Channel Activities of the Full-Length Prion and Truncated Proteins.

Prion diseases are fatal neurodegenerative disorders characterized by the conversion of the cellular prion protein (PrPC) into a misfolded prion form, which is believed to disrupt the cellular membranes. However, the exact mechanisms underlying prion toxicity, including the formation of membrane pores, are not fully understood. The prion protein consists of two domains: a globular domain (GD) and a flexible N-terminus (FT) domain. Although a proximal polybasic amino acid (FT(23-31) sequence of FT is a prerequisite for cellular membrane permeabilization, other functional domain regions may modulate its effects. Through single-channel electrical recordings and cryo-electron microscopy (cryo-EM), we discovered that the FT(23-50) fragment forms pore-shaped oligomers and plays a dominant role in membrane permeabilization within the full-length mouse prion protein (mPrP(23-230)). In contrast, the FT(51-110) domain or the C-terminal domain downregulate the channel activity of FT(23-50) and mPrP(23-230). The addition of prion mimetic antibody, POM1 significantly amplifies mPrP(23-230) membrane permeabilization, whereas POM1_Y104A, a mutant that binds to PrP but cannot elicit toxicity, has a negligible effect on membrane permeabilization. Additionally, the anti-N-terminal antibody POM2 or Cu2+ binds to the FT domain, subsequently enhancing the FT(23-110) channel activity. Importantly, our setup provides a novel approach without an external fused protein to examine the channel activity of truncated PrP in the lipid membranes. We therefore propose that the primary N-terminal residues are essential for membrane permeabilization, while other functional segments of PrP play a vital role in modulating the pathological effects of PrP-mediated neurotoxicity.

Bifunctional carbazole derivatives for simultaneous therapy and fluorescence imaging in prion disease murine cell models.

Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrPSc) of the cellular prion protein (PrPC). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrPC conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrPSc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrPC levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrPSc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds.

An optimized Western blot assay provides a comprehensive assessment of the physiological endoproteolytic processing of the prion protein.

The prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases.

Beta-endoproteolysis of the cellular prion protein by dipeptidyl peptidase-4 and fibroblast activation protein.

The cellular prion protein (PrPC) converts to alternatively folded pathogenic conformations (PrPSc) in prion infections and binds neurotoxic oligomers formed by amyloid-ß α-synuclein, and tau. ß-Endoproteolysis, which splits PrPC into N- and C-terminal fragments (N2 and C2, respectively), is of interest because a protease-resistant, C2-sized fragment (C2Sc) accumulates in the brain during prion infections, seemingly comprising the majority of PrPSc at disease endpoint in mice. However, candidates for the underlying proteolytic mechanism(s) remain unconfirmed in vivo. Here, a cell-based screen of protease inhibitors unexpectedly linked type II membrane proteins of the S9B serine peptidase subfamily to PrPC ß-cleavage. Overexpression experiments in cells and assays with recombinant proteins confirmed that fibroblast activation protein (FAP) and its paralog, dipeptidyl peptidase-4 (DPP4), cleave directly at multiple sites within PrPC's N-terminal domain. For wild-type mouse and human PrPC substrates expressed in cells, the rank orders of activity were human FAP ~ mouse FAP > mouse DPP4 > human DPP4 and human FAP > mouse FAP > mouse DPP4 >> human DPP4, respectively. C2 levels relative to total PrPC were reduced in several tissues from FAP-null mice, and, while knockout of DPP4 lacked an analogous effect, the combined DPP4/FAP inhibitor linagliptin, but not the FAP-specific inhibitor SP-13786, reduced C2Sc and total PrPSc levels in two murine cell-based models of prion infections. Thus, the net activity of the S9B peptidases FAP and DPP4 and their cognate inhibitors/modulators affect the physiology and pathogenic potential of PrPC.

Antibody binding increases the flexibility of the prion protein.

