Mechanism Of FKBP39 Regulating Nprl2/3 Expression And TORC1 Activity In Drosophila

In eukaryotes,Target of Rapamycin Complex 1(TORC1),an evolutionarily conserved serine/threonine kinase,coordinates nutrient and energy status with cell growth.Under conditions of adequate nutrition,the activity of TORC1 increases,through phosphorylation and activation of S6 kinases(S6Ks)and eIF-4E binding proteins(4E-BPs)etc.,thereby promoting protein synthesis;Under the conditions of nutrient deficiency,TORC1 activity is reduced,which promotes the occurrence of autophagy and promotes the degradation of intracellular proteins into small molecular substances.Nutrients such as amino acids mainly control the activity of TORC1 by regulating the combination of Rags GTPase RagA/B and RagC/D.When amino acids are sufficient,Rags GTPase forms a state where RagA/B binds to GTP and RagC/D binds to GDP and activates TORC1;when amino acids are lacking,a state where RagA/B binds to GDP and RagC/D binds to GTP is formed,inhibiting TORC1 activity.The GATOR1 complex is composed of three proteins:Nprl2,Nprl3 and Imll/DEPDC5.It has Rags GAP enzyme activating protein(GAP)activity.It can hydrolyze the GTP bound to RagA/B into GDP,thereby inhibiting TORC1 activity.In order to study whether nutritional status regulates the expression of GATOR1 component protein,we used Western blot in Drosophila S2 cells to detect the difference in protein expression of GATOR1 component protein Nprl3 under different nutritional status,and found that amino acid deficiency could induce endogenous Nprl3 The increase in protein level did not affect the expression of nprl3 mRNA at the transcription level,suggesting that the lack of amino acids controls the expression of Nprl3 through a post-transcriptional regulatory mechanism.In order to explore whether amino acid deficiency affects the protein stability of Nprl3,we transfected the cells with V5-tagged Nprl3 exogenous protein,and treated the cells with the protein synthesis inhibitor Cycloheximide(CHX),and found that the amino acid starvation slowed the degradation of the exogenous Nprl3 protein,Indicating that the lack of amino acids increases the protein stability of Nprl3.In order to explore the mechanism by which amino acids regulate the expression of Nprl3,we used liquid chromatography-mass spectrometry(HPLC-MS)and found that FK506 binding protein 39 kDa(FKBP39)may bind to Nprl3 protein.We have verified the binding of Nprl3 and FKBP39 in cells by co-immunoprecipitation(Co-IP).More importantly,we found that reducing the expression of FKBP39 by RNA interference can increase the expression of Nprl3 in the cell.which suggested that FKBP39 may bind to Nprl3 and promote the degradation of Nprl3 protein.By CHX treatment of S2 cells,it was found that the stability of Nprl3 protein increased under the conditions of fkbp39 RNAi.The above results indicate that under the condition of sufficient amino acids,FKBP39 binds to Nprl3 and inhibits the expression of Nprl3 by promoting the degradation of Nprl3.Lack of amino acids can relieve the promoting effect of FKBP39 on the degradation of Nprl3 protein,and increase the stability of Nprl3 protein and increase its expression.In order to verify whether the nutritional status regulates the expression of the GATOR1 component protein Nprl2 through FKBP39,we tested it in S2 cells and found that similar to the Nprl3 protein,the increase in the expression of the Nprl2 protein during amino acid starvation also depended on the FKBP39 protein.In order to explore the physiological functions of FKBP39,we constructed fkbp39 mutant drosophila by CRISPR-Cas9.Western blot analysis of the ovary of fkbp39 mutant and RNA interference confirmed that FKBP39 inhibited the expression of Nprl3.Amino acid starvation was performed on fkbp39 mutant drosophila ovary found that Nprl3 expression level infkbp39 mutant drosophila could not increase with amino acid starvation,which was different from the amino acid induced increase in Nprl3 expression in control drosophila.Through the detection of the activity of p-4EBP and p-S6K in the fkbp39 mutant,we found that the phosphorylation levels of 4EBP and S6K were reduced,indicating that the activity of TORC1 in the fkbp39 mutant drosophila was reduced.The above results indicate that FKBP39 can mediate the activity of TORC1 by regulating the expression of GATOR1 at the level of organic body.Through experiments in Drosophila cells and adults,we clarified the mechanism by which amino acid levels in the body regulate the stability of GATOR1 component protein Nprl3 through FKBP39,thereby regulating the activity of TORC1.This research lays the foundation for further analysis of the regulation mechanism of TORC1 activity by nutritional status.