Study On The Molecular Domain-mediated Interaction Of Cellular Prion Protein With Flotfllins By Using Yeast Two-hybrid System

Spongiform encephalopathy is caused by the conversion of the host cellular prion protein (PrPc)into scrapie prion protein (PrPsc). PrPc is a membrane-associated glycoprotein widely expressed on avariety of cells and involving lots of cellular activities, for example, signaling transduction. Flotillinsincluding Flotillin-1and-2are highly conserved lipid-raft-associated proteins which form their own typeof non-caveolar microdomains where PrPccan be converted into PrPscand PrPcexerts its physiologicalfunction. Flotillins form a scaffold onto which many signaling molecules are recruited to and interacteach other. In this study, we focused on to figure out the molecular domain involving in the interactionbetween PrPcand Flotillins to provide scientific information for the design of novel medicines targetingto block the conversion of PrPcto PrPsc.Open reading frame (ORF) of PrPc、Flotillin-1and-2were amplified from bovine brain tissue. Thegenes encoding mature prion protein (PrP25-241) and the protease-resistant core of PrPsc,PrP27-30(PrP102-241) were amplified and fused with GAL4yeast two-hybrid system3Prey vector pGADT7(DNA-AD). The genes encoding eight mutants of Flotillin-1(amino acid residues:36-428aa，1-331aa，1-161aa，151-428aa，1-134aa，36-161aa，1-151aa and331-428aa), the ORF of Flotillin-1and-2wereinserted into Bait vector pGBKT7(DNA-BD). The fusion Prey and Bait constructs were confirmed bysequencing. Analysis of autonomous activation and toxicity of fusion constructs: Independentlytransform all the fusion constructs into strain AH109. Assay the transformants for autonomousactivation by selecting for transformants on SD/-Trp/X-α-Gal、 SD/-Leu/X-α-Gal、SD/-His/-Trp/X-α-Gal、SD/-His/-Leu/X-α-Gal、SD/-Trp/-Ade/X-α-Gal and SD/-Leu/-Ade/X-α-Gal,respectively. If transformant colonies are blue, it means the occurence of autonomous activation. Thetransformants containing fusion prey constructs of PrP25-241or PrP102-241were inoculated intoSD/-Leu/Amp (50μg/ml) medium and the transformants containing fusion bait constructs of Flotillin-1,its mutants or Flotillin-2were inoculated into SD/-Trp/Kan(50μg/ml), respectively. The Toxicity effectsof the fusion constructs on host strain AH109were evaluated according to cell growth monitored byoptical density at600nm wavelength (OD600). Fusion prey constructs of Prion and fusion baitconstructs of Flotillins were put together by twos and transformed into strain AH109competent cells bysmall scale PEG/LiAc transformation. The transformants were assayed by selecting for them onSD/-Leu/-Trp, SD/-His/-Leu/-Trp/X-α-Gal and SD/-Ade/-His/-Leu/-Trp/X-α-Gal agar plates,respectively. The targeting genes from the blue colonies growing on SD/-Ade/-His/-Leu/-Trp/X-α-Galwere amplified and sequenced. The corresponding transformants were inoculated intoSD/-Ade/-His/-Leu/-Trp medium to grow for6hours at30℃. Then yeast proteins were extracted byurea/SDS and subjected to western-blots analysis with the monoclonal antibodies against HA (pGADT7)or Myc (pGBKT7) epitopes to confirm the expressed proteins by strain AH109after co-transformation.pCL1、pGBKT-53、pGADT7-T、pGBKT and pGADT7were performed in parallel as the positive andnegative controls.Sequencing assays indicated that genes encoding PrP25-241and PrP102-241were fused with Prey vector pGADT7and genes encoding Flotillin-1and its eight mutants and Flotillin-2were inserted intoBait vector pGBKT7in frame. Those fusion constructs could not cause toxic effects on host strainAH109and also failed to activate ADE2、HIS3and MEL1report genes. Such transformants could formblue colonies on SD/-Ade/-His/-Leu/-Trp/X-α-Gal agar plates after co-transformation of fusion preyconstruct containing gene encoding PrP25-241(pGADT7-PrP25-241) and the bait construct carryinggene encoding Flotillin-1(pGBKT7-Flotillin-1). This phenotype meant that there was strong interactionbetween PrP25-241and Flotillin-1. Those transformants could only form blue colonies onSD/-His/-Leu/-Trp/X-α-Gal after co-transformation of pGADT7-PrP25-241and the fusion baitconstruct carrying gene encoding Flotillin-2(pGBKT7-Flotillin-2). Therefore, this meant that the weakinteraction existed between PrP25-241and Flotillin-2. There were no any blue colonies forming on theSD/-His/-Leu/-Trp/X-α-Gal and SD/-Ade/-His/-Leu/-Trp/X-α-Gal after co-transformation of fusion preyconstruct containing gene encoding PrP102-241(pGADT7-PrP102-241) and pGBKT7-Flotillin-1orpGBKT7-Flotillin-2. Such phenomena showed there was no any interaction between PrP102-241andFlotillin-1or Flotillin-2. The transformants formed blue colonies on SD/-Ade/-His/-Leu/-Trp/X-α-Galafter co-transformation of pGADT7-PrP25-241and the fusion bait constructs carrying the genesencoding the Flotillin-1mutants consisting of the amino acid residues36-428aa,151-428aa,1-331aa or1-161aa. Those results indicated that PrP25-241could interact with the fragments36-428aa,151-428aa,1-331aa and1-161aa of Flotillin-1and the interaction domain was located between amino acid residues151-161of Flotillin-1. The sequencing of PCR products confirmed that the genes responsible for theinteraction proteins in strain AH109were as expected. The results of Western-blots assay furtherconfirmed that the proteins involving the interaction in strain AH109were also as expected.The strong interaction between bovine mature prion protein and Flotillin-1, and the interactiondomain were confirmed by using GAL4yeast two-hybrid system3and mutant constructs in our study.The151-161amino acid fragment responsible for the interaction is the highly conservedphosphorylation site of Flotillin-1. Our research results laid foundation for designing new medicinestargeting to inhibit or reverse PrPcto PrPscconversion.