Screening And Identification Of Proteins That Interact With S-adenosylhomocystine Hydrolase From Dunaliella Salina

S-adenosylhomocysteine hydrolase (SAHH) which catalyzes reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine (Ado) and homocystene (Hcy) is an important enzyme in the methylation pathway. SAH is a common product of all adenosylmethionine-dependent transmethylations and suppresses SAM-dependent transmethylation. Thus, fast removal of SAH is prerequisite for efficient transmethylation reactions. SAHH has been investigated well as a feedback inhibitor of transmethylation and recently has been explored as a therapeutic target for anti-parasite, cancer and AIDS therapy.To study the molecular mechanisms of SAHH in Dunaliella salina, we constructed a bait vector pGBKT7-SAHH of D.salina for screening proteins that interact with SAHH of D.salina in yeast two-hybrid system before its self-activation activity and toxic effects were evaluated. Subsequently, its interaction with SAHH was confirmed by Far-Western blotting and His pull-down in vitro, and by coimmunoprecipitation in vivo.Materials and Methods1Screening of proteins that interact with S-adenosylhomocystine hydrolase by yeast two-hybrid assayPCR were employed to obtain the open reading frame (ORF) of the SAHH gene with EcoR I and BamH, and then it was cloned into the plasmid vectors pGBKT7. The bait vector pGBKT7-SAHH was transformed to yeast strains AH109and Y187by PEG/LiAc method, and then the fusion protein toxicity and self-activation were tested.Using the bait vector pGBKT7-SAHH, the proteins that interact with SAHH of D.salina were screened from cDNA library by yeast two-hybrid system. And then the yeast prey plasmids were extracted, and cotransformed to AH109with bait vector to confirming their interaction in yeast cell.2Confirmation of the interaction between SAHH and RACKlThe full length of the RACKl cDNA was obtained by5’full RACE. The ORF of RACKl was cloned to the pGEX-6p-1vector using EcoR I and Not I. And then the vectors pGEX-6p-1-RACKl and pET28a (+)-SAHH were transformed to competent cells of E.coli BL21to express recombination protein with soluble fusion proteins GST-RACK1and followed by his-SAHH purification.The interaction of SAHH with RACKl was confirmed by Far-Western blotting and His pull-down assay in vitro. Meanwhile, the vectors pCMV-HA-SAHH and pCMV-myc-RACKl were constructed and transfected to cell COS-7. The interaction of SAHH with RAKC1was identified via co-immunoprecipitation in vivo.3Function of RACKl in flagellar regenerationFlagella of D.salina were removed by pH shock and the cells were colleted every30min during flagellar regeneration. Total RNA of D.salina were extracted, and then reversedly transcripted to cDNA. Real time quantitive PCR was used to study the changes of RACKl at transcriptional level during flagellar regeneration.Results1yeast two-hybrid assayThe bait vector pGBKT7-SAHH was successfully transformed into yeast cells Y187and AH109, and the fusion proteins were not toxic to yeast cells AH109and Y187. In yeast cells AH109and Y187, the reporter genes HIS3and ADE2were not self-activated, but MELl was self-activated. Thus, the reporter genes HIS3and ADE2are optimal to screen interacting proteins of SAHH in yeast two-hybrid system.In yeast two-hybrid screen,255clones initially selected were retested for expression of a Ga14-dependent (3-galactosidase gene and3positive clonies were screened. Three new genes of D. salina, including receptor for activated C kinase1(RACKl), methionine aminopeptidase1(MetAPl) and eukaryotic elongation factorlα (eEFlα) were obtained.In the back-cross assay, vector pGADT7-RACKl and bait vector pGBKT7-SAHH were co-transformed to yeast AH109cells. These results showed that SAHH and RACKl interacted in yeast cell. 2Confirmed the interaction of SAHH and RACKlThe full-length RACKl cDNA(Genbank No. JN540811) of1175bp from D. salina was obtained, which consists of a51bp5’UTR, a167bp3’UTR and a975bp ORF. The vector pGEX6p-l1-RACKl was successfully constructed. The soluble fusion protein of GST-RACK1and his-SAHH were purified after identification.In vitro, the results of Far-Western blotting showed that SAHH was detected on spots in the membrane, indicating that SAHH and RACKl formed a complex. For His pull-down assay, the result revealed that the compound of SAHH and RACKl was identified by the native-PAGE.In vivo, the vectors pCMV-HA-SAHH and pCMV-myc-RACKl were co-transfected to COS-7cell. The result of co-immunoprecipitation showed that SAHH and RACKl interacted with each other in COS-7cell.3Function of RACKl in flagellar regenerationThe result of real time quantitive PCR demonstrated that expression level of the SAHH genes from D. salina was increased during flagellar regeneration, but RACKl is unchanged.ConclusionIn this study, the interaction of SAHH and RACKl has been confirmed both in vitro and in vivo, and RACKl may participate in the regulation of flagellum length mediated by SAHH.