The Biochemical Mechanism Of Host Delipidation Mediated By Yersinia Effector YopT

The complex interactions between pathogen and host is the fundamental elements of infectious diseases.Studies have found that many pathogens can inject a series of toxic effectors into host cells through a special secretion system during infection.These proteins act as hydrolases and delipidate key lipidated proteins of host cells,making them lose their normal biological functions.Among the effectors,the Yersinia effector Yop T cleaves S-isopentenylated Rho GTPases and releases them from the plasma membrane,leading to destructions of host cytoskeletons and serious cytotoxicity.However,the mechanisms of how the effector Yop T recognizes and delipidates the host substrates are not clear.Therefore,studying the detailed recognition and cleavage mechanisms of Yop T’s hydrolysis of host lipidated substrates will help to deeply understand the biological significance of host delipidation by pathogens,which is crucial for the understanding and treatment discovery of infectious diseases.In this project,Yersinia effector Yop T was selected as the research target to explore the biochemical mechanisms of Yop T recognition and hydrolysis of lipidated protein K-Ras4 B.The main research methods and results of the thesis are as follows:(1)Using cell biology methods and enzyme cleavage experiments in vitro,we found that Yop T can release K-Ras4 B from the cell membrane,which proves that lipidated K-Ras4 B is a substrate of Yop T.(2)Through transfection and cleavage efficiency analysis of K-Ras4 B mutants in vitro,we revealed the specificity of Yop T to hydrolyze K-Ras4 B,that is Yop T can cleave farnesylated/geranylated K-Ras4 B,but prefers farnesylated K-Ras4B;Yop T does not distinguish the nucleotide bound conformations of K-Ras4B;the hindrance of aa X removal will destroy the hydrolysis activity of Yop T on K-Ras4 B.(3)Through transfection of truncated mutants of Yop T,we proved that the N-terminal 75-100 residues of Yop T was important for the membrane localization.(4)Through the bioinformatics structure predictions,Yop T has a broad-spectrum substrates besides Rho GTPases and K-Ras4 B in cells,among which,some proteins were selected and verified.(5)Using chemical synthesis and high performance liquid chromatography analysis,we proved that Yop T can cleave C-terminal peptides of prenylated K-Ras4 B in vitro.To sum up,the results of this thesis can lay a foundation for revealing the mechanisms of Yersinia effector protein Yop T hydrolyzing host lipidated proteins,and provide important scientific basis for anti-bacterial drug discovery against Yersinia.