Prion diseases are associated with the conversion of the cellular prion protein (PrP) into a pathogenic conformer (PrPSc). A proposed therapeutic approach to avoid the pathogenic transformation is to develop antibodies that bind to PrP and stabilize its structure. POM1 and POM6 are two monoclonal antibodies that bind the globular domain of PrP and have different biological responses, i.e., trigger neurotoxicity mimicking prion infections (POM1) or prevent neurotoxicity (POM6). The crystal structures of PrP in complex with the two antibodies show similar epitopes which seems inconsistent with the opposite phenotypes. Here, we investigate the influence of the POM1 and POM6 antibodies on the flexibility of the mouse PrP by molecular dynamics simulations. The simulations reveal that the POM6/PrP interface is less stable than the POM1/PrP interface, ascribable to localized polar mismatches at the interface, despite the former complex having a larger epitope than the latter. In the presence of any of the two antibodies, the flexibility of the globular domain increases everywhere except for the ß1-α1 loop in the POM1/PrP complex which suggests the involvement of this loop in the pathological conversion. The secondary structure of PrP is preserved whereas the polar interactions involving residues Glu146, Arg156 and Arg208 are modified upon antibody binding.

A conformational switch controlling the toxicity of the prion protein.

Prion infections cause conformational changes of the cellular prion protein (PrPC) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ('H-latch'), altering the flexibility of the α2-α3 and ß2-α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity.

Virus Infection, Genetic Mutations, and Prion Infection in Prion Protein Conversion.

Conformational conversion of the cellular isoform of prion protein, PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is an underlying pathogenic mechanism in prion diseases. The diseases manifest as sporadic, hereditary, and acquired disorders. Etiological mechanisms driving the conversion of PrPC into PrPSc are unknown in sporadic prion diseases, while prion infection and specific mutations in the PrP gene are known to cause the conversion of PrPC into PrPSc in acquired and hereditary prion diseases, respectively. We recently reported that a neurotropic strain of influenza A virus (IAV) induced the conversion of PrPC into PrPSc as well as formation of infectious prions in mouse neuroblastoma cells after infection, suggesting the causative role of the neuronal infection of IAV in sporadic prion diseases. Here, we discuss the conversion mechanism of PrPC into PrPSc in different types of prion diseases, by presenting our findings of the IAV infection-induced conversion of PrPC into PrPSc and by reviewing the so far reported transgenic animal models of hereditary prion diseases and the reverse genetic studies, which have revealed the structure-function relationship for PrPC to convert into PrPSc after prion infection.

Novel quaternary structures of the human prion protein globular domain.

Prion disease is caused by the misfolding of the cellular prion protein, PrPC, into a self-templating conformer, PrPSc. Nuclear magnetic resonance (NMR) and X-ray crystallography revealed the 3D structure of the globular domain of PrPC and the possibility of its dimerization via an interchain disulfide bridge that forms due to domain swap or by non-covalent association of two monomers. On the contrary, PrPSc is composed by a complex and heterogeneous ensemble of poorly defined conformations and quaternary arrangements that are related to different patterns of neurotoxicity. Targeting PrPC with molecules that stabilize the native conformation of its globular domain emerged as a promising approach to develop anti-prion therapies. One of the advantages of this approach is employing structure-based drug discovery methods to PrPC. Thus, it is essential to expand our structural knowledge about PrPC as much as possible to aid such drug discovery efforts. In this work, we report a crystallographic structure of the globular domain of human PrPC that shows a novel dimeric form and a novel oligomeric arrangement. We use molecular dynamics simulations to explore its structural dynamics and stability and discuss potential implications of these new quaternary structures to the conversion process.

Potential for transmission of sporadic Creutzfeldt-Jakob disease through peripheral routes.

Five sporadic Creutzfeldt-Jakob disease (CJD) strains have been identified to date, based on differences in clinicopathological features of the patients, the biochemical properties of abnormal prion proteins, and transmission properties. Recent advances in our knowledge about iatrogenic transmission of sporadic CJD have raised the possibility that the infectivity of sporadic CJD strains through peripheral routes is different from that of intracranial infection. To test this possibility, here we assessed systematically the infectivity of sporadic CJD strains through the peripheral route for the first time using a mouse model expressing human prion protein. Although the infectivity of the V2 and M1 sporadic CJD strains is almost the same in intracerebral transmission studies, the V2 strain infected more efficiently than the M1 strain through the peripheral route. The other sporadic CJD strains examined lacked infectivity. Of note, both the V2 and M1 strains showed preference for mice with the valine homozygosity at the PRNP polymorphic codon. These results indicate that the V2 strain is the most infectious sporadic CJD strain for infection through peripheral routes. In addition, these findings raise the possibility that individuals with the valine homozygosity at the PRNP polymorphic codon might have higher risks of infection through peripheral routes compared with the methionine homozygotes. Thus, preventive measures against the transmission of the V2 sporadic CJD strain will be important for the eradication of iatrogenic CJD transmission through peripheral routes.

