Lysine Acetylome Presents A Site-Specific Pattern In The Process Of Autophagy

Objective:Protein acetylation has been reported to act as a key in the regulation of related autophagy pathways during autophagy.However,up to now,the relationship between acetylome and autophagy has remained unclear.Methods:First,stable isotope labeling of amino acids in cell culture and high-throughput quantitative mass spectrometry were used to perform an acetylome analysis of rapamycin-induced autophagy in vitro.Then,with the aid of bioinformatics techniques,we analyzed the general feature of acetylome with autophagy.Finally,the effect of acetylation modification site of acetyltransferase p300 on autophagy was verified by site-directed mutagenesis and gene transfection.Results:(1)Our data revealed that 2,135 sites were quantified on 1081 proteins.During autophagy,421 sites were significantly regulated on 296 proteins,with 80.8%of sites down-regulated and 19.2%up-regulated.(2)Motif enrichment analysis revealed five main motifs were involved in autophagy,[G-K-AcK],[A-X-AcK],[G-AcK],[K-AcK]and[AcK-K].All down-regulated sites conformed to the classical functional motif of p300/CBP[G-AcK].(3)Furthermore,acetylation targeted proteins involved mainly in ribosomes,spliceosomes and AcCoA-related metabolic process.(4)In-depth analysis indicated that most of acetylation sites were either in the critical domain,functional sites or could change their enzymatic activity by acetylation,highlighting the importance of site-specific acetylation patterns.(5)Subsequently,we demonstrated that K1549 of p300 was also a functional site that could regulate the autophagic process in vitro.Conclusion:Our data reveal a deacetylation-preponderant profile with autophagy.The specificity of the related motifs and identification of site-specific acetylation patterns would assist searches for potential targets or subsequent mechanism-focused studies to elucidate site-specific protein networks in autophagy.