Structure Of Prion Protein Research Articles

Prion protein gene (PRNP) variation in German and Danish cervids

The structure of cellular prion proteins encoded by the prion protein gene (PRNP) impacts susceptibility to transmissible spongiform encephalopathies, including chronic wasting disease (CWD) in deer. The recent emergence of CWD in Northern European reindeer (Rangifer tarandus), moose (Alces alces alces) and red deer (Cervus elaphus), in parallel with the outbreak in North America, gives reason to investigate PRNP variation in European deer, to implement risk assessments and adjust CWD management for deer populations under threat. We here report PRNP-sequence data from 911 samples of German red, roe (Capreolus capreolus), sika (Cervus nippon) and fallow deer (Dama dama) as well as additional data from 26 Danish red deer close to the German border and four zoo species not native to Germany. No PRNP sequence variation was observed in roe and fallow deer, as previously described for populations across Europe. In contrast, a broad PRNP variation was detected in red deer, with non-synonymous polymorphisms at codons 98, 226 and 247 as well as synonymous mutations at codons 21, 78, 136 and 185. Moreover, a novel 24 bp deletion within the octapeptide repeat was detected. In summary, 14 genotypes were seen in red deer with significant differences in their geographical distribution and frequencies, including geographical clustering of certain genotypes, suggesting “PRNP-linages” in this species. Based on data from North American CWD and the genotyping results of the European CWD cases, we would predict that large proportions of wild cervids in Europe might be susceptible to CWD once introduced to naive populations.

Intrinsic determinants of prion protein neurotoxicity in Drosophila: from sequence to (dys)function.

Prion diseases are fatal brain disorders characterized by deposition of insoluble isoforms of the prion protein (PrP). The normal and pathogenic structures of PrP are relatively well known after decades of studies. Yet our current understanding of the intrinsic determinants regulating PrP misfolding are largely missing. A 3D subdomain of PrP comprising the β2-α2 loop and helix 3 contains high sequence and structural variability among animals and has been proposed as a key domain regulating PrP misfolding. We combined in vivo work in Drosophila with molecular dynamics (MD) simulations, which provide additional insight to assess the impact of candidate substitutions in PrP from conformational dynamics. MD simulations revealed that in human PrP WT the β2-α2 loop explores multiple β-turn conformations, whereas the Y225A (rabbit PrP-like) substitution strongly favors a 310-turn conformation, a short right-handed helix. This shift in conformational diversity correlates with lower neurotoxicity in flies. We have identified additional conformational features and candidate amino acids regulating the high toxicity of human PrP and propose a new strategy for testing candidate modifiers first in MD simulations followed by functional experiments in flies. In this review we expand on these new results to provide additional insight into the structural and functional biology of PrP through the prism of the conformational dynamics of a 3D domain in the C-terminus. We propose that the conformational dynamics of this domain is a sensitive measure of the propensity of PrP to misfold and cause toxicity. This provides renewed opportunities to identify the intrinsic determinants of PrP misfolding through the contribution of key amino acids to different conformational states by MD simulations followed by experimental validation in transgenic flies.

Identification of a novel risk factor for chronic wasting disease (CWD) in elk: S100G single nucleotide polymorphism (SNP) of the prion protein gene (PRNP)

Prion diseases are fatal and malignant infectious encephalopathies induced by the pathogenic form of prion protein (PrPSc) originating from benign prion protein (PrPC). A previous study reported that the M132L single nucleotide polymorphism (SNP) of the prion protein gene (PRNP) is associated with susceptibility to chronic wasting disease (CWD) in elk. However, a recent meta-analysis integrated previous studies that did not find an association between the M132L SNP and susceptibility to CWD. Thus, there is controversy about the effect of M132L SNP on susceptibility to CWD. In the present study, we investigated novel risk factors for CWD in elk. We investigated genetic polymorphisms of the PRNP gene by amplicon sequencing and compared genotype, allele, and haplotype frequencies between CWD-positive and CWD-negative elk. In addition, we performed a linkage disequilibrium (LD) analysis by the Haploview version 4.2 program. Furthermore, we evaluated the 3D structure and electrostatic potential of elk prion protein (PrP) according to the S100G SNP using AlphaFold and the Swiss-PdbViewer 4.1 program. Finally, we analyzed the free energy change of elk PrP according to the S100G SNP using I-mutant 3.0 and CUPSAT. We identified 23 novel SNP of the elk PRNP gene in 248 elk. We found a strong association between PRNP SNP and susceptibility to CWD in elk. Among those SNP, S100G is the only non-synonymous SNP. We identified that S100G is predicted to change the electrostatic potential and free energy of elk PrP. To the best of our knowledge, this was the first report of a novel risk factor, the S100G SNP, for CWD.