From Kuru to Alzheimer: A personal outlook.

Seventy years ago, we learned from Chris Anfinsen that the stereochemical code necessary to fold a protein is embedded into its amino acid sequence. In water, protein morphogenesis is a spontaneous reversible process leading from an ensemble of disordered structures to the ordered functionally competent protein; conforming to Aristotle's definition of substance, the synolon of matter and form. The overall process of folding is generally consistent with a two state transition between the native and the denatured protein: not only the denatured state is an ensemble of several structures, but also the native protein populates distinct functionally relevant conformational (sub)states. This two-state view should be revised, given that any globular protein can populate a peculiar third state called amyloid, characterized by an overall architecture that at variance with the native state, is by-and-large independent of the primary structure. In a nut shell, we should accept that beside the folded and unfolded states, any protein can populate a third state called amyloid which gained center stage being the hallmark of incurable neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases as well as others. These fatal diseases are characterized by clear-cut clinical differences, yet display some commonalities such as the presence in the brain of amyloid deposits constituted by one misfolded protein specific for each disease. Some aspects of this complex problem are summarized here as an excursus from the prion's fibrils observed in the brain of aborigines who died of Kuru to the amyloid detectable in the cortex of Alzheimer's patients.

Vaporized Hydrogen Peroxide and Ozone Gas Synergistically Reduce Prion Infectivity on Stainless Steel Wire.

Prions are infectious agents causing prion diseases, which include Creutzfeldt-Jakob disease (CJD) in humans. Several cases have been reported to be transmitted through medical instruments that were used for preclinical CJD patients, raising public health concerns on iatrogenic transmissions of the disease. Since preclinical CJD patients are currently difficult to identify, medical instruments need to be adequately sterilized so as not to transmit the disease. In this study, we investigated the sterilizing activity of two oxidizing agents, ozone gas and vaporized hydrogen peroxide, against prions fixed on stainless steel wires using a mouse bioassay. Mice intracerebrally implanted with prion-contaminated stainless steel wires treated with ozone gas or vaporized hydrogen peroxide developed prion disease later than those implanted with control prion-contaminated stainless steel wires, indicating that ozone gas and vaporized hydrogen peroxide could reduce prion infectivity on wires. Incubation times were further elongated in mice implanted with prion-contaminated stainless steel wires treated with ozone gas-mixed vaporized hydrogen peroxide, indicating that ozone gas mixed with vaporized hydrogen peroxide reduces prions on these wires more potently than ozone gas or vaporized hydrogen peroxide. These results suggest that ozone gas mixed with vaporized hydrogen peroxide might be more useful for prion sterilization than ozone gas or vaporized hydrogen peroxide alone.

Neurometals in the Pathogenesis of Prion Diseases.

Prion diseases are progressive and transmissive neurodegenerative diseases. The conformational conversion of normal cellular prion protein (PrPC) into abnormal pathogenic prion protein (PrPSc) is critical for its infection and pathogenesis. PrPC possesses the ability to bind to various neurometals, including copper, zinc, iron, and manganese. Moreover, increasing evidence suggests that PrPC plays essential roles in the maintenance of homeostasis of these neurometals in the synapse. In addition, trace metals are critical determinants of the conformational change and toxicity of PrPC. Here, we review our studies and other new findings that inform the current understanding of the links between trace elements and physiological functions of PrPC and the neurotoxicity of PrPSc.

Ellagic acid and pentagalloylglucose are potential inhibitors of prion protein fibrillization.

Prion diseases are fatal neurodegenerative diseases caused by the conformational transition of the cellular prion protein (PrPC) to the abnormal pathological prion protein (PrPSc). In this work, the effects of ellagic acid (EA) and pentagalloylglucose (PGG) on prion protein (PrP) fibrillization were investigated. Fluorescence quenching experiments indicated that both EA and PGG could specifically interact with native human PrP with binding affinities of 1.92 × 105 and 2.36 × 105 L·mol-1, respectively. Thioflavin-T (ThT) fluorescence assays showed that the binding of EA or PPG could effectively inhibit the nucleation and elongation of PrP fibrilization and reduce the amount of PrP fibrils generated. EA and PGG could also lead to a significant disaggregation of PrP fibrils. Circular dichroism (CD) measurements suggested that EA- or PPG-bound PrP could preserve a higher content of α-helical structures than ß-sheet-rich PrP fibrils. The PrP aggregates formed in the presence of EA or PGG showed lower resistance to proteinase K (PK) digestion. Overall, the present work reported the inhibitory effect of EA and PGG on PrP fibrillization. These two natural polyphenols could be potential prodrug molecules for the prevention and treatment of prion diseases.

