Characterization Of The Acetylome Associated With Rapamycin-induced Autophagy

Objective:To explore the characterization of acetylome of the whole protein in rapamycin-induced autophagy.Methods:First,cells were divided into three groups:negative control,DMSO control and rapamycin treatment group.Then the phosphorylation level of mTOR and mTOR's substrates were detected by immunoblot analysis,and endogenous LC3 puncta were analyzed by immunofluorescence analysis.Second,we set up a control group and 0.1?M rapamycin treatment group.Then cells were labeled in SILAC kits,and then all the proteins were digested with trypsin and affinity-enriched with anti-acetyllysin antibody.The resulting peptides were analyzed by nano-HPLC-MS/MS.Finally,the characteristic of acetylome were summarized through the GO,Motif-x,KEGG and CORUM database analysis.Results:(1)From our results,it was evident that a decrease in phosphorylation of mTOR and its substrates RPS6KB1,EIF4G1 was observed after rapamycin treatment.We found that.the ratio.of LC3-?/LC3-? Land fluorescent puncta.of LC3-? were significantly increased.(2)Through the SILAC and MS analysis,we in total identified 1808 acetylation sites on 944 proteins,among which,significnt changes of lysine acetylation on 533 sites on 397 proteins were observed.The majority of proteins contained only one acetylation site(60.4%),whereas considerable proportions of double(20.4%)and multiple(19.2%)acetylated sites in single protein were also identified.The identified proteins were mainly distributed in the cytoplasm,nucleus and mitochondria.(3)Through the motif analysis,we found that FxK*,K*xxxxK,KxxxxK*were shown to be potential autophagy-related lysine acetylated motifs.(4)GO enrichment analysis showed that macromolecular complex assembly,cell metabolism,gene expression and N-acetyltransferase activity et al were significantly enriched.(5)Further KEGG analysis found that acetylation was significantly enriched of enzymes in glycolysis,fatty acid metabolism and amino acid metabolism which is related the acetyl-CoA.(6)In autophagy,protein acetylation modification existed in the form of protein complexes,such as ribosomes,RNA spliceosomes and the ubiquitin-proteasome complex.(7)After rapamycin treatment,we observed acetyltransferase KAT7 at K155 and the multiple sites of EP300 such as K1203,K1542,K1546,K1707,were observed significantly changed.Meanwhile,the acetylation of their substrates histone such as H3 K19 and K24,H4 K16,were observed to be significantly changed.Conclusion:Protein acetylation is also an important post-translational modification that involved in autophagy regulation and multiple cellular processes,namely:transcription-dependent pathway and transcription-independent pathway such as metabolic processes.It is inseparable between autophagy and AcCoA-related metabolic pathway.It is confirmed that AcCoA is an important molecular target for rapamycin-induced autophagy.It is important protein modification for the self acetylation modification of acetyltransferase KAT7 and EP300 which involved in autophagy regulation.