Regulation Of Glycine TRNA Synthetase On Milk Protein Synthesis In Dairy Cow Mammary Epithelial Cells And Its Mechanism

The mechanism of milk protein synthesis is one of the important basic question in life science. In recent years, obvious progress on this research has been made. At present, we find that the transcription process of milk protein synthesis is regulated by prolactin via the JAK/STAT5 signaling pathways and the translation process of it was regulated by amino acids via the mTOR/S6K1 signaling pathways. But, which are other important signaling molecules involved in transcription and translation of milk protein synthesis in mammary epithelial cells? What molecule are the "molecular sensors" of amino acids and other nutrients and how the nucleus and cytoplasm reply the amino acid signal are still the important scientific question that is needed to be solved in lactation biology nowadays. In this study, a comprehensive study of glycyl-tRNA synthetase (GlyRS) regulation on the synthesis of β-casein (CSN2) as a new signal molecule to reply the methionine signal in dairy cow mammary epithelial cells was done and its mechanism were established. Both the basic theory of milk protein synthesis regulated by amino acids and the new horizon of understanding the signaling pathway network of milk protein synthesis were 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 DCMEC model was established by adding methionine (0.6mmol/L) in the culture medium. Real-time quantitative (qRT-PCR), WB and IF were used to detect the expression of CSN2 and GlyRS and the subcellular localization of GlyRS. The results showed that the expression of CSN2 and GlyRS were significantly increased (p<0.01) by methionine, and nuclear localization of GlyRS was also significantly increased (p<0.01) by methionine. It suggests that GlyRS may be an important regulatory factor in the process of CSN2 expression.Overexpression and gene silencing of GlyRS were completed. The expression of mTOR, p-mTOR, Stat5a, p-Stat5a, S6K1, p-S6K1,4EBP1, p-4EBP1 and β-casein were detected by qRT-PCR and WB and the viability, cell number and proliferation rate of cells were detected by CASY cell viability analyzer and Flow Cytometry. The results showed that in GlyRS overexpression cells, the expressions of p-mTOR, p-Stat5a, p-S6K1, p-4EBP1 and CSN2 and the viability, numbers and proliferation rate of cells were significantly increased (p<0.01),whereas in gene silencing cells, these were significantly decreased (p<0.01) but the expression of mTOR, Stat5a, S6K1 and 4EBP1 had no significant change (p>0.05) after the two treatment. After methionine stimulation (0.6mmol/L of methionine was added in cell culture medium), the expression of mTOR, p-mTOR, Stat5a, p-Stat5a, S6K1, p-S6K1,4EBP1, p-4EBP1 and CNS2 were significantly increased (p<0.01) in both normal cells and GlyRS overexpression cells. But in GlyRS gene silencing cells, methionine could not enhance the expression of p-mTOR, p-Stat5a, p-S6K1, p-4EBP1 and CNS2 and the viability, numbers and proliferation rate of cells. This result reveals that GlyRS is a positive regulator in milk protein synthesis signaling pathway, methionine controls milk protein synthesis and cell proliferation via GlyRS.His-GlyRS and GlyRS-His eukaryotic expression vector were constructed and transfected into DCMECs and the cytoplasm and nucleus proteins were extracted. Then the cytoplasm and nuclear localization and molecular shear of GlyRS were identified by WB. The result reveals that the GlyRS (GlyRS molecular weight was about 83 kDa) has three molecular forms:80-100 kDa in cytoplasm,10-15 kDa and 60-80 kDa in nucleus. The nuclear localization signal (NLS) of GlyRS was predicted through the bioinformatics analysis. The result showed that they had the sequence of RKLK (79-82) and KARKR (99-103), they are accordant with the follow classical general form of NLS sequence:K-(K/R)-X-(K/R) and R/K-X (10-12)-R-R-K-K, and predicted as a possible NLS of GlyRS. The basic amino acid (R and K) in the predicted NLS were mutated into alanine bu using amino acid point mutation technique and the NLS of GlyRS was verified by IF. The result showed that GlyRS could not enter the nucleus after any a basic amino acid in the sequence of RKLK (79-82) and KARKR (99-103) was mutated. These results suggest that there is a molecular splice process when GlyRS was guided into the nucleus and the splice site was in the N-terminal. The sequence of 79-82 and 99-103 were the NLS of GlyRS.113 proteins interacting with GlyRS in the cytoplasm and 41113 proteins interacting with GlyRS in the nucleus were identified by co-immunoprecipitation (Co-IP), phosphoprotein mass spectrometry (MALDI-TOF/TOF MS) and laser resonance energy transfer (FRET). In the nucleus, the phosphorylated sites of threonine 544 and serine704 of GlyRS were identified by MALDI-TOF/TOF MS. In the cytoplasm proteins interacting with GlyRS, eukaryotic translation elongation factor 1A1 (eEFlA1) and eukaryotic translation initiation factor 2D (eIF2D) were selected for further study. In nucleus proteins interacting with GlyRS, nuclear transcription factor NF kappa B1 (NFκB1) was selected for further study. The influences of methionine on the expression of eEFlA1, eIF2D andNFκB1 were identified by WB. The results showed that after methionine stimulation, both the expression of eIF2D and NFκB1 were significantly increased (p<0.01), whereas the expression of eEFlA1 had no significant change (p>0.05). The influences of methionine on the interaction between GlyRS and eEFlA1, eIF2D and NFκB1 were detected by quantitative Co-IP and Laser Confocal colocalization. The results showed that compared with normal cultured cells, the binding of GlyRS with eEFlA1 was significantly reduced (p<0.01) and the binding of GlyRS with eIF2D and NFκB1 were significantly increased (p<0.01) after methionine stimulation. The expression of eEFlA1l, eIF2D and NFκB1 were detected by WB after GlyRS overexpression and gene silencing. The results showed that the expression of eEFlA had no significant change (p>0.05) after GlyRS over expression and gene silencing. The expression of eIF2D and NFκB1 was significantly increased (p<0.01) and significantly decreased (p<0.01) when GlyRS was overexpressed and inhibited, respectively. The expression of CSN2 was detected by WB after NFκB1 were overexpressed. The results showed that the expression of CSN2 was significantly increased (p<0.01) when NFκB1 was overexpressed. These results reveal that GlyRS regulates milk protein synthesis via eIF2D in cytoplasm and NFκB1 in nucleus. These results suggest that in the cytoplasm, GlyRS interact with eIF2D and positively regulates the expression of CSN2 at translational level; in the nucleus, GlyRS interact with NFκB1 and positively regulates the expression of CSN2 at transcription level.According to the result of MALDI-TOF/TOFMS in the nucleus, the phosphorylated site of threonine 544 (T544) and serine704 (S704) were mutated into alanine (A) respectively. The nuclear localization of GlyRS after the phosphorylated site were mutated was detected by IF. The result showed that GlyRS could not enter the nucleus after any phosphorylated site was mutated. This result suggests that, the mechanism of the nuclear localization of GlyRS may be as follows: the T544 and S704 of GlyRS were phosphorylated by stimuli such as methionine signal, and then a part of phosphorylated GlyRS enters into the nucleus guided by NLS.Whether GlyRS and/or NFkBI can bind with the casein promoter was detected by chromatin immunoprecipitation (ChIP). The result showed that GlyRS can not bind with CSN2 promoter, whereas NFκB1 binds with casein promoter at-1065--1065 bases in the upstream of CSN2 transcription start site. These results reveal that, GlyRS positively regulate the CSN2 synthesis by interacting with NFκB1, stimulates it to bind to CSN2 promoter to increase the transcription.To sum up these experimental results, GlyRS was an important signal molecule in the process of milk protein synthesis in DCMECs. The mechanism of GlyRS is as follows:GlyRS combines with eIF2D in the cytoplasm and positively regulates the expression of CSN2 at translational level, GlyRS also phosphorylated by stimuli such as methionine signal in the cytoplasm and enters into the nucleus guided by NLS, and these GlyRS were spliced in the nucleus. Then, the C-terminal part of the spliced GlyRS binds NFκB1 in the nucleus and promotes the combination of NFκB1 with CSN2 promoter, and finally raised the expression of CSN2 at transcription level.