Mechanisms For Zinc Modification On Prion Protein Misfolding

Prion diseases are a group of transmissible fatal neurodegenerative disease which caused by misfolding of prion proteins. It may happen on many mammals, including bovine spongiform encephalopathy, scrapie, human creutzfeldt-jakob diseases and so on. Prion diseases are caused by the conformational change of cellular prion protein PrPc into pathological prion protein PrPSc, and PrPSc could convert PrPC to PrPSc. At last, the accumulation of PrPSc could lead to neurodegenerative diseases. Because prion diseases could be transmissible like viruses, the infectious source is called prion. According to decades of study, though it has been proved that the main constitute of prion is PrPSc, there are still many problems remained to solve in this field, like how PrPc convert to PrPSc, what other factors could affect the misfolding of PrPc, how prion strains form and propagate. As both zinc and prion protein could accumulate in the synaptic gap, which could be a big relationship, we studied how zinc affects the aggregation of human prion protein.The first part of the work is focused on how zinc affects the aggregation of human prion protein in vitro. PrPSc can form amyloid fibrils in the brain of patients, and recombinant prion protein can also form amyloid fibrils in vitro by different methods. They have many similar points, so it is normal to use in vitro methods to study the misfolding of prion proteins. As revealed by thioflavin T binding assays, Sarkosyl-soluble SDS-PAGE. and transmission electron microscopy, aggregation of wild-type PrP in the absence of Zn2+ undergoes four steps:amorphous aggregates, profibrils, mature fibrils, and fragmented fibrils. In the presence of 100 ?M Zn2+, however, aggregation of wild-type PrP undergoes another pathway in which wild-type PrP forms oligomers quickly and then forms short-rod aggregates. Unlike wild-type PrP, the octarepeats deletion mutant PrP?octa forms typical mature fibrils either with or without zinc. As evidenced by isothermal titration calorimetry, Fourier transform infrared spectroscopy, and proteinase K digestion assays, Zn2+ strongly binds to wild-type PrP monomers with the first binding constant exceeding 107 M-1 under denaturing conditions, and changes the conformation of wild-type PrP aggregates remarkably, but weakly binds to PrP?octa with binding affinity around 104 M-1and has no obvious effects on the conformation of PrP?octa aggregates. Our data demonstrate that zinc significantly changes the aggregation pathway and the conformation of wild-type PrP aggregates mainly via interaction with its octarepeat region.In the second part of the work we have studied how zinc affects the aggregation of prion protein in cells. Through the observation of confocal and detection of Western Blot, we have found that 200?M zinc could induce the aggregation of human prion protein in cells. Furthermore, we confirm by immune electron microscopy that the aggregates formed in cells are mainly oligomers, which are similar to those quickly formed in vitro in the presence of 100 ?M zinc. By adding a high concentration of vitamin c into the cells to induce cell death, we have preliminarily found the relationship between prion protein aggregation caused by zinc and cell death. Cell death is a necessary but not a sufficient condition for prion protein aggregation.Our work has studied how zinc affects prion protein aggregation, and elucidated the mechanism. The abnormal zinc can accelerate the aggregation of prion protein and change the aggregation pathway. Our findings could explain how zinc modifies pathological PrP conformation associated with prion diseases and link abnormal aggregation of prion protein modified by zinc to pathogenesis of prion diseases and prion strain diversity.