Dissection Of Leucine-regulated MTORC1 And Fatty Acid ?-Oxidation

Leucine is a multifunctional essential amino acid that plays crucial roles in various cellular processes.However,the integral effect of leucine on the cellular proteome remains largely unknown.In addition,leucine has been found to play a crucial role in the regulation of mTORC1(mechanistic target of rapamycin complex 1)signaling,but the mechanism has not been fully understood.To address these two questions,we conducted three studies: we firstly applied an isobaric tags for relative and absolute quantification(iTRAQ)-based comparative proteomics strategy to investigate the proteome alteration induced by leucine deprivation and re-supplementation in human liver hepatocellular carcinoma(HepG2)cells,by which we found that leucine deprivation activates the fatty acid ?-oxidation pathway.Next we explored the possible mechanism underlying which leucine regulates the fatty acid ?-oxidation pathway.At last we explored the mechanim of leucine-regulated mTORC1 pathway.The main results were as below: 1.KAT7-mediated calnexin crotonylation regulates leucine-stimulated mTORC1 activityLeucine plays crucial roles in the mTORC1 pathway.However,the underlying mechanisms are not fully understood.Here,we establish a cell-free system to mimic the activation of mTORC1,by which we identify calnexin(CANX)as an essential regulator for leucine-stimulated mTORC1 pathway.Western and confocal assays indicated that CANX translocates to the lysosomes after leucine deprivation.Further functional exploration found that CANX loss-of-function renders either the mTORC1 activity or the lysosomal translocation of mTOR insensitive to leucine deprivation.While the mTORC1 activity was still sensitive to serum starvation,suggested that CANX is essential for leucine-stimulated mTORC1 activity,and CANX is likely to regulate mTORC1 by affecting its lysosomal translocation.By constructing a RAGA/RAGB double knockout and a CANX/RAGA/RAGB triple knockout cell lines,we further found that CANX regulates mTORC1 activity upstream of the Rag GTPases.By combining immunoprecipitation(IP)with mass spectrum(MS)analysis,we found that lysosome-associated membrane glycoprotein 2(LAMP2)binds to CANX after leucine deprivation and is essential for leucine-stimulated lysosomal translocation of CANX and mTOR.Moreover,leucine deprivation promotes the lysine(K)525 crotonylation of CANX,which is another essential condition for the lysosomal translocation of CANX.Finally,we find that KAT7(lysine acetyltransferase 7)mediates the K525 crotonylation of CANX.Loss of KAT7 renders the mTORC1 insensitivity to leucine deprivation.Our findings provide new insights for the regulatory mechanism of leucine-stimulated mTORC1 pathway.2.Quantitative proteomics profiling reveals leucine deprivation activates the fatty acid ?-oxidation pathwayTo investigate the intergral effects of leucine deprivation or supplementation on the hepatic proteome,we used iTRAQ based comparative proteomics strategy to profile the proteome alteration after leucine deprivation and resupplementation.By iTRAQ analysis,a total of 3066 proteins were quantified,189 and 178 proteins were identified as significantly differentially expressed proteins in the-Leu/Normal and +Leu/-Leu,respectively.Further bioinformatics analysis showed the differentially expressed proteins were involved in various metabolic processes including amino acid and lipid metabolism.Furthermore,several fatty acid ?-oxidation pathway related proteins,including ACSL1,ACOX1,and ACADS,were highly up-regulated by leucine deprivation,while down-regulated by 5 mM leucine re-supplementation.Leucine deprivation also leads to the reduction of cellular triglycerides in HepG2 cells.These results may provide new insights into the regulatory function of leucine in multiple cellular processes,especially fatty acid as well as lipid metabolism 3.Autophagy and PPAR? are involved in leucine deprivation-activiated fatty acid ?-oxidation pathwayThis study aimed to investigate the potential mechanism of leucine deprivation-activated fatty acid ?-oxidation pathway.We first proved that leucine deprivation activates the fatty acid ?-oxidation pathway in a mouse primary liver cell line AML12 by up-regulating the abundance of Acads,Acox1 and Acsl1.In addition,leucine deprivation decreased the content of triglyceride,while increased the ATP content in AML12 cells.Q-PCR assays indicated that the mRNA abundance of Acads,Acox1 and Acsl1 was also increased under leucine deprivation,suggesting that a transcriptional mechanism might be involved in the leucine deprivation-induced activation of fatty acid ?-oxidation pathway.By bioinformatics upstream predication,we found that the transcriptional factor PPAR? might be involved in the transcriptional regulation of these three genes.Inhibition of PPAR? redered the protein or mRNA abundance of Acads,Acox1,and Acsl1 insensitive to leucine deprivation,suggesting that leucine deprivation is likely to activate fatty acid ?-oxidation pathway by inducing PPAR? to transcriptionally regulate the fatty acid ?-oxidation-related genes.Further investigation found that the treatment of rapamycin,the inhibitor of mTORC1,rendered the fatty acid ?-oxidation activity insensitive to leucine deprivation.In addition,in ATG5 KO autophagy deficient cells,the abundance of fatty acid ?-oxidation-related proteins was not differential under leucine deprivation.These results suggest that the mTORC1-dependent autophagy is also involved in the regulation of leucine deprivation-activated fatty acid ?-oxidation pathway.This study provides new insights into the mechanism of leucine-regulated fatty acid ?-oxidation pathway,as well as the function of leucine-regulated autophagy.