Identification Of The Candidate Proteins Participated In Autophagy Pathway Mediated By Leucine Via Cell-free System And ITRAQ

Amino acids are not only substrates for protein synthesis,but also signaling molecules(especially leucine) which regulate m TORC1 signaling pathway and autophagy. Amino acids starvation can induce autophagy, whereas amino acids sufficiency inhibit autophagy. We have gained much knowledge about the mechanism through which amino acids regulate m TORC1 and autophagy, due to the identification of several molecules which functions upstream of m TORC1 signaling pathway. However, it has not been elucidated clearly. There are still some unknown proteins need to be further investigated, which participate in the regulation of autophagy by amino acids. Thus, how to effectively screen the unknown proteins which participate in autophagy regulation in response to amino acids is one of the research hotspots.In consideration of the above scientific questions, we plan to establish a cell-free system and add single amino acid into this system to validate whether this amino acid can regulate autophagy, and identify some key molecules which participate in the regulation of autophagy by amino acid via comparative proteomics research methods( isobaric tag for relative and absolute quantitation, i TRAQ). We constructed a cell-free system for studying autophagy signaling payhway, which is composed of crude autophagosome fraction, S100(cytosol) and purified Atg14-Flag. We had proved that it has better effectiveness. We added single amino acid into this system after successfully establishing the cell-free system, it revealed that autophagy may be regulated by leucine and methionine in the cell-free system. So that, we preliminarily select leucine as a research object, we detected the protein in the supernatant and pellet of cell-free system after adding leucine by applying i TRAQ assay. 4811 proteins had been identified in our experiments. Among all of the identified proteins, 243 proteins was differentially regulated in the supernatant(S2/S1), 134 proteins was differentially regulated in the pellet(P2/P1)(assessment criteria of differentially regulated proteins is fold-change ≥1.2 or ≤0.83, and p-value<0.05).The most significantly enriched pathway of differentially regulated proteins in the supernatant was aminoacyl-t RNA biosynthesis, it suggested that this pathway may be correlatedwithautophagy regulation by leucine. One of the significantly enriched pathways of differentially regulated proteins in the pellet was oxidative phosphorylation, it suggested that the proteins participated in this pathway may also be correlated with autophagy regulation by leucine. The number of down-regulated proteins in the supernatant and up-regulated proteins in the pellet is 12(VPS35, CAND1, PKM, PYGL, XRCC6, IPO7, ILF2, GNPDA1, NCDN, NME1, SRP9, SEPHS1), however, the number of up-regulated proteins in the supernatant and down-regulated proteins in the pellet is 0. We speculate that VPS35 and CAND1 most probably take parts in the regulation of autophagy by leucine through bioinformatic analysis of the above 12 proteins.In conclusion,(1) We successfully established a cell-free system which can be applied to study autophagy signaling pathway, this system has better effectiveness.(2) Autophagy may be regulated by leucine and methionine in the cell-free system.(3) The proteins which most probably participate in the regulation of autophagy by leucine are VPS35 and CAND1.