First report of structural characteristics and polymorphisms of the prion protein gene in raccoon dogs: The possibility of prion disease-resistance.

Prion diseases are fatal degenerative encephalopathies caused by misfolded prion protein (PrPSc) converted from normal prion protein (PrPC). Previous studies have reported that genetic polymorphisms of the prion protein gene (PRNP) play a critical role in susceptibility to prion diseases. In addition, prion disease-resistant animals showed unique structural features of prion protein (PrP) related to species-specific amino acids. However, investigations of genetic polymorphisms of the PRNP gene and structural characteristics of PrP have not been performed in raccoon dogs thus far. We investigated genetic polymorphisms of PRNP in 87 raccoon dogs using amplicon sequencing and analyzed the genotype, allele, haplotype frequencies, and linkage disequilibrium (LD) using Haploview version 4.2. In addition, we performed phylogenetic analysis and multiple sequence alignment (MSA) using MEGA X version 10.1.8 and Clustal X version 2.1, respectively. We estimated the impact of raccoon dog and Canidae family-specific amino acids using PolyPhen-2, PROVEAN, and AMYCO. Furthermore, we analyzed the effect of raccoon dog and Canidae family-specific amino acids using the AlphaFold2 and Swiss-PdbViewer programs. We found 4 novel single nucleotide polymorphisms (SNPs) of the raccoon dog PRNP gene. In addition, the raccoon dog PrP showed 99.61% identity and the closest genetic distance to dog PrP. Among the substitutions of Canidae-specific amino acids with interspecific amino acids, D163N showed increased amyloidogenic propensity, and R181H showed alterations of hydrogen bonds. Furthermore, electrostatic potentials were changed according to the substitutions of D163N and R181H. By comparing PrP between raccoon dogs and raccoons, R168K and K224R were found to be related to changes in hydrogen bonds, and K224R altered the electrostatic potential of raccoon dog PrP. In the present study, we first reported 4 novel synonymous SNPs of the raccoon dog PRNP gene. We also identified that the PrP of raccoon dog has high homology (99.61%) with PrP of dog, which is a prion-resistant animal. In addition, raccoon dog PrP-specific amino acids are related to low amyloid propensity and inherent characteristics of 3D structure of raccoon dog PrP compared to the PrP of prion-susceptible species.

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 β1-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.

Novel Polymorphisms and Genetic Characteristics of the Prion Protein Gene in Pheasants.

Transmissible spongiform encephalopathies (TSEs) also known as prion diseases, are fatal neurodegenerative diseases. Prion diseases are caused by abnormal prion protein (PrPSc) derived from normal prion protein (PrPC), which is encoded by the prion protein gene (PRNP). Prion diseases have been reported in several mammals. Notably, chickens, one species of bird, have not been reported to develop prion diseases and showed resistance to bovine spongiform encephalopathy (BSE) infection. However, genetic polymorphisms of the PRNP gene and protein structure of the prion protein (PrP) related to vulnerability to prion diseases have not been investigated in pheasants, another species of bird. We performed amplicon sequencing of the pheasant PRNP gene to identify genetic polymorphisms in 148 pheasants. We analyzed the genotype, allele and haplotype frequencies of the pheasant PRNP polymorphisms. In addition, we evaluated the effect of genetic polymorphisms of the pheasant PRNP gene on pheasant PrP by the AMYCO, PROVEAN, PolyPhen-2 and PANTHER softwares. Furthermore, we compared the amino acid sequences of tandem repeat domains and secondary and tertiary structures of prion proteins (PrPs) among several animals. Finally, we investigated the impact of non-synonymous single nucleotide polymorphisms (SNPs) on hydrogen bonds and tertiary structures of pheasant PrP by Swiss PDB viewer software. We identified 34 novel genetic polymorphisms of the pheasant PRNP gene including 8 non-synonymous SNPs and 6 insertion/deletion polymorphisms. Among the non-synonymous SNPs, the L23F, G33C and R177Q SNPs showed that they could have a deleterious effect on pheasant PrP. In addition, the R177Q SNP was predicted to show an increase in amyloid propensity and a reduction in hydrogen bonds of pheasant PrP. Among the insertion/deletion polymorphisms, c.163_180delAACCCGGGGTATCCCCAC showed that it could have a detrimental effect on pheasant PrP. Furthermore, secondary and tertiary structures of pheasant PrP were predicted to have structures similar to those of chicken PrP. To the best of our knowledge, this is the first study on genetic polymorphisms of the pheasant PRNP gene.

