Study On Hsp104 Inhibite PrP 106-126 Fibril Formation In Vitro

Objectives:(1) Observations of prion-like behavior from other systems may be informative for how prion is functioning as well as yeast. Thus, can Hsp104 prevent or reverse amyloidoses or aggregation of PrP106-126? It will help clarify whether PrPSc aggregation is protective or toxic in prion disease. (2) We employ PrP106-126 which has characteristic of amyloidlike fibrils and neurotoxic activity to delineate how Hsp104 modulates PrP106-126 aggregation and amyloidlike fibrils.Methods:(1) In this study, recombinant full-length yeast Hsp104 was expressed using prokaryotic expression system.(2) Fiber assembly or disassembly was determined by ThT fluorescence, ATM, or EM. For fiber assembly reactions, PrP106-126 peptides (50μM) were incubated in 50 mM Tris, pH 7.4, and 150 mM KCl with ATP (10 mM) in the presence of or absence of 5μM Hsp104 at 37℃for Oh,12h, 24h,48h,72h and 96h. Various samples were differently prepared for ThT fluorescence, ATM, or EM.Result:Recombinant full-length yeast Hsp104 was expressed using prokaryotic expression system.The effect of Hsp104 on PrP106-126 fibril formation were investigated thioflavin T (Th T) assay, atomic force microscopy, transmission electron microscope, circular dichroism and cell viability assay were performed Our results showed PrP106-126 may format fibrils which including various forms, and with prolongation of incubation time, fibrils became thicker and thicker and looked like net. PrP106-126 peptide had similar toxicity on different cell such as both HeLa and SK cells from our experiment results; however, we found PrP106-126 fibrils appear lower toxicity than its peptide on cell. we found that Hsp104 can obviously habits PrP106-126 fibril formation by assays of thioflavin T binding, atomic force microscopy and transmission electron microscope. When in presence of Hsp104, PrP106-126 peptide will keep its toxicity on cell from our results of cell viability assay. Our results show the similar phenomenon that PrP106-126 may undergoes aβ-sheet transition change the second structure PrP106-126 fromβ-sheet to random coil during fibril formation, as observed here by circular dichroism in the presence of Hsp104. Hsp104 antagonizing fibrils formation against amyloid change.Conclusions:(1) Hsp 104 inhibits PrP106-126 forms fibrils.(2) Hsp104 disaggregates PrP106-126 fibril formation and induce cytotoxicity...