The Study Of SELENOF Biological Function Using SELENOF Recombinant Expressed Proteins And Knock-out Mice

Selenium,as an essential trace element in living organisms,has the functions of enhancing human immunity,reducing viral infection,and preventing cancer.In vivo,the biological function of selenium(Se)is mainly achieved in the form of selenoprotein.So far,25 selenoproteins have been found in the human body.In vivo,selenocysteine plays an important role in redox reactions because selenocysteine is more active and avid than cysteine.Thus,the redox function of selenoprotein has been well studied,especially the most well-known selenoprotein families Trx Rs(thioredoxin oxidoreductase family),GPXs(glutathione peroxidase family)and DIOs(deiodinase family)..In contrast,little is known about the exact structure and function of other selenoproteins.This is also the case with SELENOF(selenoprotein F).Previous studies reported that SELENOF is a member of the Trx-like folding superfamily,which can bind UGGT(UDP-glucose: glycoprotein glucosyltransferase)tightly through its N-terminal Cys-rich region and enhance UGGT's enzymatic activity.It is speculated that SELENOF and UGGT can participate in the processing of glycoprotein folding in ER(endoplasmic reticulum).Becides SELENOF is structurally similar to the PDI(disulfide isomerase),and has a surface containing Redox-active motif(-Cx U-)of Sec(selenocysteine).Although the inquiry into the SELENOF structure has become more and more complete,its potential function has not yet been verified and its biological effects have not yet been discovered.In order to verify the function of SELENOF and to explore the potential role of SELENOF,this article first expressed and purified SELENOF(cysteine in place of selenocysteine,Sec?Cys)and tested for thioredoxin-related activity.Then we used protein in vitro binding assays(Pull-down)to find interacting proteins and performed bioinformatics analysis.In order to further clarify its role in vivo,we performed proteomic studies on a SELENOF knockout mouse model.Using relative and absolute quantitative isotopes(i TRAQ)method,the differentially expressed proteins in the hippocampus and liver tissue of KO mice were detected and bioinformatics analysis was performed.Finally,we used PET/CT,tissue section staining and other methods to further validate the i TRAQ results.Also,differentially expressed proteins from i TRAQ and proteins identified in the Pull-down results were compared to provide experimental data for the SELENOF biological function study.After successful purification of the SELENOF(sec ? cys)protein in vitro,the activity assay showed that SELENOF was a substrate for Trx R and Trx,respectively,also it interacted with GSSG and hydrogen peroxide.These indicated that SELENOF is functionally similar to the Trx superfamily and has redox activity.The Trx superfamily mainly affects protein folding through redox disulfide bonds,and we found SELENOF does not possess this function from the SELENOF and insulin response experiments.Therefore,SELENOF is suspected to participate in protein quality control mechanisms through other means.A total of 109 proteins interacting with SELENOF were identified in Pull-down experiments.A total of 86 differentially expressed proteins were screened in the i TRAQ results of hippocampal tissues,of which 50 were up-regulated and 36 were down-regulated.A total of 69 differentially expressed proteins were screened in the liver tissue,of which 17 were up-regulated and 52 were down-regulated.The bioinformatics analysis showed that the proteins identified in Pull-down experiments were involved in...The knockout of SELENOF caused up-regulation of the pentose phosphate pathway in the hippocampus,while the level of glucose metabolism in the liver was down-regulated.Overall,the KO mice exhibited a decrease in blood glucose tolerance,indicating that SELENOF affects the glucose metabolism process including the pentose phosphate pathway.At the same time,SELENOF plays a role in lipid metabolism by affecting biological processes such as cholesterol synthesis and apolipoprotein synthesis.In the KO mice sebaceous lipid metabolism was reduced,and abnormal fat symptoms were observed after high-fat diet.This suggested that the knockout of SELENOF will lead to a decrease in lipid metabolism,which will lead to more accumulation of fat.Furthermore,proteins in the phagocytosis related pathways were down-regulated in SELENOF deficient mice.These results suggested that SELENOF mainly affects proteins involved in protein andribose synthesis,endocytosis,glucose metabolism,lipid metabolism and other biological processes.In summary,this study verified the structure-based redox function of SELENOF,systematic identified and compared the SELENOF interaction proteins and differentially expressed proteins caused by gene knockout.Our results provide foundation for the future SELENOF functional research.