First report of a strong association between genetic polymorphisms of the prion protein gene (PRNP) and susceptibility to chronic wasting disease in sika deer (Cervus nippon).

Prion diseases are incurable neurodegenerative disorders caused by proteinase K-resistant prion protein (PrPSc ) derived from normal prion protein (PrPC ) encoded by the prion protein gene (PRNP). Although the cervid PRNP gene plays a pivotal role in the pathological mechanism of chronic wasting disease (CWD), there is no existing association analysis between susceptibility to CWD and genetic polymorphisms of the PRNP gene in sika deer. We investigated genetic polymorphisms of the PRNP gene using amplicon sequencing in sika deer. In addition, to identify a genetic susceptibility factor, we compared the genotype, allele and haplotype frequencies of the PRNP gene between CWD-positive and CWD-negative sika deer. Furthermore, to assess the effect of the genetic polymorphisms on sika deer prion protein (PrP), we performed in silico analysis using PolyPhen-2, PROVEAN and AMYCO. Finally, we analysed the tertiary structure and electrostatic potential of sika deer PrP based on single nucleotide polymorphisms (SNPs) using the SWISS-MODEL and Swiss-PdbViewer programs. We found a total of 24 SNPs of the PRNP gene, including 22 novel SNPs (10 synonymous SNPs and 12 nonsynonymous SNPs), in sika deer. Among the nonsynonymous SNPs, we found a strong association of susceptibility to CWD with c.56G>A (Ser19Asn). In addition, we found that c.56G>A (Ser19Asn), c.296A>T (His99Leu) and c.560T>A (Val187Asp) were predicted to have damaging effects on sika deer PrP. Furthermore, we observed significant alterations in the electrostatic potential of sika deer PrP by genetic polymorphisms of the 187Asp allele. To the best of our knowledge, this was the first association study between genetic polymorphisms of the PRNP gene and susceptibility to CWD in sika deer.

Synthetic Sulfated Polymers Control Amyloid Aggregation of Ovine Prion Protein and Decrease Its Toxicity.

Amyloid aggregation, including aggregation and propagation of prion protein, is a key factor in numerous human diseases, so-called amyloidosis, with a very poor ability for treatment or prevention. The present work describes the effect of sulfated or sulfonated polymers (sodium dextran sulfate, polystyrene sulfonate, polyanethole sulfonate, and polyvinyl sulfate) on different stages of amyloidogenic conversion and aggregation of the prion protein, which is associated with prionopathies in humans and animals. All tested polymers turned out to induce amyloid conversion of the ovine prion protein. As suggested from molecular dynamics simulations, this effect probably arises from destabilization of the native prion protein structure by the polymers. Short polymers enhanced its further aggregation, whereas addition of high-molecular poly(styrene sulfonate) inhibited amyloid fibrils formation. According to the seeding experiments, the protein–polymer complexes formed after incubation with poly(styrene sulfonate) exhibited significantly lower amyloidogenic capacity compared with the control fibrils of the free prion protein. The cytotoxicity of soluble oligomers was completely inhibited by treatment with poly(styrene sulfonate). To summarize, sulfonated polymers are a promising platform for the formulation of a new class of anti-prion and anti-amyloidosis therapeutics.

