Screening Of Key Candidates For Leucine-Regulated Protein Synthesis Signaling Pathway Via Cell-Free System Assays

Activation of the m TORC1(mammalian target of rapamycin complex 1)can not only promote the synthesis of the proteins,it can also inhibit autophagy,which is a way of protein degradation.Amino acids,especially leucine are not only substrates for protein synthesis,but also signaling molecules which activate mTORC1.In the past few years,more and more proteins have been found to participate in the process of the mTORC1 activation by amino acids.However,it is currently unclear how amino acid sufficiency or limitation is sensed to modulate mTORC1 activity.There are many unknown proteins that may have a role in mTORC1 activation by amino acids.We constructed a cell-free system for investigating mTORC1 signaling pathway,which combined with comparative proteomics research methods iTRAQ(isobaric tags for relative and absolute quantification)and bioinformatics analysis to select some key molecules which participate in the regulation of mTORC1 signaling pathway by leucine.The cell-free system is composed of crude lysosome fraction(including P20(+)and P20(-)),cytosol(including S100(+)and S100(-))and purified HA-raptor(a component of the mTORC1).We had showed that in a cell-free system that contained S100(-)and P20(-),leucine could promote raptor lysosome association.After successfully establishing the cell-free system,we applied iTRAQ assay for detecting the supernatant and pellet of both the control(S1,P1)and leucine(S2,P2)group.We had identified 6292 proteins in the iTRAQ assay and the assessment criteria of differential proteins is fold-change >1.2 or <0.83,and Q-value<0.05.In P2-VS-P1,the number of the up-regulated proteins was 208 and the number of the down-regulated proteins was 190.In S2-VS-S1,the number of the up-regulated proteins was 12 and the number of the down-regulated proteins was 3.Bioinformatics analysis(including GO categories,COG annotation and pathway annotation)results indicated that the differential proteins mainly localized in organelles and participated in metabolic process.We also found that the proteins that participated in lipid metabolism and associated with neurodegenerative disease might participate in themTORC1 activation by leucine.In order to select some key molecules which participate in the regulation of mTORC1 signaling pathway by leucine,we have to consider the differential proteins in supernatant and pellet.The number of down-regulated proteins in the supernatant and up-regulated proteins in the pellet is 0,while the number of up-regulated proteins in the supernatant and down-regulated proteins in the pellet is 6,we speculate the 6proteins(APOA1、CFH、FGA、ERH、CANX、F2)take part in the regulation of mTORC1 signaling pathway by leucine.TO validate the iTRAQ data,we preliminarily select CANX as a research object,the distribution of the CANX in the cell-free system was consistent with the iTRAQ data,moreover,confocal fluorescence analysis supported that leuncine could promote the translocation of the CANX from the lysosome to the cytosol.In conclusion,(1)We successfully established a cell-free system for investigating the regulation of mTORC1 signaling pathway by leucine for the first time,which is effective and stable.(2)Bioinformatics analysis of the iTRAQ data showed that APOA1、CFH、FGA、ERH、CANX、F2 might participate in the regulation of mTORC1 signaling pathway by leucine.