Strain-Dependent Prion Infection in Mice Expressing Prion Protein with Deletion of Central Residues 91-106.

Conformational conversion of the cellular prion protein, PrPC, into the abnormally folded isoform, PrPSc, is a key pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Transgenic mice expressing PrP with a deletion of the central residues 91-106 were generated in the absence of endogenous PrPC, designated Tg(PrP∆91-106)/Prnp0/0 mice and intracerebrally inoculated with various prions. Tg(PrP∆91-106)/Prnp0/0 mice were resistant to RML, 22L and FK-1 prions, neither producing PrPSc∆91-106 or prions in the brain nor developing disease after inoculation. However, they remained marginally susceptible to bovine spongiform encephalopathy (BSE) prions, developing disease after elongated incubation times and accumulating PrPSc∆91-106 and prions in the brain after inoculation with BSE prions. Recombinant PrP∆91-104 converted into PrPSc∆91-104 after incubation with BSE-PrPSc-prions but not with RML- and 22L-PrPSc-prions, in a protein misfolding cyclic amplification assay. However, digitonin and heparin stimulated the conversion of PrP∆91-104 into PrPSc∆91-104 even after incubation with RML- and 22L-PrPSc-prions. These results suggest that residues 91-106 or 91-104 of PrPC are crucially involved in prion pathogenesis in a strain-dependent manner and may play a similar role to digitonin and heparin in the conversion of PrPC into PrPSc.

Soluble Prion Peptide 107-120 Protects Neuroblastoma SH-SY5Y Cells against Oligomers Associated with Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent form of dementia and soluble amyloid ß (Aß) oligomers are thought to play a critical role in AD pathogenesis. Cellular prion protein (PrPC) is a high-affinity receptor for Aß oligomers and mediates some of their toxic effects. The N-terminal region of PrPC can interact with Aß, particularly the region encompassing residues 95-110. In this study, we identified a soluble and unstructured prion-derived peptide (PrP107-120) that is external to this region of the sequence and was found to successfully reduce the mitochondrial impairment, intracellular ROS generation and cytosolic Ca2+ uptake induced by oligomeric Aß42 ADDLs in neuroblastoma SH-SY5Y cells. PrP107-120 was also found to rescue SH-SY5Y cells from Aß42 ADDL internalization. The peptide did not change the structure and aggregation pathway of Aß42 ADDLs, did not show co-localization with Aß42 ADDLs in the cells and showed a partial colocalization with the endogenous cellular PrPC. As a sequence region that is not involved in Aß binding but in PrP self-recognition, the peptide was suggested to protect against the toxicity of Aß42 oligomers by interfering with cellular PrPC and/or activating a signaling that protected the cells. These results strongly suggest that PrP107-120 has therapeutic potential for AD.

Incomplete glycosylation during prion infection unmasks a prion protein epitope that facilitates prion detection and strain discrimination.

The causative factors underlying conformational conversion of cellular prion protein (PrPC) into its infectious counterpart (PrPSc) during prion infection remain undetermined, in part because of a lack of monoclonal antibodies (mAbs) that can distinguish these conformational isoforms. Here we show that the anti-PrP mAb PRC7 recognizes an epitope that is shielded from detection when glycans are attached to Asn-196. We observed that whereas PrPC is predisposed to full glycosylation and is therefore refractory to PRC7 detection, prion infection leads to diminished PrPSc glycosylation at Asn-196, resulting in an unshielded PRC7 epitope that is amenable to mAb recognition upon renaturation. Detection of PRC7-reactive PrPSc in experimental and natural infections with various mouse-adapted scrapie strains and with prions causing deer and elk chronic wasting disease and transmissible mink encephalopathy uncovered that incomplete PrPSc glycosylation is a consistent feature of prion pathogenesis. We also show that interrogating the conformational properties of the PRC7 epitope affords a direct means of distinguishing different prion strains. Because the specificity of our approach for prion detection and strain discrimination relies on the extent to which N-linked glycosylation shields or unshields PrP epitopes from antibody recognition, it dispenses with the requirement for additional standard manipulations to distinguish PrPSc from PrPC, including evaluation of protease resistance. Our findings not only highlight an innovative and facile strategy for prion detection and strain differentiation, but are also consistent with a mechanism of prion replication in which structural instability of incompletely glycosylated PrP contributes to the conformational conversion of PrPC to PrPSc.

