Study On Regulatory Effects Of Selenium Compounds On The Structure And Function Of Methionine-Containing Proteins And Glutathione Peroxidase 4

To establish new methods of photoanalytical chemistry for exploring the regulatory effects of small molecules on key proteins in life processes has great significance,which is also a trend and hotspot in the field of analytical chemistry in life science.Proteins are one of the most vitally organic substances and basiclly functional units that constitute organisms which play multiple roles in life activity such as transportation of substances(hemoglobin's carrying oxygen),catalysis of life reactions(enzymes),formation of biological materials(muscle,bone,hair,etc.)and occurrence of immune response.Generallly,proteins are capable to bind to small-molecule ligands specifically,such as substrates,inhibitors,cofactors,carbohydrates,etc.,or bind to other proteins.Therefore,to determining and analyzing the interactions between proteins and small molecules are crucial issues to be solved in the fields of biochemistry,drug development and biosensing.Methionine,an essential amino acid,plays significant biological functions in organisms.Methionine participates in protein synthesis,such as bovine serum albumin(BSA),calmodulin(Ca M),and?-Lactalbumin(?-La).In addition,methionine residues form S=N bonds with lysine residues,which contribute to the stability of collagen ?.Moreover,methionine is also involved in cells'physiological functions,including serving as the precursor of glutathione,forming polyamines spermine and spermidine,presenting as the major source of methyl groups for methylation of DNA and other molecules.Recently,it was found that dietary methionine restriction in animals and cell culture media provides metabolic benefits such as suppressing inflammatory response,reducing adiposity,decreasing oxidative stress,increasing insulin sensitivity,and extending lifespan.However,methionine cannot be generated endogenously in living organism,but only from the diet.Therefore,rationalizing the intake of methionine in diet and timely tracking the physiological activities of methionine participated in living system will be of great significance for sustaining healthy life.In mammals,more than 20 kinds of proteins contain selenocysteine(Sec)which named selenoproteins.Notebly,glutathione peroxidase 4(GPX4)has been widely studied as a type of selenoprotein.GPX4 is discovered in 1982 which served as a cytoplasmic peroxidation inhibitor and regarded as a specific target for new drug treatments to activating or inhibiting cell death in cancer or degenerative diseases,especially in ferroptosis.Ferroptosis was created by Dixon et al.in 2012 which is a regulated form of cell death induced by erastin and characterized by the accumulation of iron-dependent lipid peroxides to lethal levels.Erastin-induced ferroptosis was mainly regulated by inhibiting the input of cystine,then leading to the depletion of glutathione(GSH)and the inactivation of GPX4,which ultimately leads to cell death.Based on the significant fuction of methionine-containing proteins and GPX4,we believe that deep and accurate analytical-methods for exploring the regulatory effects of chemical small-molecules on these proteins are urgent needed during life process.Selenium plays an important role in preventing cancer,cardiovascular and neurodegenerative diseases.In organisms,selenium compounds usually include selenite,selenomethionine,methylselenocysteine and selenocysteine(Sec)which exerted their biological activities depend on their different forms and metabolites.Moreover,the metabolic pathway of selenium compounds and the relative abundance of their metabolites are both closely related to the efficacy of selenium compounds in disease prevention and treatment.Significantly,all dietary selenium compounds in orginasims can generate selenoproteins and methylated selenium compounds for excretion,and the metabolic pathways of selenium compounds intersect with the common metabolite which is generally refered to hydrogen selenide(H₂Se).Therefore,H₂Se is considered as a key small-molecule for selenium to acting significant functions in life processes.Hence,this research intends to focusing on the regulatory effects of two selenium-containing compounds(H₂Se and Sec)on the structure and function of methionine-containing proteins and