Quantitative Acetylomics Investigate Acetylation-Mediated Molecular Pathway Network Changes In Human Nonfunctional Pituitary Neuroendocrine Tumors

Background:Pituitary neuroendocrine tumors(PitNETs)are the second most common primary central nervous system tumors in adults.Because of the occult of early symptoms,the lack of effective drug therapy,the invasiveness and the higher recurrace rate after surgery,the cure of nonfunctional pituitary neuroendocrine tumors(NF-PitNETs)has become a challenging clinical problem.Acetylation is a reversible post-translational modification at lysine residues,which is able to regulate many cellular biological processes related to tumors.This study focused on the investigation of acetylated protein profile alterations and acetylation-mediated molecular pathway changes in human NF-PitNETs,in order to clarify the biological significance of protein acetylation in NF-PitNETs,and provide new clues to find out biomarkers used for it early diagnosis and target trearment.Methods:(1)Human NF-PitNETs and normal pituitary tissue samples were collected,followed by the anti-acetyl antibody-based label-free quantitative proteomics to analyze acetylated peptides and acetylation sites,and to identify differentially acetylated proteins(DAPs)(p<0.05),the main steps include:proteolysis,recognizing and enriching acetylated peptides using anti Ac-K antibody,liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis and data analysis.(2)Acetylated proteomics data and ubiquitinated proteomics data in NF-PitNETs vs the controls were compared to identify lysine sites that simutaneously modified by acetyl and ubiquitin.(3)NF-PitNETs DAP data and the differentially expressed genes data of invasive NF-PitNETs were integrated to find out the DAPs related to NF-PitNETs invasiveness.(4)The DAVID database was used to perform GO analysis and KEGG analysis,respectively,to reveal functional characteristics of DAPs and the invasive DAPs,and signaling pathways which DAPs and the invasive DAPs involved in.These functional characteristics and signaling pathways were further grouped by the Cluster analysis.(5)Finally,immunoprecipitation(IP)and Western blotting(WB)was used to verify important DAPs.Results:(1)A total of 296 acetylated proteins with 517 acetylation sites,and 166 DAPs with 292 differentially acetylated sites(p<0.05 or only be quantified in NF-PitNETs/controls)were identified,and the majority of which were significantly down-acetylated in NF-PitNETs.(2)These acetylated proteins extensivly function in various cellular biological processes and signaling pathways,and the most of which are associated with metabolism and energy yield,such as carbon metabolism,glycolysis/gluconeogenesis,citrate cycle,biosynthesis of amino acids,pyruvate metabolism,the metabolism of glyoxylate and dicarboxylate,oxidative phosphorylation,and the degradation of valine,leucine and isoleucine.(3)Among these acetylated proteins,15 lysine residues within 14 proteins were down-acetylated and simultaneously up-ubiquitinated in NF-PitNETs,demonstrating a direct competition relationship between acetylation and ubiquitination.(4)A total of 26 DAPs were identified that related to NF-PitNETs invasiveness,which mainly involved in metabolism-associated pathways—carbon metabolism,glycolysis/gluconeogenesis,oxidative phosphorylation,biosynthesis of amino acids,and the metabolism of fructose and mannose,indicating that acetylation-mediated metabolic reprogramming might be the molecular mechanism that affects the NF-PitNETs invasiveness.(5)Phosphoglycerate kinase 1(PGK1)is randomly selected from 166DAPs of NF-PitNETs to verify differences in acetylation between NF-PitNETs and the controls.LC-MS/MS identified that the lysine sites(K)291,K146,K156 and K361 of PGK1 were only acetylated in the controls.IP/WB analysis showed that compared with the control pituitary tissues,the acetylation level of PGK1 decreased in NF-PitNETs,which were consistent with quantitative acetylated proteomics based on LC-MS/MS.Conclusions:The human acetylated proteomic profiling was identified by antibody enrichment-based label-free quantitative acetylomics in this study,and their functional characteristics and acetylation-mediated signaling pathways were identified by bioinformation analysis.The acetylation levels of more than half acetylated proteins were decreased in NF-PitNETs,and the most of signaling pathways that DAPs involved in were associated with cellular metabolism and energy generation.The lysines co-regulated by acetylation and ubiquitination were down-acetylated but up-ubiquitinated in NF-PitNETs,which indicated that acetyl and ubiquitin directly competed for the same lysine.The DAPs related to invasiveness mainly involved in metabolism-related pathways,thus acetylation-mediated metabolic reprogramming might be considered as one of the underlying mechanisms in tumorigenesis and invasiveness of NF-PitNETs.These findings reveal acetylation-mediated molecular pathway network changes in NF-PitNETs,and provide experimental data for the further discovery of new biomarkers and therapeutic targets.