Neddylation Modification And Regulation Of Succinate Dehydrogenase Subunit A

Background and objective:SDHA is a major catalytic subunit of succinate dehydrogenase,a complex of the mitochondrial respiratory chain.It participates in both the citric acid cycle and the electron transportchain and plays a key role in the process of energy metabolism.Mutations in this gene have been associated with a fatal inherited neurometabolic disorder known as Leigh Syndrome.The gene defect and abnormal expression of SDHA have been found in wild-type gastrointestinal stromal tumors,renal carcinoma,p araganglioma and pheochromocytoma.These studies indicate that the adjustment disorders of the protein stability of SDHA have been closely related to neurological diseases and many kinds of tumors,but the regulatory mechanisms of the protein stability of SDHA have not been reported so far.Some clinical studies have found that functional defects of SDHA mainly affect organs which have a vigorous energy metabolism,such as brain and muscle.The liver plays a major role in energy metabolism,of which normal mitochondria function is important for maintaining the normal physiological function,growth and development of the hepatocytes,however,the role of SDHA in it is unknown.BNL CL.2 cell,a mouse fetal liver cell line,is often used studying function of the hepatocytes.This subject adopts a trick called RNA interference mediated by the lentivirus to knockdown Sdha gene,and then investigates its effects on biological function of hepatocytes,developing our cognition of the imporotance of SDHA.Neddylation has been a hot area of research on post-translational modification for recent years.It has been involved in many regulatory processes in the cell,such as cell cycle,apoptosis,cell proliferation and differentiation,inflammation,DNA-repair and stress response.The main steps of the Neddylation pathway include NEDD8 precursor processing,activation by the E1(UBA3-APPBP1 heterodimer),loading onto the E2(UbcI2),conjugation to a substrate by an E3 and recycling of NEDD8 by an isopeptidase.In our previous work,we found that SDHA is a potential substrate of Neddylation,our subject verifies if SDHA can be neddylated by further study and investigates the regulatory effects of Neddylation on SDHA,thus to reveal regulatory mechanism of the protein stability of SDHA.Methods:1.The BNL CL.2 cells were transfected by two kinds of Sdha-shRNA lentivirus to knockdown Sdha gene.Then we observed the changes of cell proliferation rate,percentage of cells in every period of cell cycle and apoptosis.2.Expression vectors of Flag-NEDD8 and Myc-SDHA were transfected into BNL CL.2 cells,then we detected the modification of exogenous SDHA by immunoprecipitation and Western boltting.We detected the modification of endogenous SDHA in hepatocytes of wide-type mice and the mice lacking UBA3 by immunoprecipitation and Western boltting.3.We constructed four mutants of Sdha gene(Mut 1,Mut 2,Mut 3 and Mut 4),making lysines in one structural domain of SDHA unchanged and lysines in other structural domains mutate to arginines in turn,and then transfected them with Flag-NEDD8 into BNL CL.2 cells respectively,at last we detected the modification of mutants by immunoprecipitation and Western boltting to define which structural domain the modified sites reside in.4.First,we transfected HA-Ub with Myc-SDHA into BNL CL.2 cells,and then transfected Flag-NEDD8 or pcDNA3.1,after that,we investigated the effects of Neddylation on SDHA ubiquitylation by immunoprecipitation and Western boltting.The BNL CL.2 cells were divided into two groups,the control group was given DMSO,the other group was given a specific inhibitor of Neddylation called MLN4924,and an inhibitor of protein synthesis known as cycloheximid(CHX)was added to both of groups in time gradient,then we compared the half-life of SDHA in these two groups of BNL CL.2 cells by Western blotting,which can make us clear at effects of Neddylation on the protein stability of SDHA;We transfected Myc-SDHA into BNL CL.2 cells,adding DMSO into one of group,adding MLN4924 into the other group of cells for 24 hours,then we observed the subcellular localization of SDHA in two groups by immunofluorescence assay.Results and analysis:The reduction of SDHA expression can inhibit cell proliferataion of BNL CL.2 by arresting cell cycle;Both exogenous and endogenous SDHA can be modified by NEDD8,and the modified sites reside in the second structural domain of protein;Neddylation of SDHA has antagonistic effects on its ubiquitination,which can also stabilize SDHA,but can not change the subcellular localization of protein.In conclusion,this paper revealed that SDHA is important for maintaining biological functions of hepatocytes for the first time,and attempted to provide theoretical basis for the prevention and treatment of liver-related diseases.Besides,we found a new substrate of Neddylation——SDHA,mitochondrial protein,and we had a primary confirmation that which structural domain the modified sites reside in,which can be of important significance to kown the new function of neddylation;This paper illustrated the regulatory mechanism of protein stability of SDHA for the first time,and investigated other regulatory effects on SDHA,which can remind us of the complex functionalities of neddylation when we intend to target this pathway to treat relative diseases.