| The objective of this study was to determine the functional role of seryl-tRNA synthetase(SARS)in methionine(Met)regulating casein synthesis,and deeply evaluate its molecular and cellular mechanisms during this process in bovine mammary gland epithelial cells(BMECs).This study totally contained four parts.In Part 1,RNA-seq-based transcriptomic and iTRAQ-based proteomic techniques were utilized to investigate and integrate the differences of molecular pathways and biological processes[especially focus on aminoacyl-tRNA synthetase(AARS)-related processes]in the mammary tissues(MG)from dairy cows fed different quality forages.In Part 2,the expression patterns of SARS in response to Met in BMECs were determined.Part 3 was conducted to determine the effects of SARS,Met supply and their interaction on casein synthesis in BMECs.Part 4 was conducted to determine the effects of SARS-mediated Met on protein turnover,casein synthesis and the related regulatory signaling pathways in BMECs.Knowledge of the SARS mediating Met to regulate casein synthesis may provide new insights into milk protein synthesis in bovine mammary gland,and achieve the final aim to improve milk protein production in dairy cows.1 Effects of different quality forages on the mammary transcriptomic and proteomic profiles in dairy cowsThis study was to investigate the differences of molecular pathways and biological processes in the MG tissues collected from lactating cows fed corn stover(CS,low-quality)vs.alfalfa hay(AH,high-quality).Here we focused on the differentially expressed AARS profiles from the MG of the CS-and AH-fed dairy cows.Twelve multiparous Holstein dairy cows were blocked and were randomly assigned to CS-(n=6)and AH-based diets(n=6),respectively.Diets were completely the same except different forage sources(on a DM basis):(1)23%AH and 7%Chinese wild rye hay,(2)30%CS.The experiment was conducted over 14-weeks,and the bovine MG tissues were collected at the end of the experiment.The RNA-seq transcriptomic and iTRAQproteomic techniques were utilized to explore the differentially expressed transcripts(DET)and proteins(DEP)profiles of bovine MG from dairy cows fed CS vs.AH-based diets.A total of 1,631 DET(1,046 up-regulated and 585 down-regulated)and 346 DEP(138 increased and 208 decreased)were detected in the MG between the CS-and AH-fed animals.Expression patterns of 33 DEP(18 increased and 15 decreased)were consistent with the expression of their mRNAs,and 7 DEP were inconsistent with the expression of their mRNAs.Compared with the MG of AH-fed cows,the marked expression changes found in the MG of CS group were for genes involved in reduced mammary growth/development,less oxidative phosphorylation,less aminoacyl-tRNA biosynthesis(eg.SARS),enhanced lipid uptake/metabolism,more active fatty acid beta-oxidation,less amino acid/protein transport,reduced protein translation,more proteasome-and ubiquitin-mediated protein degradation,and more protein disassembly-related enzymes.Results indicated that the lower milk production in the CS-fed dairy cows compared with the AH-fed cows was associated with a network of mammary gene expression changes,importantly,the prime factors include decreased energy metabolism,attenuated protein synthesis,enhanced protein degradation,and the lower mammary cell growth.Besides,the AARS(including SARS,KARS,YARS,TARS etc.)were found significantly up-regulated in AH group compared to CS group,which suggested that AARS may play a crucial role in milk protein synthesis of bovine mammary glands.The present study provides insights into the effects of the varying quality of forages on mammary metabolisms,which can help the improvement of strategies in feeding dairy cows with CS-based diet.2 Study of SARS response patterns to Met in BMECs2.1 Effects of Met on SARS expression in BMECsThe object of this study was to investigate the expression patterns of SARS in response to Met in BMECs.The BMECs were cultured in custom medium to 90%confluency,serum-starved overnight to avoid cell differentiation differences before all treatments.First we conducted the subcellular location analysis of SARS within BMECs by Western blot and immunofluorescent assay;to study the effects of Met on the gene expression of AARS in BMECs,cells were treated with Met-deficient and Met-sufficient medium for 24 h,and RT-PCR method was applied to determine the mRNA abundance of AARS;to explore the effects of Met on the protein abundance of SARS,cells were cultured in the above Met concentrations(0,0.1,0.2,0.4,0.6,0.8,1.6 and 2.4 mM)for 24 h,and proteins were collected for Western blot assay;to determine the effects of Met-deprivation and Met-supplement treatment time on the protein abundance of SARS,cells