Zn(II) binding causes interdomain changes in the structure and flexibility of the human prion protein

The cellular prion protein (PrPC) is a mainly α-helical 208-residue protein located in the pre- and postsynaptic membranes. For unknown reasons, PrPC can undergo a structural transition into a toxic, β-sheet rich scrapie isoform (PrPSc) that is responsible for transmissible spongiform encephalopathies (TSEs). Metal ions seem to play an important role in the structural conversion. PrPC binds Zn(II) ions and may be involved in metal ion transport and zinc homeostasis. Here, we use multiple biophysical techniques including optical and NMR spectroscopy, molecular dynamics simulations, and small angle X-ray scattering to characterize interactions between human PrPC and Zn(II) ions. Binding of a single Zn(II) ion to the PrPC N-terminal domain via four His residues from the octarepeat region induces a structural transition in the C-terminal α-helices 2 and 3, promotes interaction between the N-terminal and C-terminal domains, reduces the folded protein size, and modifies the internal structural dynamics. As our results suggest that PrPC can bind Zn(II) under physiological conditions, these effects could be important for the physiological function of PrPC.

Volatile Anesthetic Sevoflurane Precursor 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP) Exerts an Anti-Prion Activity in Prion-Infected Culture Cells

Prion disease is a neurodegenerative disorder with progressive neurologic symptoms and accelerated cognitive decline. The causative protein of prion disease is the prion protein (PrP), and structural transition of PrP from the normal helix rich form (PrPC) to the abnormal β-sheet rich form (PrPSc) occurs in prion disease. While so far numerous therapeutic agents for prion diseases have been developed, none of them are still useful. A fluorinated alcohol, hexafluoro isopropanol (HFIP), is a precursor to the inhalational anesthetic sevoflurane and its metabolites. HFIP is also known as a robust α-helix inducer and is widely used as a solvent for highly aggregated peptides. Here we show that the α-helix-inducing activity of HFIP caused the conformational transformation of the fibrous structure of PrP into amorphous aggregates in vitro. HFIP added to the ScN2a cell medium, which continuously expresses PrPSc, reduced PrPSc protease resistance after 24-h incubation. It was also clarified that ScN2a cells are more susceptible to HFIP than any of the cells being compared. Based on these findings, HFIP is expected to develop as a therapeutic agent for prion disease.

Pathologic Prion Protein Forms a Beta‐Solenoid Structure Initiated By Misfolding of Its Flexible N‐terminal Region

Prion diseases, such as Creutzfeld-Jakob disease, Gerstman-Sträussle-Scheinker syndrome, fatal familial insomnia, and kuru, are neurodegenerative diseases characterized by neuronal loss, the proliferation of glial cells, absence of an inflammatory response, and a spongiform appearance of neural elements. They occur due to an abnormal accumulation of an isoform of a host cell protein, the prion protein (PrP) found in neuronal and non-neuronal cells. In this process, PrP that is membrane-bound, designated as PrPC, is converted to a pathologic form, PrPSc. This conversion results from a conformational change in PrPC rather than a chemical modification. The Summit Country Day School MSOE Center for BioMolecular Modeling MAPS Team used 3D modeling and printing technology to examine structure-function relationships of the PrP and its pathogenic form, PrPSc. PrPC exists primarily in an alpha-helical conformation with a flexible N-terminal region and a C-terminal globular domain. The C-terminus consists of two β-sheets, three α-helices, and two potential sites for N-linked glycosylation. The N-terminus of PrPC is composed of approximately 125 amino acids, with octarepeat regions. In contrast, PrPSc forms highly complex structures characterized by a beta-solenoid, a protein-fold composed of repeating beta-subunits. The insoluble beta-solenoid structure of PrPSc forms amyloid fibrils, is prone to aggregation, and is thought to be both pathogenic and infective. The end of the PrPSc aggregate can form hydrogen bonds with additional PrP molecules, elongating the pathogenic PrPSc structure. Murine experiments point to a model of antibody-binding to the globular C-terminal domain of PrPC, which causes the PrPC flexible N-terminal region to misfold and transmit a pathogenic signal to neurons. The murine model mimics the interaction of PrPSc with PrPC. This project demonstrates the difference in structure between PrPC and PrPSc and highlights possible mechanisms for its neurotoxicity and propagation. Understanding the structure of PrP will elucidate potential targets in preventing pathology and infectivity in prion diseases.

