Identification Of Disulfide Bonds Mediate Arabidopsis Cryptochrome2 Dimerization And Functional Analysis Of Cryptochrome2 Inhibitors BICs

Cryptochrome,also known as blue light/UV receptor,is a homologous protein of E.coli photolyase with FAD(flavin adenine dinucleotide)as its prosthetic group.Arabidopsis CRY protein has two domains:The N-terminal domain sequence is similar to that of E.coli photolyase,but it does not possess DNA repair activity,so it is also known as PHR(Photolyase-Homologous Region)domain.C-terminal domain is also called CCE domain(Cryptochrome C-terminal Extension),the domain shows a instability,less conservative,is the intramolecular signal output of the region,also known as the effector domain.At present,the accurate molecular mechanism of cryptochrome initial photoreactions such as the detailed molecular mechanism of photoactivation,the mechanism by which BICs,a inhibitor of CRY2,suppress the CRY2 dimerization and the mechanism of cryptochromes involved in the regulation of circadian clock network remain to be further studied.Studies have shown that the PHR domain is a homodimerisation region of CRY and that photoactivated CRY exerts its physiological function as a dimer,therefore,in-depth study of the dimerization mechanism of CRY in Arabidopsis is of great significance for understanding the mechanism of photoactivation of CRY and its downstream signaling pathways.Previous research in laboratory is based on the construction of the mutation of all cysteines of CRY2 and the use of various redox agents(?-mercaptoethanol,N-ethylmaleimide,polyethylene glycol maleimide)which was chemical modification demonstrated the formation of blue light-induced CRY2 dimerization and oligomerization was formed by cysteine-mediated disulfide bonds.This paper is based on the above research on the disulfide bonds which regulate the formation of blue light-induced CRY2 dimerization and oligomerization was identified.By using highly efficient and convenient HEK293T cells as a primary research tool,combined with the improved Split-Luciferase(luciferase complementation)system to analyze the expression of fluorescence values and BiFC(bimolecular fluorescence complementary technology)technology to observe the formation of blue light-induced CRY2 photobodies,identify the disulfide bonds mediate the CRY2 dimerization.The cysteine sites forming disulfide bonds that mediate the formation of CRY2 dimers and oligomers were further verified in Arabidopsis plants by the AGROBEST method.Disulfide bonds play an important role in maintaining the advanced structure of the protein and its functional activity,accurate identification of disulfide bonds in protein and protein complexes also requires mass spectrometry and software analysis,in this study,the disulfide bonds mediating CRY2 dimer formation was also preliminarily analysis and identified by IP-MS tandem assay.BICs as negative regulators of CRY2 photosensitivity has been demonstrated that BIC1 interacts directly with CRY2,There is evidence BIC1 binds to CRY2 to suppress the blue light-dependent dimerization,photobody formation,phosphorylation,degradation,and physiological activities of CRY2.In this study,the action mechanism of BICs and CRY2 was preliminarily studied by constructing BICs-CRY2 expression vectors in HEK293T cells,this study provides the basis for further study of photoactivation and inactivation of CRY2.Through the above research,the following conclusions are obtained:1.Through optimized HEK293T cell expression system,all the 11 cysteine single mutation of Arabidopsis CRY2 were constructed,CRY2 39th,CRY2 89th,CRY2 158th,CRY2 187th and CRY2 583th cysteines were identified the disulfide bonds formed by these five cysteines participate in the formation of CRY2 dimer,and the CRY2 89th cysteine is the key to forming the disulfide bond of CRY2 dimer.2.The BiFC experiment was used to observe the formation of blue light-induced CRY2 photobodies.It was found that CRY2 39th,CRY2 89th,CRY2 158th,CRY2 187th and CRY2 583th cysteines these five cysteines not only regulate the formation of CRY2 dimers and oligomers,but also participate in the formation of photobodies.3.Constructing plant expression vector of Split-Luciferase system and infecting cry2 mutant Arabidopsis by AGROBEST method,the results showed that the fluorescence values of CRY2 C39S,CRY2 C89S,CRY2 C158S,CRY2 C187S and CRY2 C583S cysteine single mutation has decreased to varying degree.These results confirmed in vivo from Arabidopsis plants that the disulfide bonds formed by these 5 cysteines mediate CRY2 dimerization.4.Dimerization of CRY2 is inhibited by BIC1 in a dose-dependent manner within a certain range,and it was identified that the interaction between BICs and CRY2 was not formed by cysteine-mediated competitive disulfide bond,the detailed molecular mechanism needs further study.