The Study Of Conformational Changes Of Autophagy-related Protein LC3B Using SDSL-EPR Technology

Autophagy is a catabolic pathway to maintain the requirements of metabolism and the renewals of organelles through degradation and recycling of cellular macromolecules and organelles in eukaryotic cells,which plays a critical role in various biological activities of organism.Autophagy induced by ionizing radiation via damage of mitochondria and accumulation of reactive oxygen species?ROS?,fulfills cytoprotective and cytotoxic function on cell homeostasis and fate.The study of autophagy involving a mechanism for effects of ROS is an important topic in recent years.Microtubule-associated protein 1 light chain 3B?MAP1LC3B/LC3B?,derived from mammalian cells,is associated with regulation of autophagy by contributing to autophagosome development and maturation and is used to monitor autophagic activity.The study of the conformational characteristics of LC3B protein and on which the effects of ROS will be helpful to understand the regulatory mechanisms of autophagy induced by radiation/ROS.Site-directed spin labeling-electron paramagnetic resonance?SDSL-EPR?technology,having such characters as that molecular weight is not limited and it is capable of studying proteins in solution,etc,could measure the mobility and dynamic structural characteristics of biomacromolecules.Currently,this technology is one of the important methods to analyze structures and mobility of proteins in physiological environments.To obtain the conformational features of specific amino acid residue sites in physiological environment by SDSL-EPR technology,will contribute to reveal the molecular mechanisms of interaction between proteins.The structure and conformational changes of protein molecule are closely related to the characteristics and functions of protein.It is more practical significant to obtain structural information of protein molecules in solution.At present,more studies have involved the biological functions of LC3B protein,but its structure and the dynamic changes of conformation in physiological environment are still unclear.Therefore,in order to study the relationships between function and structure of LC3B protein in solution by means of SDSL-EPR technology and to lay foundation for exploring the molecular mechanism of autophagy,the main aim of this work is to establish site mutantion models of LC3B protein thereby to spin label the specific sites and analyze their conformational characteristics.Methods:?1?Construction of the LC3B-I of specific sites mutantion protein models:Based on the crystal structure of LC3B protein,single/double cysteine?Cys?mutation sites were designed in the functionally active domain;According to wild-type human-derived LC3B gene,specific residue sites of LC3B were mutated to Cys by using sequence overlapped extension PCR?SOE-PCR?and cloned into p ET-22b prokaryotic expression vector and transferred into E.coli BL21?DE3?for protein expression.The LC3B mutants were then purified by STREP column affinity chromatography;Protein activity was identified to select out mutants with biological activity.?2?Construction of the LC3B-II of specific sites mutantion protein model in vitro:The p ET28a-ATG3 prokaryotic vector was transferred into BL21?DE3?to express E2-like enzyme ATG3,and ATG3 protein was then purified by nickel column affinity chromatography;The p ET28b-Tat-ATG7 prokaryotic vector with the HIV-1 virus-encoded trans-activator transduction?Tat?core peptide?‘GRKKRRQRRR'?was constructed and transferred into BL21?DE3?to express E1-like enzyme ATG7,and ATG7 protein was then purified by nickel column affinity chromatography;The concentrations of ATP,liposome and each protein and the conditions of mixed incubation were optimized to construct LC3B-II mutants in vitro;Reconstituted LC3B-II mutants were purified according to sucrose gradient centrifugation.?3?The effect of ROS on LC3B protein:Different residue sites of LC3B-I mutants were spin labeled with2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate?MTSL?and measured by EPR before and after adding with a final concentration of 50?1000 u M H₂O₂;In vitro reconstitution of LC3B-II protein,H₂O₂ were added into the reaction mixture at different concentrations for having a knowledge of influences on the reconstitution efficiency of LC3B-II protein in vitro by ROS;The spectrum fitting program based on Easy Spin package was used to analyze the different components and motility parameters of the spectra in order to study the characteristics of the mobility of LC3B protein.Results:?1?A series of single/double Cys mutation sites were designed around the HP1 and HP2 domain as well as between the?1 helix and NHD domain in the LC3B protein.The LC3B-I mutants were expressed and purified and soluble LC3B-I mutant protein model was successfully constructed.?2?The p ET28b-Tat-ATG7 vector was constructed to solve the problem of insoluble expression of ATG7 protein in the form of inclusion bodies by the prokaryotic expression system.The ATG3 and ATG7 proteins,which are the key auxiliary proteins during the conversion from LC3B-I protein to LC3B-II protein,were efficiently expressed and purified in soluble status;An in vitro reconstitution system of LC3B-II protein model was constructed thereby the LC3B-II protein was successfully purified.?3?The selected residue sites were successfully spin labeled and analyzed with EPR for motion correlation time in LC3B-I protein Therefore the conformational information of the selected residue sites in solution was obtained in LC3B-I protein.The preliminary results suggested that there might exist 2-3 different conformational characteristics in some selected sites of the functional domain in LC3B protein in solution.?4?No obvious effect was found on the conformational feature of mutation site in LC3B-I protein or on the efficiency reconstitution of LC3B-II protein in vitro after certain concentration of ROS was introduced.Conclusion:In this work,it was the first attempt to study the conformational characteristics of specific sites in the autophagy-related protein LC3B in solution using SDSL-EPR technology,which has laid an experimental foundation and provided a novel method for deeply exploring the structural basis and mechanism of regulating autophagy of LC3B protein;The preliminary results of this study could provide a reference for understanding the mechanism of autophagy from the perspective of LC3B protein.