Transmissible Spongiform Encephalopathies: An Underrecognized Infection Control Issue

Background: Transmissible spongiform encephalopathies comprise a class of rapidly progressive and inevitably fatal degenerative brain disorders. The pathogenesis of these diseases is thought to be due to a change in the structure of the normal prion protein to an abnormal structure, leading to propagation of the abnormal protein. This abnormal protein is highly transmissible; thus, appropriate infection control measures should be put in place if the diagnosis is suspected. However, the diagnosis is often not considered at all, and many hospitals do not have protocols in place. Our hospital missed a case of familial fatal insomnia in a 45-year-old male. He was diagnosed with fatal familial insomnia by autopsy. The autopsy was performed without appropriate infection control measures, leading to costly contamination of medical instruments and exposure of multiple staff. This occurrence led our institution to re-evaluate hospital protocols and guidelines regarding workup and management of transmissible spongiform encephalopathies (TSEs). Methods: We reviewed cases of TSEs or Creutzfeldt-Jakob Disease (CJD)-like illness presenting to our hospital over a 30-month period. Patients were considered for inclusion based on clinical suspicion. CDC diagnostic criteria were used. Infection control measures were employed, including an alert in the EMR. MRI was then performed. If clinical or diagnostic suspicion was high, the patient underwent lumbar puncture. CSF results were reviewed based on criteria Creutzfeldt-Jakob Disease Foundation criteria. Infection control measures were maintained throughout hospitalization. Results: In total, 34 patients met the inclusion criteria: 8 patients had confirmed CJD and 25 were negative. Medical records were not available for 1 patient, who was excluded. Lumbar puncture was performed on all suspected cases. Of those confirmed cases, the 7 patients who underwent lumber puncture had a positive result for 14-3-3 protein. Also, 5 patients underwent RT-QuIC testing and were found to have a positive result. No further cases of contamination occurred using our protocol. Additionally, 1 patient with suspected CJD underwent a brain biopsy with appropriate precautions after an inconclusive lumbar puncture. Although biospy was negative, the case exemplifies how the initiation of a protocol can optimize the workflow and prevent potentially dangerous exposure. Conclusion: Diagnosis of TSEs remains difficult and is often missed. In our case, lack of suspicion for TSE led to a waste of resources and unnecessary exposure of staff member. It is of utmost importance to consider TSEs in rapidly progressive dementia and to employ appropriate sterile guidelines to prevent contamination of equipment and potential subsequent transmission. Healthcare providers should consider a similar protocol in cases suspicious for TSEs.Funding: NoneDisclosures: None

Structural effects of the highly protective V127 polymorphism on human prion protein

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease.

Novel Polymorphisms and Genetic Characteristics of the Prion Protein Gene (PRNP) in Dogs—A Resistant Animal of Prion Disease

Transmissible spongiform encephalopathies (TSEs) have been reported in a wide range of species. However, TSE infection in natural cases has never been reported in dogs. Previous studies have reported that polymorphisms of the prion protein gene (PRNP) have a direct impact on the susceptibility of TSE. However, studies on polymorphisms of the canine PRNP gene are very rare in dogs. We examined the genotype, allele, and haplotype frequencies of canine PRNP in 204 dogs using direct sequencing and analyzed linkage disequilibrium (LD) using Haploview version 4.2. In addition, to evaluate the impact of nonsynonymous polymorphisms on the function of prion protein (PrP), we carried out in silico analysis using PolyPhen-2, PROVEAN, and PANTHER. Furthermore, we analyzed the structure of PrP and hydrogen bonds according to alleles of nonsynonymous single nucleotide polymorphisms (SNPs) using the Swiss-Pdb Viewer program. Finally, we predicted the impact of the polymorphisms on the aggregation propensity of dog PrP using AMYCO. We identified a total of eight polymorphisms, including five novel SNPs and one insertion/deletion polymorphism, and found strong LDs and six major haplotypes among eight polymorphisms. In addition, we identified significantly different distribution of haplotypes among eight dog breeds, however, the kinds of identified polymorphisms were different among each dog breed. We predicted that p.64_71del HGGGWGQP, Asp182Gly, and Asp182Glu polymorphisms can impact the function and/or structure of dog PrP. Furthermore, the number of hydrogen bonds of dog PrP with the Glu182 and Gly182 alleles were predicted to be less than those with the Asp182 allele. Finally, Asp163Glu and Asp182Gly showed more aggregation propensity than wild-type dog PrP. These results suggest that nonsynonymous SNPs, Asp182Glu and Asp182Gly, can influence the stability of dog PrP and confer the possibility of TSE infection in dogs.