GPX4,and will be divided into the following parts:(1)The sulfilimine bond(S=N),found in the collagen ? scaffold,significantly stabilizes the architecture via the formation of sulfilimine cross-links.However,precisely governing the formation and breakup process of the sulfilimine bond in living organisms for better life functions still remains a challenge.Hence,we established a new way to regulate the breaking and formation of the sulfilimine bond through hydrogen selenide(H₂Se)and hypobromous acid(HOBr),which can be easily controlled at simulated physiological conditions.This novel strategy provides a circulation regulation system to modulate the sulfilimine bond in peptides and NC1 hexamers,which can offer a substantial system for further study of the physiological function of collagen ?.(2)Sodium selenite(Na₂SeO₃)has alleviating effects on liver fibrosis;however,its therapeutic molecular mechanism remains unclear.Herein,hydrogen selenide(H₂Se),a major metabolite of Na₂SeO₃,was tested to uncouple the sulfilimine bond in collagen ?,the biomarker of liver fibrosis.A mouse model of liver fibrosis was constructed via a CCl₄-induced method,followed by the administration of 0.2 mg kg^-1 Na₂SeO₃via gavage three times per week for 4 weeks.Changes in H₂Se,NADPH,and H₂O₂levels were monitored in real time by using NIR-H₂Se,DCI-MQ-NADPH,and H₂O₂probes in vivo,respectively.H₂Se continuously accumulated in the liver throughout the Na₂SeO₃treatment period,but the levels of NADPH and H₂O₂decreased.The expression of collagen ? was analyzed through western blot and liquid chromatography-mass spectrometry.Results confirmed that the sulfilimine bond of collagen ? in the fibrotic mouse livers could be broken by H₂Se with the Na₂SeO₃treatment.Therefore,the therapeutic effect of Na₂SeO₃on liver fibrosis could be mainly attributed to H₂Se that uncoupled the sulfilimine bond to induce collagen ? degradation.This study provided a reasonable explanation for the molecular mechanism of the in vivo function of Na₂SeO₃and the prevention of liver fibrosis by administering inorganic selenium.(3)To develop a simple,fast and low-toxic method for identification and separation of specific proteins from complex living systems remains a great challenge.Herein,a nanocomposite(Fe₃O₄@Au-Se-peptide)was designed and synthesized to fish out methionine-containing proteins based on a non-enzyme biochemical reaction,which couples amino of lysine with S-methyl of methionine in the presence of HOBr.Peptides which contains four lysine(Lys-Lys-Lys-Lys-{Se-Cys})linked to the Fe₃O₄@Au nanocomposites were used to capture methionine residues efficiently via a S=N crosslinking.The methionine-containing protein was obtained by magnetic separation,and released from the Fe₃O₄@Au-Se-peptide nanocomposites with the influence of H₂Se.The HRMS and SDS-PAGE results confirmed the methionine-containing protein could be successfully fished out from a complex biological system.This work provides a significant strategy for recognition and separation of specific proteins from complex living systems.(4)Glutathione peroxidase 4(GPX4)is a significant regulatory protein duiring ferroptosis process.It is still a great challenge to develop accurate methods to achieve visual characterization of GPX4.In this chapter,by realizing the fluorescence imaging of the active molecule of GPX4,selenocysteine(Sec),we established a correlation between fluorescence signals of Sec and functional expressions of GPX4,and thus providing an intuitional and visual method for monitoring GPX4.In this work,Au-Se bonds nanoprobes were constructed to achieve precise detection of Sec,and the fluorescence signal of Sec can directly indicate the expression of GPX4before and after ferroptosis in cells.The visualization results show that the fluorescence signal of Sec is positively correlated with the functional expression of GPX4.Meanwhile,the Au-S bonds nanoprobes were desigened to detect GSH during this process.Fluorescence imaging results show that in normal cells,the presence of GSH cannot up-regulate the expression of GPX4;after ferroptosis of cells,supplementation of GSH can stabilize the functional and expressional level of GPX4.This study provides a fluorescent method to monitor the intracellular GPX4 and supports an intuitional and effective method for prediction the functional expression of intracellular GPX4.