stimulates protein synthesis independently of the Akt/m TOR pathway, the specific mechanism is unclear. Akt/m TOR pathway as a core path for regulating protein synthesis, mainly act by the regulation of the eukaryotic initiation factors(e IFs). Therefore, the aim of our trial is to eclucidate the mechanism that transcription factor Jun B regulates protein synthesis in both cellular and molecular levels by staying the relationship between Jun B and translation initiation /elongation.The contents of the research were mainly composed of two parts: the first part was about developing SUn SET as a method can not only detect the protein synthesis rate but also be able to study protein translation initiation and elongation; the second part was the mechanism reaserch, the use of overexpression and interference Jun B plasmid, transfected C2C12 myoblasts, detected the influence on the rate of protein synthesis by SUn SET, and then detecting translation initiation, translation elongation, ribosomes generation to clarify the mechanism of regulation of protein synthesis by Jun B.The first part:application of a nonradioactive method of measuring protein synthesis and studying protein translation. The main results are as follows:1. It was determined incubated C2C12 myotubes with 1 μg/m L puromycin 30 min best. In the concentration range of 0~5 μg/m L, with the puromycin concentration increasing, the corresponding protein increased, but when the concentration reached 10 μg/m L protein has decreased.2. SUn SET method could be used for the detection changes in the rate of protein synthesis caused by translation initiation / translation elongation changes.Respectively, promote and reduced m TOR, S6K1 protein phosphorylation after insulin and rapamycin treatment C2C12 myotubes, indicating a change of translation initiation, corresponding SUn SET were detected to increase and decrease the rate of protein synthesis; After C2C12 treated by CCCP 2 min, m TOR, S6K1, e IF2α phosphorylation did not change, only e EF2 protein phosphorylation increased, indicating no change in translation initiation, but translation elongation was inhibited, SUn SET detected corresponding to the protein synthesis rate reduction; After PBS and amino acid starvation, m TOR, S6K1, e IF2α, e EF2 protein phosphorylation have changed, indicating that translation initiation and translation elongation changes have occurred, SUn SET detected changes in the rate of protein synthesis is consistent.3. SUn SET method could be used for the detection of C2C12 myoblasts, C3H10T1/2 cellular protein synthesis rate. C3H10T1/2cells were treated by PBS, rapamycin, amino acid starvation, adding amino acids after amino acid starvation, using SUn SET method for detecting protein synthesis rate, the result was consistent with the previous results obtained in C2C12 cells.The second part: mechanisms in transcription factor Jun B regulating of muscle protein synthesis in C2C12. The main results are as follows:1. Jun B could regulate protein synthesis. Protein synthesis increased by overexpression, and reduced by interference Jun B in C2C12 myoblasts.2. Jun B didn’t affect the m TOR signaling pathway and e IF2α phosphorylation, and the 18 S and 28 S ribosomal r RNA production, but affected the e EF2 protein phosphorylation and myostatin gene expression. Overexpression and interference Jun B in C2C12 myoblasts, m TOR, S6K1 phosphorylation did not change, e IF2α phosphorylation did not change, 18 S and 28 S ribosomal r RNA generation didn’t change, but overexpression of Jun B detected e EF2 protein phosphorylation reduced and interference of Jun B detected e EF2 protein phosphorylation increased, consistent to changes in protein synthesis. Similarly overexpression of Jun B detected myostatin gene downregulation, interference of Jun B detected myostatin gene upregulation. In summary, the conclusions of this study are:1. SUn SET nonradioactive method could be used to detect protein synthesis caused by translation initiation / elongation rate changes, what’s more, together with the traditional characterization of protein translation initiation / lengthening of m TOR, S6K1, e IF2a, changes e EF2 protein activity, could be used to study protein translation initiation and elongation.2. myostatin-Smad signaling pathway inhibites protein synthesis by inhibiting protein translation elongation;3. Jun B regulated of protein synthesis independent of m TOR signaling pathways, on one hand by regulating e EF2 protein activity to regulate translation elongation, on the other hand by regulating myostatin signaling pathways to regulate protein synthesis rate.