Unique Structural Features of Mule Deer Prion Protein Provide Insights into Chronic Wasting Disease.

Chronic wasting disease (CWD) is a highly infectious prion disease of cervids. Accumulation of prions, the disease-specific structural conformers of the cellular prion protein (PrPC), in the central nervous system, is the key pathological event of the disorder. The analysis of cervid PrPC sequences revealed the existence of polymorphism at position 226, in which deer PrP contains glutamine (Q), whereas elk PrP contains glutamate (E). The effects of this polymorphism on CWD are still unknown. We determined the high-resolution nuclear magnetic resonance structure of the mule deer prion protein that was compared to previously published PrP structures of elk and white-tailed deer. We found that the polymorphism Q226E could influence the long-range intramolecular interactions and packing of the β2−α2 loop and the C-terminus of the α3 helix of cervid PrP structures. This solvent-accessible epitope is believed to be involved in prion conversion. Additional differences were observed at the beginning of the well-defined C-terminus domain, in the α2−α3 region, and in its interactions with the α1 helix. Here, we highlight the importance of the PrP structure in prion susceptibility and how single amino acid differences might influence the overall protein folding.

Transient multimers modulate conformer abundances of prion protein monomer through conformational selection

Prions are known to be involved in neurodegenerative pathologies such as Creutzfeld-Jakob disease. Current models point to a molecular event which rely on a transmissible structural change that leads to the production of β-sheet-rich prion conformer (PrPSc). PrPSc itself has the capability to trigger the structural rearrangement of the ubiquitously present prion (PrPc) substrate in a self-perpetuating cascade. In this article, we demonstrate that recombinant PrPc exists in a conformational equilibrium. The conformers’ abundances were shown to be dependent on PrPc concentration through the formation of transient multimers leading to conformational selection. The study of PrPc mutants that follow dedicated oligomerization pathways demonstrated that the conformers’ relative abundances are modified, thus reinforcing the assertion that the nature of conformers’ interactions orient the oligomerization pathways. Further this result can be viewed as the “signature” of an aborted oligomerization process. This discovery sheds a new light on the possible origin of prion protein diseases, namely that a change in prion protein structure could be transmitted through the formation of transient multimers having different conformer compositions. This could explain the selection of a transient multimeric type that could be viewed as the precursor of PrPSc responsible for structural information transmission, and strain apparition.

Protective Role of the Host Innate Immune System in Prion Pathogenesis

Prion diseases, including human Creutzfeldt-Jakob disease, are infectious, intractable central neurodegenerative diseases, which are also zoonoses that commonly infect not only higher organisms but also a wide variety of animals. Pathogenic prions induce abnormal prion protein (PrP), which is produced by structural conversion of normal PrP, a beta-sheet-rich structure with high aggregation propensity. Thus, it is believed that the host is immunotolerant against prions because there is no difference in the primary structure of normal and abnormal PrP, and prions do not induce a marked immune response. Recently, using mutated Toll-like receptor (TLR) 4-transgenic mice, a bioassay after prion inoculation has intriguingly found that the TLR4-signaling pathway may have a protective role against prion infection. Meanwhile, we reported that a transcription factor, interferon regulatory factor-3 (IRF-3), located downstream of TLR4 signaling, showed resistance to prions. IRF-3-inducing type I interferon (I-IFN) is a critical factor for the host defense against pathogen invasion. These findings indicate that the TLR-signaling pathway of the innate immune system might regulate prion invasion. However, the details have not been fully determined. In this symposium, we will introduce new findings including the relationship between I-IFN and prions.

