Effects Of Protein Disulfide Isomerase On Secretion Of Amyloidogenic Protein From Pichia Pastoris

Amyloid deposition disease is a disease caused by the misfolding of certain amyloidogenic protein.Instead of forming their natural and active structures,amyloidogenic proteins abnormally deposit in tissues and organs,causing disease eventually.These diseases include Parkinson's disease,Alzheimer's disease,cerebral amyloid angiopathy,etc.Pichia pastoris is the simplest eukaryote and the biomedical applications of this kind of yeast include production of therapeutic proteins and subunit vaccines.Protein disulfide isomerase(PDI)play a role of molecular chaperon and it can also help proteins to form disulfide bonds.Our preliminary results show that Pichia pastoris overexpressing PDI can significantly increase the expression of ?-syn and chicken cystatin C(c C),but little is known about the effect of knocking out or knocking down PDI on amyloidogenic protein expression.Studies shown that the PDI gene is an essential gene for yeast.Knockout of PDI will cause death of Saccharomyces cerevisiae.Therefore,MPDI gene,a PDI family member which has a high structural similarity with PDI,was selected to be knock out so as to avoid the lethality of yeast.In this study,a knock-out vector was constructed by homologous recombination,and was transformed into Pichia pastoris by electroporation to let homologous recombination spontaneously happen in the yeast cell,thus inactivate the target gene.Next,the recombinant plasmid vectors p PICZ?A-?-syn and p PICZ?A-c C for expression of foreign proteins were transferred to the normal Pichia pastoris GS115 strain and the MPDI knockout Pichia pastoris GS115-?MPDI strain by electroporation,totally,four recombinant strains were obtained.The expression difference of these two amyloid proteins in two Pichia strains was detected by immunoblotting reaction.On the other hand,to find out the key genes involved in protein expression and protein degradation pathways,RNA sequencing technology was used to analyze the differences in the expression levels of wild-type chicken cystatin C(WT),its amyloid mutant I66 Q,and unstable truncated mutant ?W in the normal Pichia pastoris strain and the strain overexpressing PDI.The results showed that when the MPDI-knockout Pichia pastoris strain expressed exogenous ?-syn,its intracellular amount was lower than that in wild-type Pichia pastoris,and no expression of ?-syn was detected extracellularly.The expression level of c C showed similar trend.146,468 and 368 differentially expressed genes were screened by pair comparison between the normal strains that express WT c C,I66 Q and?W and the PDI overexpression strains,among which 157,315 and 303 differentially expressed genes were annotated to the KEGG biological pathway,respectively.In a cell,when a protein is misfolded and its structure is altered,it can't form its natural conformation,the protein-degradation pathway in cell's stress protection system would be activated to degrade the misfolded protein.Among them,the ubiquitin-proteasome degradation pathway plays an important role.Therefore,when screening differential genes,we focused on genes involved in the ubiquitination pathway.In the WT VS PDIWT group,we did not find any genes involved in this pathway.In the I66 Q VS PDII66 Q group,we found that Gene3818 was involved in this pathway.Gene3818 and Gene4516 were found to be involved in this pathway in the ?W VS PDI-?W group.In conclusion,knocking out of MPDI reduced the expression level of ?-syn and c C,suggestting that Mpd1 cooperates with PDI to play the role of molecular chaperone.In the screening of key genes,although I66 Q is an amyloidogenic mutant,only one amino acid change occurred.Compared with wild-type c C,its conformation change was relatively small,so only gene3818 was activated under stress conditions for protein degradation.As for ?W,this protein is a ?-Heix2 shortened mutant,and the structural changes are relatively large,so gene3818 and gene4516 are activated under stress conditions to degrade the misfolded protein.The protein-degrading genes screened in this study can provide clues for further understanding of the degradation of amyloid and misfolded proteins,thus providing potential new molecular chaperone drug targets for the treatment of amyloid disease.