Computational Analysis Of Yeast Autophagy Kinase Atg1 Based On Multi-omics Integration

Autophagy is a highly conserved biological process in eukaryotes that degrades intracellular material through lysosomes(mammal)/vacuoles(yeast,plant).Autophagy is closely related to the processes such as immunity,development,aging and even life span,as well as the occurrence of diseases such as neurodegenerative diseases and tumors.There are many autophagy-related(Atg)proteins involved in the process of autophagy.Atg1,a conserved serine/threonine kinase,is the first autophagy-related protein to be identified,and its mammalian homology is ULK1(UNC-51-like kinase 1).As the core kinase of autophagy,Atg1 plays a crucial role in autophagy regulation.The N-terminal of Atg1 is the kinase domain,which regulates autophagy through phosphorylation.The integral membrane protein Atg9 is an important substrate of Atg1.There are two microtubule interaction and transport(MIT)domains located at the C-terminal of Atg1,which interact with Atg13 and Atg17 and perform non-catalytic functions independent of the kinase domain.Since autophagy starts with the assembly of the Atg1 complex,the C-terminal domain is also dubbed the early autophagy targeting(EAT)domain.Previous studies have shown that Atg1 participates in autophagy regulation through kinase activity and interactions,but its downstream specific molecular regulation mechanism is still unclear,and new functional phosphorylated substrates and interacting molecules need to be discoveredIn this study,for a comprehensive grasp of the Atg1-dependent molecular landscapes,we prepared cell samples of wild type(WT),atg1 gene knockout(atg1?)and ATG1^D211A(Atg1-KD)of Saccharomyces cerevisiae BY4741 strains cultured under nitrogen starvation for 0,1 and 4 hours.The samples used for time-series identification of transcriptomes,quantitative proteomes and quantitative phosphoproteomes.The differential expression analysis of atg1?explained that 409 and 441 genes were markedly regulated by ATG1 at the transcriptional and translational level,respectively;and there are2,583 differentially regulated phosphorylation sites.And the differential expression analysis of Atg1-KD showed that the transcription of 250 genes and the translation of 563genes were significantly related to the kinase activity of Atg1,and the phosphorylated levels of 2,463 sites were significantly changed due to the lack of Atg1 kinase activity.In order to identify new interacting proteins and phosphorylated substrates of Atg1involved in autophagy regulation,we made potentially reasonable assumptions on the changes in transcriptional?translational and phosphorylation levels of Atg1 potential interacting proteins and substrates in atg1?and Atg1-KD strains according to the characteristics of structural and functional of Atg1.According to the hypothesis modeling,an“inference of phosphorylated substrates and interacting partners”(i PSIP)algorithm based on multi omics integration was developed.Using the i PSIP algorithm,12 potential Atg1 interactions and 17 potential Atg1 phosphorylated substrates that may play a role in autophagy regulation were predicted.Finally,in order to get more accurate prediction results,we screened and verified the potential functional interactions and substrates of Atg1 using the biochemical experimental methods of autophagy research.First,Western blotting was used to detect the degradation of Atg8,and the function of potential proteins in autophagy regulation was initially identified and screened.It was found that RGD1 and WHI5 genes knockout significantly inhibited autophagy activity.Then,the functions of Rgd1 and Whi5 in autophagy regulation were verified by Western blotting and confocal microscopy based on GFP-Atg8.At the same time,their interaction with Atg1 was verified by immunoprecipitation,and finally the site mutation technology was used to identify the functional phosphorylation sites of Whi5.The preliminary results indicate that Rgd1 is a new interacting partners of Atg1 involved in autophagy regulation,and Whi5 is a potential functional phosphorylated substrate of Atg1.In summary,this project not only developed a method for predicting protein phosphorylated substrates and interacting proteins based on the integration of multi-omics data,but also discovered new autophagy regulators of Atg1 downstream.