Arg177 and Asp159 from dog prion protein slow liquid-liquid phase separation and inhibit amyloid formation of human prion protein.

Prion diseases are a group of transmissible neurodegenerative diseases primarily caused by the conformational conversion of prion protein (PrP) from α-helix-dominant cellular prion protein (PrPC) to ß-sheet-rich pathological aggregated form of PrPSc in many mammalian species. Dogs exhibit resistance to prion diseases, but the mechanism behind the phenomenon remains poorly understood. Compared with human PrP and mouse PrP, dog PrP has two unique amino acid residues, Arg177 and Asp159. Because PrPC contains a low-complexity and intrinsically disordered region in its N-terminal domain, it undergoes liquid-liquid phase separation (LLPS) in vitro and forms protein condensates. However, little is known about whether these two unique residues modulate the formation of PrPC condensates. Here, using confocal microscopy, fluorescence recovery after photobleaching assays, thioflavin T binding assays, and transmission electron microscopy, we report that Arg177 and Asp159 from the dog PrP slow the LLPS of full-length human PrPC, shifting the equilibrium phase boundary to higher protein concentrations and inhibit amyloid formation of the human protein. In sharp contrast, His177 and Asn159 from the human PrP enhance the LLPS of full-length dog PrPC, shifting the equilibrium phase boundary to lower protein concentrations, and promote fibril formation of the canid protein. Collectively, these results demonstrate how LLPS and amyloid formation of PrP are inhibited by a single residue Arg177 or Asp159 associated with prion disease resistance, and how LLPS and fibril formation of PrP are promoted by a single residue His177 or Asn159. Therefore, Arg177/His177 and Asp159/Asn159 are key residues in modulating PrPC liquid-phase condensation.

Cryo-EM structure of an amyloid fibril formed by full-length human prion protein.

Prion diseases are caused by the misfolding of prion protein (PrP). Misfolded PrP forms protease-resistant aggregates in vivo (PrPSc) that are able to template the conversion of the native form of the protein (PrPC), a property shared by in vitro-produced PrP fibrils. Here we produced amyloid fibrils in vitro from recombinant, full-length human PrPC (residues 23-231) and determined their structure using cryo-EM, building a model for the fibril core comprising residues 170-229. The PrP fibril consists of two protofibrils intertwined in a left-handed helix. Lys194 and Glu196 from opposing subunits form salt bridges, creating a hydrophilic cavity at the interface of the two protofibrils. By comparison with the structure of PrPC, we propose that two α-helices in the C-terminal domain of PrPC are converted into ß-strands stabilized by a disulfide bond in the PrP fibril. Our data suggest that different PrP mutations may play distinct roles in modulating the conformational conversion.

Author Correction: Glycosylation Significantly Inhibits the Aggregation of Human Prion Protein and Decreases Its Cytotoxicity.

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Glycosylation Significantly Inhibits the Aggregation of Human Prion Protein and Decreases Its Cytotoxicity.

Prion diseases are primarily caused by the misfolding of prion proteins in humans, cattle, sheep, and cervid species. The effects of glycosylation on prion protein (PrP) structure and function have not been thoroughly elucidated to date. In this study, we attempt to elucidate the effects of glycosylation on the aggregation and toxicity of human PrP. As revealed by immunocytochemical staining, wild-type PrP and its monoglycosylated mutants N181D, N197D, and T199N/N181D/N197D are primarily attached to the plasma membrane. In contrast, PrP F198S, a pathological mutant with an altered residue within the glycosylation site, and an unglycosylated PrP mutant, N181D/N197D, primarily exist in the cytoplasm. In the pathological mutant V180I, there is an equal mix of membranous and cytoplasmic PrP, indicating that N-linked glycosylation deficiency impairs the correct localization of human PrP at the plasma membrane. As shown by immunoblotting and flow cytometry, human PrP located in the cytoplasm displays considerably greater PK resistance and aggregation ability and is associated with considerably higher cellular ROS levels than PrP located on the plasma membrane. Furthermore, glycosylation deficiency enhances human PrP cytotoxicity induced by MG132 or the toxic prion peptide PrP 106-126. Therefore, we propose that glycosylation acts as a necessary cofactor in determining PrP localization on the plasma membrane and that it significantly inhibits the aggregation of human PrP and decreases its cytotoxicity.

Zinc significantly changes the aggregation pathway and the conformation of aggregates of human prion protein.

Prion diseases are caused by the conformational change of cellular prion protein PrP(C) into pathological prion protein PrP(Sc). Here we study the effect of zinc on the aggregation and conformational change of human prion protein (PrP). As revealed by thioflavin T binding assays, Sarkosyl-soluble SDS-PAGE, and transmission electron microscopy, aggregation of wild-type PrP in the absence of Zn(2+) undergoes four steps: amorphous aggregates, profibrils, mature fibrils, and fragmented fibrils. When the molar ratio of Zn(2+) to PrP was 9:1, 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 PrPΔocta forms typical mature fibrils either with or without zinc. As evidenced by isothermal titration calorimetry, Fourier transform infrared spectroscopy, and proteinase K digestion assays, Zn(2+) strongly binds to wild-type PrP monomers with the first binding constant exceeding 10(7)M(-1) under denaturing conditions, and changes the conformation of wild-type PrP aggregates remarkably, but weakly binds to PrPΔocta with binding affinity around 10(4)M(-1) and has no obvious effects on the conformation of PrPΔocta 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. Our findings could explain how zinc modifies pathological PrP conformation associated with prion diseases.

Recent progress in prion and prion-like protein aggregation.

Prion diseases and prion-like protein misfolding diseases involve the accumulation of abnormally aggregated forms of the normal host proteins, such as prion protein and Tau protein. These proteins are special because of their self-duplicating and transmissible characteristics. Such abnormally aggregated proteins mainly formed in neurons, cause the neurons dysfunction, and finally lead to invariably fatal neurodegenerative diseases. Prion diseases appear not only in animals, such as bovine spongiform encephalopathy in cattle and scrapie in sheep, but also in humans, such as Creutzfeldt-Jacob disease, and even the same prion or prion-like proteins can have many different phenotypes. A lot of biological evidence has suggested that the molecular basis for different strains of prions could be hidden in protein conformations, and the misfolded proteins with conformations different from the normal proteins have been proved to be the main cause for protein aggregation. Crowded physiological environments can be imitated in vitro to study how the misfolding of these proteins leads to the diseases in vivo. In this review, we provide an overview of the existing structural information for prion and prion-like proteins, and discuss the post-translational modifications of prion proteins and the difference between prion and other infectious pathogens. We also discuss what makes a misfolded protein become an infectious agent, and show some examples of prion-like protein aggregation, such as Tau protein aggregation and superoxide dismutase 1 aggregation, as well as some cases of prion-like protein aggregation in crowded physiological environments.