Application Of An Aspirin Probe And Chemoproteomics In The Study Of Protein Acetylation Regulation

Aspirin is a commonly used non-steroidal anti-inflammatory drug with antipyretic,analgesic,anti-inflammatory,antithrombotic and other functions.One of its earliest discovered mechanisms is to inhibit the activity of cyclooxygenase(COX)by acetylating a serine residue on the active site of COX,thereby inhibiting the synthesis of prostaglandins and thromboxane,achieving anti-inflammatory and antiplatelet agglutination effect.Acetylation is a reversible protein post-translational modification,and its most well-known functions focus on the effect of histone acetylation on the structure of cell chromosomes and the activation of transcriptional regulators in the nucleus.At the same time,a variety of cytoplasmic proteins,metabolic enzymes,this non-histone proteins also have acetylation modifications.In addition to COX,aspirin can also acetylate some other proteins.Understanding the acetylation modification of aspirin on proteins is of great significance for studying other potential mechanisms of aspirin.On one hand,various mechanisms of aspirin have not yet been elucidated,some of which may be achieved by acetylation of target proteins.It is meaningful to study the regulation of acetylation at the whole proteome level by aspirin.On the other hand,protein acetylation metabolism plays a key role in cell cycle,metabolism,epigenetics,etc.However,the acetylation level of proteins is generally low,and the analysis is difficult.The enrichment and analysis of acetylated proteins by small molecular probes is helpful to study the mechanism of deacetylase inhibitors and lay a foundation for the research of related drugs.In recent years,proteomics studies based on small molecular probes have been more and more widely used in the field of drug analysis.In this thesis,an aspirin-based probe was used to study the regulation of protein acetylation by aspirin.The aspirin probe was used in combination with proteomic methods to analyze cellular protein acetylation sites.In order to study the regulation of protein acetylation by aspirin,we first screened conditions for the aspirin probe labeling of cellular proteins through cytotoxicity experiments and in-gel fluorescent imaging.Under the optimized labeling conditions,we analyzed the probe-labeled proteins with proteomic methods based on mass spectrometry.The COX2 protein and some previously reported proteins that could be acetylated by aspirin were identified from the mass spectrometry results.Among the identified proteins,fructosebisphosphate aldolase A(ALDOA)is an important glycolytic enzyme and gluconeogenic enzyme.Through experiments,we found that aspirin has certain selectivity to the acetylation modification of K13,K14 and K42 of ALDOA,and hyperacetylation can inhibit the activity of ALDOA.At the same time,we developed a new probe,pentynoic anhydride,for the analysis of protein acetylation.According to the in-gel fluorescent imaging,we found that it has a much stronger labeling efficiency on cellular proteins compared with the aspirin probe.Combining sirtuin inhibitors with an acetylating reagent could increase the level of protein acetylation in cells.This new probe could be further utilized in the study of SIRT inhibitors combining with quantitative proteomic methods.