Peptide transporter 2(PepT2)transports short peptides from the blood into bovine mammary epithelial cells(BMEC)to stimulate milk protein synthesis.Despite the fact that the effect of PepT2 is acknowledged in BMEC,litt...Peptide transporter 2(PepT2)transports short peptides from the blood into bovine mammary epithelial cells(BMEC)to stimulate milk protein synthesis.Despite the fact that the effect of PepT2 is acknowledged in BMEC,little is known about its regulation.This study was completed to investigate the role of mammalian target of the rapamycin(mTOR)signaling in regulating the expression and function of PepT2 in BMEC.The regulation of PepT2 by mTOR in BMEC was studied in vitro using peptide transport assay,gene silencing,Western blot.The membrane expression of PepT2 and the uptake of b-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid(b-Ala-Lys-AMCA),a model dipeptide,in BMEC were reduced by rapamycin(a mTOR inhibitor)and silencing of either mTOR complex 1(mTORC1)or mTOR complex 2(mTORC2),stimulated by DEP domain-containing mTOR-interacting protein(DEPTOR,endogenous inhibitor of mTORC1 and mTORC2)silencing.The trafficking of PepT2 to the membrane and the uptake of b-Ala-Lys-AMCA was promoted by neuronal precursor cell-expressed developmentally down-regulated 4 isoform 2(Nedd4-2)silencing.The effects of knockdown of mTORC1,but not mTORC2,on cell membrane expression and transport activity of PepT2 was abolished by Nedd4-2 silencing.With immunofluorescence staining,PepT2 was identified to be interacting with Nedd4-2.The Nedd4-2 expression and the interaction between PepT2 and Nedd4-2 was increased through mTORC1 knockdown,indicating an increased ubiquitination of PepT2.The results revealed that mTORC1 can regulate the expression and function of PepT2 through Nedd4-2 in BMEC.展开更多
基金the National Natural Science Foundation of China 32072756 and 31872989.
文摘Peptide transporter 2(PepT2)transports short peptides from the blood into bovine mammary epithelial cells(BMEC)to stimulate milk protein synthesis.Despite the fact that the effect of PepT2 is acknowledged in BMEC,little is known about its regulation.This study was completed to investigate the role of mammalian target of the rapamycin(mTOR)signaling in regulating the expression and function of PepT2 in BMEC.The regulation of PepT2 by mTOR in BMEC was studied in vitro using peptide transport assay,gene silencing,Western blot.The membrane expression of PepT2 and the uptake of b-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid(b-Ala-Lys-AMCA),a model dipeptide,in BMEC were reduced by rapamycin(a mTOR inhibitor)and silencing of either mTOR complex 1(mTORC1)or mTOR complex 2(mTORC2),stimulated by DEP domain-containing mTOR-interacting protein(DEPTOR,endogenous inhibitor of mTORC1 and mTORC2)silencing.The trafficking of PepT2 to the membrane and the uptake of b-Ala-Lys-AMCA was promoted by neuronal precursor cell-expressed developmentally down-regulated 4 isoform 2(Nedd4-2)silencing.The effects of knockdown of mTORC1,but not mTORC2,on cell membrane expression and transport activity of PepT2 was abolished by Nedd4-2 silencing.With immunofluorescence staining,PepT2 was identified to be interacting with Nedd4-2.The Nedd4-2 expression and the interaction between PepT2 and Nedd4-2 was increased through mTORC1 knockdown,indicating an increased ubiquitination of PepT2.The results revealed that mTORC1 can regulate the expression and function of PepT2 through Nedd4-2 in BMEC.
