Dissection Of The Posttranslational Modifications In Leucine-Regulated Autophagy

As an evolutionarily conserved protein degradation pathway,autophagy plays a key role in maintaining cell homeostasis and cell survival.Leucine as a signaling molecule can regulate autophagy,protein post-translational modification,as an important regulatory method,is involved in the regulation of almost all biological processes.However,little is known about how post-translational modifications regulate leucine depletion induced autophagy.This study first verified the relationship between leucine deletion and autophagy.The relative abundance of LC-II was maximized in AML12 cultured with leucine-free medium for 2 hours(h).After transfection of GFP-LC3 plasmid in AML12 cells,the number of spotted GFP-LC3 was significantly increased in cells cultured with leucine-free medium compared with control,and the same phenomenon was observed in HeLa cells stably transfected with GFP-LC3.The above results show that leucine deprivation activates autophagy.The leucine-regulated post-translational modification was screened by Western blot.The results showed that the level of acetylation,ubiquitination,and succinylation in AML12 cells did not change significantly after leucine deprivation.Only some bands were different.Whereas,the crotonylation modification level was significantly up-regulated after leucine deprivation.Laser confocal microscopy(Confocal)experiments also demonstrated that leucine depletion significantly up-regulated protein crotonylation.These results demonstrate that leucine deficiency activates autophagy and increases crotonylation.Western and Confocal experiments have shown for the first time that sodium crotonate(NaCr)treatment improves crotonylation and activates autophagy in AML12 and HeLa cells,suggesting that lysine crotonylation correlated with cellular autophagy positively.To screen leucine-regulated crotonylation proteins and sites in AML12 cells further,we performed proteomics and crotonylation proteomics based on TMT labeling technology.Samples were divided into normal group and leucine deprivation group,with three replicates in each group.Proteomics identified a total of 5644 proteins in AML12,of which there were 5026 quantifiable proteins.Screening for crotonylome in AML12 cells,a total of 1898 crotonylation sites were identified in 654 proteins in 6 samples.Of these,1,501 sites on 536 proteins could be quantified.After the leucine-free medium treatment,the hyper-modification abundance site of crotonylation was significantly increased,and the low-modification abundance site was significantly reduced.Analysis of amino acid motifs(Motif)revealed that ±1 position of the modification site was significantly enriched in two acidic amino acids,aspartic acid and glutamic acid.Using the full proteomic data to normalize the crotonylation data,a difference in multiples of >1.5 or <0.67 and a p-value of <0.05 was used as the screening threshold value,and 326 crotonylation sites on 208 proteins showed significant differences.Among them,298 sites on 189 proteins were significantly up-regulated,and 28 sites on 19 proteins were significantly down-regulated.The functional enrichment analysis of the differential crotonylation protein revealed that the differential proteins are mainly distributed in the cytoplasm(50%),nucleus(31%)and mitochondria(6%),and involved in a series of signaling pathways,such as the PI3K-AKT pathway,and the protein domains enriched in the differential crotonylation sites include 14-3-3 domains,type II aminoacyl tRNA synthetases,ubiquitin-related domains,and so on.In comparison with the Autophagy Database,12 autophagy-related crotonylation sites were screened and 18 differential crotonylation sites were present on 12 proteins.These proteins and sites may be involved in leucine-regulated autophagy.In summary,(1)this study demonstrated that leucine depletion activates cellular autophagy;(2)Leucine depletion has no significant effect on protein acetylation,ubiquitination,and succinylation in AML12 cells,but can significantly increase protein crotonylation(3)Sodium crotonate as a crotonylation-modifying activator can activate cellular autophagy;(4)this study identified a series of brightened by proteomics and crotonylomics studies.The leucine-regulated proteins and crotonylated sites provide the basis for subsequent studies.(5)The 14-3-3 ? K73 and K78 crotonylation sites may be the key candidate crotonylation sites involved in the regulation of leucine deletion-induced autophagy.