GlyRS Regulates The Milk Protein Synthesis By Interacting With ELP4 In Dairy Cow Mammary Epithelial Cells

The mechanism of milk protein synthesis is one of the important basic theories in life science. In recent years, obvious progress on this research has been made. At present, we find that the transcription progress of milk protein synthesis is regulated by prolactin via the JAK2/STAT5 signaling pathways and amino acids regulates the translation process of milk protein process via the mTOR/S6K1 signal pathways. The early stage of the laboratory experiment found that Gly RS positively regulates the expression of CSN2 in the nucleus. However,the mechaniam is unclear. To clarify this mechaniam will be helpful to reveal the detailed molecular mechanism of milk protein synthesis. In this study, we make a comprehensive study and find that Gly RS regulates the milk protein synthesis by interacting with ELP4 in dairy cow mammary epithelial cells under the stimuli such as methionine signal. Both the basic theory of milk protein synthesis regulated by ELP4 and Gly RS and the new horizon of understanding the signal pathway network of milk protein synthesis are provided by this study.The dairy cow mammary epithelial cells(DCMECs) were cultured and purified by tissue culture method. The purity and function of milk protein synthesis of cells were identified by testing the expression of keratin 18(CK18) and CSN2 by using western blotting(WB) and immunofluorescence(IF). And the DCMECs model was established by adding methionine(0.6mmol/L) in the culture medium. WB was used to detect the expression of ELP4. The results showed that the expression of ELP4 were significantly increased(p<0.01) by methionine. By studying the influence of ELP4 overexpression, WB was used to detect the expression of CSN2 in the culture medium. The results showed that in ELP4 overexpression cells, the expressions of CSN2 were significantly increased(p<0.01). It suggests that ELP4 may be an important regulatory factor in the process of CSN2 expression.The interaction between ELP4 and Gly RS were identified by co-immunoprecipitation(Co-IP) in the DCMECs. Eukaryotic expression vector ELP4-EGFP and Gly RS-RFP were constructed and transfected into DCMECs in this study. The influences of methionine on the interaction between ELP4 and Gly RS were detected by laser resonance energy transfer(FRET), quantitative Co-IP and laser confocal colocalization. The results showed that compared with normal cultured cells, the binding of ELP4 with Gly RS was significantly increased(p<0.01) after methionine stimulation. By the experiments of overexpression and inhibition of GlyRS, we examined the expression and synthesis of ELP4 and CSN2 by WB. The results showed that the expression of ELP4 and CSN2 were significantly increased(p<0.01) after Gly RS over expression and significantly decreased(p<0.01) when Gly RS was inhibited. These results reveal that ELP4 combines with Gly RS and positively regulate the CSN2 synthesis under the stimuli such as methionine signal. By the experiments of overexpression and inhibition of GlyRS, we examined the binding between NFκB1 with the promoter of ELP4. The results showed that the binding between NFκB1 with the promoter of ELP4 was significantly increased(p<0.01) after Gly RS over expression and significantly decreased(p<0.01) when Gly RS was inhibited. These results reveal that, Gly RS positively regulate the ELP4 synthesis, stimulates p-NFκB1 to bind to ELP4 promoter to increase the transcription.