Substrate Profiling And Functional Studies Of β-hydroxybutyrate-induced β-hydroxybutyrylation And ULK1-regulated Phosphorylation Modification

Protein post-translational modifications(PTMs)are covalent processing events by adding modifying groups on specific amino acid residues,which change the properties of proteins.As the main undertaker of life activities,proteins exert their biological activities and corresponding functions through PTMs.Common PTMs include methylation,acetylation,ubiquitination,phosphorylation,and glycosylation.With the development of quantitative proteomics technology,a wide array of novel protein PTMs has been discovered,such as lysine benzoylation(Kbz),lysine lactylation(Kla),and lysine β-hydroxybutyrylation(Kbhb).However,the biological functions of these newly identified modifications remain to be explored.The autophagy kinase unc-51-like kinase 1(ULK1)regulates various tumor processes by phosphorylating downstream substrates,while its phosphorylation substrate sites are still very limited.Since the detection of the global substrates and modification sites is crucial for better characterization of the functions and regulatory mechanisms governed by PTMs,we performed Kbhb proteome and ULK1-mediated phosphorylation proteome by a variety of quantitative proteomic techniques in this paper.Besides,we also explored their physiological functions by bioinformatics analysis.First,we identified and analyzed the substrate profile of Kbhb converted fromβ-hydroxybutyrate(BHB)in mouse embryonic fibroblasts(MEF)cells by quantitative proteomics using stable isotope labeling by amino acids in cell culture(SILAC).The results showed that a total of 840 unique Kbhb sites on 429 proteins were identified in MEF cells after the stimulation of BHB,of which 42 sites on 39 proteins were increased by more than 50% compared to the normal MEF cells without stimulation.Moreover,the bioinformatics analysis indicated that the up-regulated Kbhb is involved in aminoacyl-t RNA biosynthesis,2-oxocarboxylic acid metabolism,citric acid cycle,pyruvate metabolism and other biological pathways.However,the up-regulated Kac sites and down-regulated Kac sites were almost the same in MEF cells after BHB induction,and the up-regulated lysine acetylation(Kac)proteins were mainly associated with focal adhesions,proteoglycan and RNA transport processes in cancer.Together,these findings broad the Kbhb substrate profile and lay the foundation for Kbhb functional studies.Second,we identified and analyzed the substrate profile of ULK1-mediated phosphorylation.As an autophagy-initiating switch,ULK1 has a complex and unknown mechanism in regulating tumor growth.Through label-free quantitative proteomics technology,we found that the phosphorylation levels of pyruvate dehydrogenase E1 component subunit alpha(PDHA1),minichromosome maintenance deficient 3(MCM3)and other proteins closely related to tumors were significantly decreased when the ULK1 kinase activity was inhibited.In addition,proteins specially regulated by ULK1 were widely distributed in many organelles.Moreover,the functional enrichment analysis showed that these significantly changed proteins affected by ULK1 were related to various biological functions such as DNA transcription,biosynthesis and metabolism,suggesting different functions more than autophagy.Next,the phosphorylated substrates which changed significantly when knockdown of ULK1 in mammalian cells were identified using SILAC method and the functional annotation analysis suggested that they were involved in a wide range of biological processes such as energy metabolism,cell division,signal transduction,and immune responses.Furthermore,we validated thatβ-Tubulin 4A(TUBB4A)were interacted with ULK1 by Co-immunoprecipitation(Co-IP),indicating the close relationship between ULK1 and occurrence of tumors and other diseases.Overall,our study provides new targets and directions for tumor therapy.