were treated with Met-deprivation(-Met)followed by Met re-supplement(+Met)treatment for 10 min,0.5 h,1 h,6 h and 12 h;in a different set of plates,the treatment order(Met deprivation and Met supplement treatments)was inter-changed.Subsequently,proteins were immediately collected from BMECs and the protein abundance of SARS was analyzed by Western blot method.The above results showed SARS mainly existed in cytoplasm and a small amount was in mitochondria;the mRNA abundance of SARS,MARS and CARS was significantly enhanced by the supplement of Met(P<0.05);the protein abundance of SARS reached the highest when Met was at 0.6 mM/L in BMECs;at 6 h of Met re-supplement and re-deprivation,the protein abundance of SARS was significantly changed compared to those within the other time points(P<0.05).In summary,Met had a remarkable effect on the protein expression of SARS,and the levels of these expression patterns in response to Met were related to Met concentrations and treatment time.2.2 Effects of SARS-mediated Met on casein synthesis and cell status in BMECsThe object of this study was to determine the effects of SARS-mediated Met on casein synthesis,cell viability and cell cycle in BMECs.Cells were cultured in custom medium to 90%confluency,serum-starved overnight to avoid the cell differentiation differences before all treatments.First,we establish the SARS-knockdown model in response to Met in BMECs by using RNAi methods.Subsequently,cells were treated with Met-deprived and 0.6 mM/L Met-sufficient medium combined with SARS knock-down treatment,then casein expression,cell viability and cell cycle were measured by Western blot method,CCK-8 assay and flow cytometry assay,respectively.Results showed Met supplement significantly increased the protein abundance of SARS inBMECs(P<0.05)compared to Met-deficient group;the supplement of Met also significantly enhanced the protein abundance of ?-casein(P<0.05)and cell viability(P<0.05),promoted cell cycle progression(P<0.05)and reduced cell cycle arrest(P<0.05)in BMECs;within Met treatment,SARS inhibition significantly reduced cell viability(P<0.05),decreased the mammary cell number in S phase(P<0.05)but increased the cell number in G1 phase(P<0.05).These suggested Met could promote casein synthesis via increasing cell viability and cell cycle progression;SARS further strengthened Met-stimulated cell proliferation,subsequently resulting in higher casein production.Overall,this study revealed that SARS could act as a signaling molecule,and promoted casein synthesis through cell proliferation in response to Met in BMECs.3 Effects of SARS,Met transporter ASCT2 and their interaction on casein synthesis3.1 Study of Met transporters in BMECsThe object of this study was to determine the relative specific amino acid(AA)transporter for Met in BMECs.To avoid the cell differentiation differences before all treatments,cells were cultured in custom medium to 90%confluency,serum-starved overnight.To examine the effects of Met on the gene expression of AA transporters,cells were treated with Met-deprived and 0.6 mM/L Met-sufficient medium for 6 h,and the mRNA and protein abundance of AA transporters were determined by RT-PCR and Western blot assay,respectively;to explore the effects of GPNA on protein expression of ASCT2,cells were treated by culture medium containing different concentrations of GPNA(0,5,50,500 and 1000 ?M/L)for 24 h,proteins were immediately collected from BMECs and the protein abundance of ASCT2 was analyzed by Western blot method;to further examine the effects of Met and GPNA on the protein expression on ASCT2 and P-casein,cells were treated with Met-deprived and 0.6 mM/L Met-sufficient medium combined with 500 ?M/L GPNA for 6 h,and the protein abundance of ASCT2 and ?-casein was measured by Western blot assay.Results showed Met exerted the most remarkable effect on A A transporter AS CT2 in BMECs(P<0.05);the protein abundance of ASCT2 was significantly decreased by GPNA(P<0.05),and when GPNA was at 500 ?M/L,the protein abundance of ASCT2 reached the lowest;under Met supplement,the protein abundance of ASCT2 was significantly increased(P<0.05);500?M GPNA significantly reduced the protein abundance of ?