目的 探讨PrP蛋白和PrP106-126多肽对14-3-3蛋白二聚体形成的影响 方法 将重组表达纯化的0.25 μg、0.5 μg和1μg PrP蛋白、人工合成的PrP06-126肽分别与重组的14-3-3β共孵育,分别通过非变性聚丙烯酰胺凝胶的Western Blott检测PrP蛋白和PrP106-126肽对14-3-3β二聚体和多聚体的形成影响.将不同浓度的PrP与250 μmol/L的PrP106-126肽和14-3-3蛋白共孵育,检测PrP蛋白对PrP106-126肽对14-3-3β二聚体和多聚体的形成影响.随后,将200μmol/L、100 μmol/L、50 μmol/L的PrP106-126多肽分别作用于HeLa细胞8h后,检测细胞内的14-3-3二聚体形成.结果 PrP蛋白与14-3-3蛋白共孵育后,14-3-3二聚体和多聚体的信号随着PrP浓度的增加明显增强；PrP106-126多肽共孵育时,14-3-3二聚体和多聚体的信号随着PrP浓度的增加而减弱；不同浓度的PrP蛋白可以拮抗PrP106-126多肽对14-3-3β二聚体的形成.而且PrP106-126多肽可以干扰HeLa细胞中的二聚体形成.结论 PrP蛋白促进14-3-3二聚体的形成,而PrP106-126多肽干扰二聚体的形成,且PrP蛋白具有拮抗PrP106-126肽干扰14-3-3二聚体形成的作用.%Objective To investigate both PrP and PrP106-126 peptide effect on 14-3-3β dimeration.Methods 14-3-3β were incubated with different does recombinant PrP or PrP106-126 peptide,both 14-3-3β dimer and polymer were separated 15％ non-denaturing polyacrylamide gel electrophoresis (PAGE) and the 14-3-3 dimers were evaluated using 14-3-3β-specific Western blotting.And then,14-3-3β dimeration buffer were incubated with different does recombinant PrP and 250 μmol/L PrP106-126 peptide,14-3-3β dimer and polymer were detected by above methods.Cellular 14-3-3 dimer were also detected after PrP106-126 peptide were added to HeLa cell for 8 hours.Results Recombinant full-length PrP facilitated the dimerization of 14-3-3β and PrP106-126 disturbed 14-3-3β dimeration as both have dose dependence effect.PrP antagonized PrP106-126-induced 14-3-3β dimer with PrP protein increase in vitro.Cellular 14-3-3 dimerization also decreased after treatment of peptide PrP106-126 on HeLa cells for 8 hours.Conculsion Dimerization process of 14-3-3β was promoted by full-length PrP (PrP23-231) but inhibited by peptide PrP106-126 in vitro.PrP agonized PrP106-126-induced inhibition of 14-3-3 dimeration.PrP106-126 inhibited cellular 14-3-3 dimerization.
展开▼
