Regulation Of Sestrin2 Gene On Amino Acid Mediated Casein Synthesis In Cow Mammary Epithelial Cells And Its Mechanism

In cow mammary epithelial cells(CMEC),Amino acid(AA)is important regulators of casein synthesis.Mammalian target of rapamycin complex 1(mTORCl)pathway is the main signaling pathway that mediates AA-induced casein synthesis,but the mechanism of mTORCl upstream pathways regulated by AA is not yet clear.The purpose of this study is to determine the regulation of Sestrin2(SESN2)in AA mediated casein synthesis,and clarify the mechanism of casein synthesis.SESN2 negatively regulates AA-mediated casein synthesisCMEC were treated with AA absence,only essential AA presences and AA presences.The result showed that cell proliferation,casein expression,and activation of mTORCl pathway were increased,but SESN2 expression was decreased in response to AA presences;CMEC were treated with SESN2 overexpression or inhibition.The result showed that cell proliferation,casein expression,and activation of mTORC1 pathway were decreased in response to SESN2 overexpression.Furthermore,the increase in cell proliferation,casein expression,and activation of the mTORCl pathway in response to AA supply was inhibited by overexpressing SESN2,and vice versa.This result suggested that SESN2 is an important inhibitor of mTORCl in CMEC blocking AA-mediated casein synthesis.SESN2 negatively regulates AA-mediated casein synthesis via SH3BP4Interaction proteins of SESN2 were identified by Co-immunoprecipitation(Co-IP)and QE-MS.The result showed that 108 proteins were identified and some were the regulators of mTORCl upstream pathways such as LAMTOR4,SH3BP4 and TSC1.SH3-domain binding protein 4(SH3BP4)was selected for further study;To investigate the affect of SH3BP4 on mTORCl pathway regulated by SESN2,CMEC were treated with SH3BP4 overexpression or inhibition.The result showed that the decreased of activation of mTORCl pathway in response to SESN2 overexpression was restored by inhibiting SH3BP4,and those effects were reversed by overexpressing SH3BP4;To investigate the affect of SESN2 on the interaction of SH3BP4 and Rag GTPase,CMEC were treated with SESN2 overexpression or inhibition.The result showed that the interaction of SH3BP4 and Rag GTPase was increased by overexpressing SESN2,and vice versa.This result suggested that SESN2 inhibits mTORC1 pathway by promoting the interaction of SH3BP4 and Rag GTPase;CMEC were treated with ATF4 overexpression or inhibition.The result showed that AA starvation promoted the expression of SESN2 by ATF4.SESN2 negatively regulates AA-mediated casein synthesis by SH3BP4Lysosomal membrane proteins of CMEC were identified by SWATH-MS.The result showed that GNG12 is related to AA-mediated casein synthesis;CMEC were treated with GNG12 overexpression or inhibition,the result showed that AA-mediated cell proliferation,casein synthesis and activation of mTORC1 were promoted by GNG12;Interaction proteins of GNG12 were identified by Co-IP,The result showed that Ragulator was the interaction protein of GNG12;CMEC were treated with p18(one subunit of Ragulator)overexpression or inhibition,the result showed that GNG12 promoted mTORCl through Ragulator;CMEC were treated with SESN2 overexpression or inhibition,the result showed that the expression and lysosomal localization of GNG12 was inhibited by SESN2.In summary,in CMEC,SESN2 is an important inhibitor.AA starvation promotes the expression and nuclear localization of ATF4,and then the expression of SESN2 is promoted by ATF4,the expressed SESN2 inhibited the mTORCl pathway and casein synthesis via two pathway.On one hand SESN2 interacts with SH3BP4,and then promotes the inhibition of SH3BP4 on Rag GTPase,on the other hand SESN2 inhibits the AA-mediated expression of GNG12,and then inhibits the activation of Ragulator induced by GNG12.