Mechanisms For The Effect Of Condon 127 Polymorphism On Human Prion Protein Misfolding

Prions,meaning“protein infectious agents,”were initially described by Stanley B.Prusiner,who won the Nobel Prize in 1997.Prion protein（Pr P）misfolding in some mammals,including humans,causes prion diseases or transmissible spongiform encephalopathies（TSEs）.Prion diseases are mainly caused by the conformational change of the cellular Pr P,Pr P^C,into the pathological Pr P,Pr P^Sc,and the key steps for this conversion are the formation of a Pr P^C-Pr P^Sc heterodimer and its transformation into a Pr P^Sc-Pr P^Sc homodimer.The primary structure of Pr P^Sc is the same as that of Pr P^C,but there are many differences between Pr P^C and Pr^PSc,such as theirβ-sheet content,solubility in detergents,resistance to protease activity,and mitochondrial function/malfunction.A naturally occurring mutant of human Pr P,G127V,is found exclusively in people who lived in the region where kuru was prevalent and has been recently reported to completely prevent prion diseases.Very recently,it has been found that G127V is not favorable for the formation of both dimers and fibrils of human Pr P,possibly due to the steric hindrance offered by the hydrophobic side chain of Val-127.G126V,an equivalent to the G127V human Pr P mutant,is not favorable for fibril formation of mouse Pr P.However,the mechanisms by which G127V protein is intrinsically resistant to prion conversion and propagation have not been fully illuminated.In this work,we studied the mechanisms the effects of condon 127 polymorphism on human Pr P misfolding.First of all,we constructed mutiple mutants such as G127A,G127W,G127E,G127K,G127V,G127I and G127L.We found that only G127V,G127I and G127L could prevent the formation of amyloid fibrils.Next,we focused on influences of G127V and two alternative mutations,G127I and G127L,on fibril formation and neurotoxicity of human Pr P.We used thioflavin T（Th T）binding assays,Sarkosyl-soluble SDS-PAGE,8-anilino-1-naphthalene-sulfonic acid（ANS）binding assays,Fourier transform infrared（FTIR）spectroscopy,circular dichroism（CD）,proteinase K（PK）digestion assays,transmission electron microscopy（TEM）,Western blotting,confocal microscopy,and flow cytometry,and our study could be divided into two levels:molecular biology and cell biology.We demonstrate that the lag time of fibril formation of G127V,G127I,and G127L Pr Ps is significantly longer than that of wild-type Pr P,indicating that the long-chain hydrophobic aliphatic amino acids at position 127,Val-127,Ile-127,and Leu-127,significantly inhibit fibril formation of human Pr P.Cellular G127V,G127I,and G127L Pr Ps have significantly weaker protease-resistant activity and aggregation ability than wild-type Pr P.Notably,Val-127,Ile-127,and Leu-127 prevent the impairment of the correct localization of Pr P at the cell surface and protect against the high cellular ROS levels,mitochondrial damage,and human Pr P neurotoxicity caused by prion peptide Pr P 106-126.Hence,these data show that G127V,G127I,and G127L Pr Ps act as potent inhibitors of human Pr P aggregation and that long-chain hydrophobic aliphatic amino acids at position 127 in human Pr P protect against prion toxicity.Our findings help explain how prions transformation,propagation and infection,and are beneficial in developing novel drugs for prion diseases.