| With the development of science and technology,proteomics has entered the scientific era of post-translational modification.Post-translational modification,as the key at protein level,alters protein activity or mutual interaction through dynamic,reversible chemical modification of specific amino acid residues in proteins.It also serves an important role in a number of key physiological activities such as the growth and metabolism of bacteria,signal transduction,stress resistance and drug resistance.At present,among the nearly 600 kinds of post-translational modification,the most frequently-used protein acylation modification belongs to lysine residues,including methylation,propionylation,malonation,butyrylation,myristoylation,acetylation and succinylation,etc.In order to further explore the acylation modification among different bacteria and its biological functions,this study,by using antibody affinity enrichment and high resolution mass spectrometry method,verified the distribution of modifications after protein translation in aquatic pathogen Aeromonas hydrophila,constructed the two acylation protein group spectrum of this bacteria respectively and discussed these modification sites and the function of modified proteins.Furthermore,by combining with bioinformatics,statistical analysis,immunology,molecular biology and other biological methods,this study compared and demonstrated from various angles of the distribution rules when the post-translational modification of these two proteins participates and modulates in several important metabolic pathways and the influence on protein conformation and related functions.Detailed research is carried out in the following aspects:First of all,this study applied antibody affinity technology to enrich and obtain the lysine-succinylated peptide of Aeromonas hydrophila,and identified a total of 2174 modification sites,covering 666 related proteins which composed of 16.2%of this protein species in database.The highly modified proteins are involved in the important regulating factors and energy supply hub when prokaryotes enter into translation process.Through the results obtained by bioinformatics study,8 modified conservative models were found,in which sequence Ksuc....R and Ksuc....K combination model scored higher and had certain preference to adjacent hydrophobic amino acids.According to the GO function enrichment and the subcellular localization analysis,it was found that the modified proteins mostly exist in the cytoplasm,and most of them are involved in the important functions such as core energy metabolism,biosynthesis,translation process and binding.In addition,the protein structure domain of most modified and enriched proteins were closely related to GO function and KEGG metabolic pathway and protein-protein interactions,including energy metabolism pathway,pentose phosphate metabolism,TCA cycle,glycolysis,and the carbon metabolism,pyrimidine metabolism,methane metabolism,ribosome,degradation of RNA,protein efflux of other key metabolic pathways.Besides,the stability of the modified protein was confirmed by the immunoprecipitation experiments of some proteins.These indicates that this species is likely to direct or indirectly regulate important metabolic pathways through post-translational modifications,and thereby plays a role in bacterial growth and adaptation to the external environment.Secondly,In order to further investigate the related protein level under other post-translational modification of Aeromonas hydrophila,by enrichment of the lysine-acetylated proteins,a total of 1058 aceylated lysine related proteins were identified,including 3451 peptides with this modification,which composed of 25.67%of the total protein database;Compared with succinylated protein result,the highly modified proteins have good consistency;The analysis results obtained by the study of bioinformatics,amont the 9 acetylated Motif models,sequence EKace and DKace combination model scored higher and have the preference to combine with the K,R that bear positive charge.Through the secondary structure analysis,it was found that both types of the acylation modification have the preference to certain domains of the alpha helix structure while surface affinity was lower than the succinylation modification.GO functional classification and KEGG enrichment analysis showed metabolic pathways that exist differences with succinylation pathway are:antibiotic biosynthesis,biosynthesis and metabolism of amino acid,fatty acid metabolism and degradation,terpenoid skeleton biosynthesis and degradation of benzoic acid.From these results,it is concluded that it is not coincidental for the occurance of the two kinds of modification to have good connections.It may play a role in the protein function complementary.The existence of the differences also indicates its important role in organisms including Aeromonas hydrophila growth,energy supply and other metabolic pathway in this study.Eventually,through the comparison of the two kinds of modification,S-ribosylhomocysteine lyase(LuxS)was used as quorum sensing type ? molecular signals as an example to investigate its function and acylation sites.In the study of this protein,two types of modifications were found on 3 lysine positions including:K23(Suc),K30(Suc)and K165(Suc,Ace),and K23 was predicted to be a polynucleotide binding region,while K30 and K165 are protein binding regions,which has certain effect on the overall protein hydrophilicity and hydrophobicity.In order to further investigate its protein biological function,Aeromonas hydrophila luxS gene deletion mutant was obtained by homologous recombination.After comparison with the wild type,its characteristics was identified such as the slow trend in growth,significant reduction of type ? autoinducers activity,biofilm formation,activity of the secretion of extracellular protease and antibiotic sensitivity,and the relatively enhancement of hemolysis.Further toxicity of detection studies on model organism;Mice and zebrafish showed that under high dose viable injection conditions,its pathogenicity was higher in trend than that of the wild type non-mutant.In order to test the influence on the function of the targeted protein that are modified with acylation,further experiments were performed to construct luxS gene complemented strains and complete the one by one single point mutation modification of the corresponding sites.Through the testing of the type ? signal molecule activity,it was found that K23 and K30 positions in E.coli under non-modified conditions has higher influences on the LuxS protein function compared to K165.However,in Aeromonas hydrophila mutant,K23 and K165 have higher influence than K30.This result indicates that different host bacteria dictates the position for modification and have certain effect on the related protein function.In addition,the sites mutation of LuxS also affects the formation of biofilm and the ability to compete with other bacteria.As conclusion,this research applied the proteomics methods to systematically compare and analyze the rules of the lysine succinylation and acetylation position in Aeromonas hydrophila,modified protein distribution and the difference in participating the related metabolite pathway.Furthermore,through the study of modified protein LuxS,its important role in the biological functions was disclosed.These findings not only deepen the understanding for the common acylation modification in bacteria,but also provide theoretical bases for the study of the functions and its participated metabolite pathway of the modified proteins.It could lead to potentially new strategy and new ideas for the control of diseases caused by these bacteria. |
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