Molecular Mechanism Of The Interaction Between Selenoprotein TrxR1 And Caveolin-1

Thioredoxin reductase(TrxR)is involved in multiple biological processes by regulating the redox state of thioredoxin(Trx).Caveolin-1 is the core protein during caveolae formation.Moreover,caveolin-1 is included in cell signal transduction,cell metabolism,and cholesterol homeostasis.In the previous study,Caveolin-1 interacts with TrxR1 and induces premature senescence under oxidative stress.However,the structural basis and the molecular mechanism of the interaction between Caveolin-1 and TrxR1 still unclear.In this paper,we examined the inhibition of TrxR1 by the CSD peptide in vitro.Meanwhile,using recombinant rat TrxR1,we carried out the characterizations TrxR1 in the catalytic mechanism and screened out a natural irreversible inhibitor of TrxR1.Compared with the negative control CAV-X,CSD peptide significantly inhibited TrxR1 activity,including DTNB,9,10 PQ and Juglone reducing activity.To identify the critical amino acid of TrxR1 inhibited by CSD polypeptide,we took a single point mutant of aromatic amino acids in the TrxR CBM domain,including Y402 A,Y402F,F405 A,F406A,W407 A,W407F,W411 A,and W411 F.Using TrxR1 variants,we found that some variants,like Y402 A,F405A,F406 A,W411A,and W411 F enhanced TrxR1's resistance on CSD polypeptide,suggesting Y402,F405,F406,and W411 may take part in the interaction between TrxR1 and CSD peptide.What's more,mutation at W407 did not affect the inhibitory effect.The TrxR1 CBM region are located at positions of 402-411,which belongs to the unique‘guiding bar' structure of TrxR1.Therefore,we tested whether the TrxR1 variants could affect the reductive half of the catalytic cycle.The results showed that the TrxR1 CBM region mutation reduced TrxR1 DTNB reduction activity,especially the F406 A mutation,the enzyme activity was only 20% of the wild type enzyme,which indicated that ‘guiding bar' plays a vital role in TrxR1 catalytic.In addition,we explored whether the addition of chelating agents could protect or rescue TrxR1 activity which was impaired by trace metal ions.Based on the previous study,we propose to add 20 m M EDTA during the purification process to protect ADP-Sepharose chromatography column as well as TrxR1 activity and improve the quality of selenoprotein preparation.Besides,using recombinant TrxR1,we explored the molecular mechanism of TrxR1 mediated menadione reduction and revealed the role of TrxR1's catalytic sites in the reduction of menadione and the redox cycle of menadione is asssciated with oxygen and generates reactive oxygen species.Finally,we found that the natural product chlorophyll and the synthetic pigment chlorophyll copper sodium are irreversible inhibitors of TrxR1 which screening form natural pigments.In summary,we found(1)CSD peptide inhibits TrxR1 activity and depends on aromatic amino acids at positions 402,405,406,and 411.Mutation of these four positions to alanine will enhance TrxR1's resistance to CSD peptide;(2)Chelating agents such as EDTA could protect TrxR from metal ion damage,and adding chelating agents could improve the quality of selenoprotein preparation;(3)TrxR1 mediated menadione reduction is in a sec-independent manner and produces superoxide anion;(4)Sodium copper chlorophyllin irreversibly inhibits TrxR1 activity.This study explored the interaction mechanism of TrxR1-CAV1 and provided a theoretical basis for further revealing the biological functions of TrxR1 and related research.