-casein(P<0.05).These suggested that Met was mainly transported by ASCT2 in BMECs and it could promote casein synthesis through enhancing Met uptake and absorption into BMECs.3.2 Effects of SARS,ASCT2 and their interaction on casein synthesisThe object of this study was to determine the effects of SARS,Met supply and their interaction on casein synthesis.The BMECs were cultured in custom medium to 90%confluency,serum-starved overnight to avoid the cell differentiation differences before all treatments.To investigate the effects of Met and ASCT2 on the protein expression of SARS,cells were treated with Met-deprived and 0.6 mM/L Met-sufficient medium combined with 500 ?M/L GPNA for 6 h,and the protein abundance of SARS was measured by Western blot assay;to determine the effects of SARS-mediated Met on the protein expression of ASCT2 in BMECs,cells were treated with Met-deprived and 0.6 mM/L Met-sufficient medium combined with SARS knockdown treatment,and the protein abundance of ASCT2 was measured by Western blot assay;to investigate the effects of Met on the interaction between SARS and ASCT2 in BMECs,cells were treated with Met-deprived and 0.6 mM/L Met-sufficient medium for 6 h,and the colocalization between SARS and ASCT2 in BMECs was determined by the immune-fluorescent assay.Results showed under Met deprivation,by using 500 ?M GPNA to inhibit the expression of ASCT2,the protein abundance of SARS was significantly decreased(P<0.05)in BMECs;SARS inhibition significantly reduced the protein abundance of ASCT2 especially when Met was deficient(P<0.05)in BMECs;Met supplement significantly enhanced the colocalization between SARS and ASCT2 in BMECs(P<0.05).Results revealed that there existed a close relationship between SARS and ASCT2 proteins in BMECs,and Met could promote this interaction.In summary,SARS could act as a signaling molecule,and promoted Met-stimulated caseinsynthesis through more active interaction with ASCT2 and the subsequent higher Met uptake into BMECs.4 Study of the utilization mechanism of SARS-mediated Met regulating casein synthesis in BMECs4.1 Effects of SARS-mediated Met on protein turnover and casein synthesis in BMECsThis study was to explore the effects of SARS-mediated Met on protein turnover and casein synthesis in BMECs.To avoid the cell differentiation differences before all treatments,BMECs were cultured in custom medium to 90%confluency,serum-starved overnight.To measure protein turnover,cells were treated with Met-deprived and 0.6 mM/L Met-sufficient medium combined with SARS knockdown treatment,the isotope tracing technique(L-[ring-3H5]Phe)was applied to measure cellular protein turnover of BMECs after 2 days of treatments;to study the effects of SARS-mediated Met in BMECs,the mRNA and protein abundance of 4 caseins was measured by RT-PCR and Western blot methods,respectively.Results showed that Met supplement significantly increased total protein synthesis ratio(P<0.05)but decreased protein degradation ratio(P<0.05);Met supplement also significantly enhanced the mRNA and protein abundance of 4 caseins(P<0.05);SARS further strengthened Met-stimulated total protein synthesis,casein synthesis,but prevented Met-inhibited protein degradation(P<0.05).These results indicated that SARS could act as a crucial signaling molecule,and it could enhance Met-stimulated protein turnover and casein synthesis.4.2 Effects of SARS-mediated Met on related signaling pathways in BMECsThis study was to investigate the effects of SARS-mediated Met on related signaling pathways in BMECs.To avoid the cell differentiation differences before all treatments,BMECs were cultured in complete medium to 90%confluency,serum-starved overnight.The treatments were completely the same as those in the experiment of 2.2.After 6 h treatments,RT-PCR and Western blot methods were used to determine the effects of SARS-mediated Met on the gene and protein expression of related genes/proteins involved in signaling pathways(mTOR,JAK2-STAT5 and GCN2)in BMECs.Results showed Met supplement significantly promoted the mRNA and protein abundance of mTOR,S6K1,4EBP1 and their phosphorylated proteins(P<0.05),and JAK2,STAT5 and their phosphorylated proteins(P<0.05),but reduced the mRNA and protein abundance of GCN2,ATF4 and P-eIF2a(P<0.05);SARS further strengthened Met-stimulated the abundances of related genes/proteins above involved in the mTOR and JAK2-STAT5 signaling pathways(P<0.05),but prevented Met-inhibited abundances of GCN2,ATF4 and P-eIF2a(P<0.05).These suggested SARS could act as a crucial signaling molecule,and it could promote Met-stimulated casein synthesis by activating JAK2-STAT5 and mTOR signaling pathway and inhibiting GCN2 signaling pathway in BMECs.In summary,SARS acts as a crucial signaling molecule,and could sense Met signals,and it may promote Met-stimulated casein synthesis through the following 3 regulatory mechanisms:1)increase of the mammary cell population via cell viability and cell cycle;2)enhance of its interaction with Met transporter ASCT2 to increase mammary cellular Met availability for milk protein synthesis;and 3)activation of mTOR and JAK2-STAT5 signaling pathways but inhibition of GCN2 signaling pathway. |
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