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.

α2,3 linkage of sialic acid to a GPI anchor and an unpredicted GPI attachment site in human prion protein.

Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrPSc) after conversion of the cellular prion protein (PrPC). The glycosylphosphatidylinositol (GPI) anchor of PrPC is involved in prion disease pathogenesis, and especially sialic acid in a GPI side chain reportedly affects PrPC conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrPC Moreover, the sialic acid linkage type in the GPI side chain has not been determined for any GPI-anchored protein. Here we report GPI glycan structures of human PrPC isolated from human brains and from brains of a knock-in mouse model in which the mouse prion protein (Prnp) gene was replaced with the human PRNP gene. LC-electrospray ionization-MS analysis of human PrPC from both biological sources indicated that Gly229 is the ω site in PrPC to which GPI is attached. Gly229 in human PrPC does not correspond to Ser231, the previously reported ω site of Syrian hamster PrPC We found that ∼41% and 28% of GPI anchors in human PrPCs from human and knock-in mouse brains, respectively, have N-acetylneuraminic acid in the side chain. Using a sialic acid linkage-specific alkylamidation method to discriminate α2,3 linkage from α2,6 linkage, we found that N-acetylneuraminic acid in PrPC's GPI side chain is linked to galactose through an α2,3 linkage. In summary, we report the GPI glycan structure of human PrPC, including the ω-site amino acid for GPI attachment and the sialic acid linkage type.

Amplification Techniques for the Detection of Misfolded Prion Proteins in Experimental and Clinical Samples.

This article describes two methods for amplifying prions present in experimental and clinical samples: the protein misfolding cyclic amplification (PMCA) assay and the real-time quaking-induced conversion (RT-QuIC) assay. Protocols for preparation of amplification substrate and analysis of results are included in addition to those for the individual assays. For each assay, control and suspect samples are mixed with appropriate amplification substrate, which is whole brains from mice in the case of PMCA and recombinant prion protein produced in bacteria for RT-QuIC, followed by cyclic amplification over a number of cycles of sonication (PMCA) or shaking (RT-QuIC) at a consistent incubation temperature. The resultant amplification products are then assessed either by western blotting (PMCA) or based on fluorescent emissions (RT-QuIC). The equipment and expertise necessary for successfully performing either assay vary and will be important factors for individual laboratories to consider when identifying which assay is more appropriate for their experimental design. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Prion amplification via protein misfolding cyclic amplification Support Protocol 1: Collection of whole brains from mice and preparation of normal brain homogenate Basic Protocol 2: Prion amplification via real-time quaking-induced conversion Support Protocol 2: Preparation of recombinant truncated white-tailed-deer prion protein.

Prion protein-Semisynthetic prion protein (PrP) variants with posttranslational modifications.

Deciphering the pathophysiologic events in prion diseases is challenging, and the role of posttranslational modifications (PTMs) such as glypidation and glycosylation remains elusive due to the lack of homogeneous protein preparations. So far, experimental studies have been limited in directly analyzing the earliest events of the conformational change of cellular prion protein (PrPC ) into scrapie prion protein (PrPSc ) that further propagates PrPC misfolding and aggregation at the cellular membrane, the initial site of prion infection, and PrP misfolding, by a lack of suitably modified PrP variants. PTMs of PrP, especially attachment of the glycosylphosphatidylinositol (GPI) anchor, have been shown to be crucially involved in the PrPSc formation. To this end, semisynthesis offers a unique possibility to understand PrP behavior invitro and invivo as it provides access to defined site-selectively modified PrP variants. This approach relies on the production and chemoselective linkage of peptide segments, amenable to chemical modifications, with recombinantly produced protein segments. In this article, advances in understanding PrP conversion using semisynthesis as a tool to obtain homogeneous posttranslationally modified PrP will be discussed.