In silico Analyses of the Binding of Experimentally Tested or Potentially Anti‐Prion Drugs to the PrPC Structures of Several Species of Mammals

Transmissible Spongiform Encephalopathies (TSE's) or prion diseases are a group of rare neurodegenerative diseases that include Creutzfeldt–Jacob Syndrome in humans and Bovine Spongiform Encephalopathy (BSE) in cattle, and which are inevitably fatal. We have made in silico analyses on the docking of several drugs with either experimentally determined or potential anti‐prion activity, to the experimentally determined structures of the cellular isoform of the prion protein (PrPC) from several species of mammals, including humans.ObjectivesThe objectives of our study are, in the first place to identify differences, if any, between the predicted affinities and/or binding regions of the drugs tested to the PrPC structures from several species of mammals including humans, cattle, mouse, rabbit, and the Bank vole and, secondly, to determine whether these differences, if found, have any correlation with the different susceptibilities to infection with scrapie (PrPSc) prion protein from other species that different mammals exhibit.MethodsWe used free web–based software in our analyses, especially AUTODOCK VINA to model the binding of the drugs tested to experimentally determined crystal or NMR structures that are already available in the Protein Data Bank (PDB). We also used the web–based PRODRG platform to generate the structures of the drugs tested in PDB format, the NCBI Blast software for sequence alignments, and the Statdisk software to perform statistical analyses.ResultsSo far, we have not found yet any significant differences in the predicted affinities and/or binding regions for the drugs tested between binding to the Bank vole prion protein structure and binding to PrPC structures from other mammals which are less susceptible to infection with PrPSc from other species.ConclusionsWe have successfully modeled the binding of several drugs with potential anti‐prion activity to the experimentally determined NMR or crystal structures of the cellular prion protein of several mammals, including humans. Our results suggest that the differential susceptibilities to infection between the Bank vole and other species of mammals that have been found by experiments cannot be explained, at least alone, by structural differences between their PrPC isoforms.Support or Funding InformationThe author is a PhD candidate at the Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, and has received financial support for his studies from the grants NIMHD‐G12MD007600 and 8U54MD007587 to the University of Puerto Rico, and also from grant NIGMS‐RISE‐R25GM061838 and NIGMS‐INBRE.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prediction of Secondary Structures of Human Prion Proteins using Miscellaneous Methods

The contemporary study is an attempt to predict the secondary structures of proteins, from the dataset of human prion proteins which has been acquired from NCBI repository. In this context, we have exploited PSIPRED server which is considered to be proficient and impulsive method to protein structure prediction where users can submit the query sequence of their desire and receive the results of prediction both textually and eloquently. Furthermore, Phyre2 was applied across the amino acid sequence which is among the most widely server deployed for generating consistent protein models characterizing the prediction of protein structures. Moreover, two feed forward neural networks with a sole hidden layer which was tested and trained on a 10 fold cross validation mechanism in MATLAB, and subsequently significant prediction accuracy of 71.73% and minimum mean absolute error of 12.3% was achieved.

Changes in Secondary Structure of Human Prion Protein Peptide 30-90 under the Influence of Selected Dicationic and Anionic Surfactants

The peptide (PrP30-90), which was used in this study, was designed on the basis of amino acid sequence of the unstructured N-terminal domain of human prion protein. Flexibility of this moiety and physicochemical properties of PrP N-terminal domain are related to the ability of prion protein to bind metal cations and are probably linked with the pathological conformational changes and formation insoluble deposits of this protein in the brain, which were observed in transmissible spongiform encephalopathies (e.g. Creutzfeldt-Jakob disease, CJD) [1]. This peptide, as well as N-terminal domain of PrP, possesses octarepeat sequences containing four histidine residues which overlap with binding sites of metal ions (copper, zinc etc). Interactions with surfactants probably could influence secondary structure of N-terminal domain or even full human prion protein. The present study was aimed at determination of the effects of cationic gemini imidazolium surfactant and anionic surfactant with different spacer groups on secondary structure of human prion protein at different pH values. The studies were carried out in MES buffer (pH 5.5) and phosphate buffer (pH 7,4) using circular dichroism (CD, Fourier transform infrared spectroscopy (FTIR) spectroscopy, NMR diffusometry and transmission electron microscopy (TEM). This work was supported by the funds from the National Science Centre (Poland) granted on the basis of decision no. 2016/23/N/ST5/02013. [1] Cobb N.J., Surewicz W.K. (2009) Biochemistry. 48(12), 2574-2585.