The Region Approximately between Amino Acids 81 and 137 of Proteinase K-Resistant PrPSc Is Critical for the Infectivity of the Chandler Prion Strain

ABSTRACT Although the major component of the prion is believed to be the oligomer of PrPSc, little information is available concerning regions on the PrPSc molecule that affect prion infectivity. During the analysis of PrPSc molecules from various prion strains, we found that PrPSc of the Chandler strain showed a unique property in the conformational-stability assay, and this property appeared to be useful for studying the relationship between regions of the PrPSc molecule and prion infectivity. Thus, we analyzed PrPSc of the Chandler strain in detail and analyzed the infectivities of the N-terminally denatured and truncated forms of proteinase K-resistant PrP. The N-terminal region of PrPSc of the Chandler strain showed region-dependent resistance to guanidine hydrochloride (GdnHCl) treatment. The region approximately between amino acids (aa) 81 and 137 began to be denatured by treatment with 1.5 M GdnHCl. Within this stretch, the region comprising approximately aa 81 to 90 was denatured almost completely by 2 M GdnHCl. Furthermore, the region approximately between aa 90 and 137 was denatured completely by 3 M GdnHCl. However, the C-terminal region thereafter was extremely resistant to the GdnHCl treatment. This property was not observed in PrPSc molecules of other prion strains. Denaturation of the region between aa 81 and 137 by 3 M GdnHCl significantly prolonged the incubation periods in mice compared to that for the untreated control. More strikingly, the denaturation and removal of this region nearly abolished the infectivity. This finding suggests that the conformation of the region between aa 81 and 137 of the Chandler strain PrPSc molecule is directly associated with prion infectivity.

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Mouse-Adapted Ovine Scrapie Prion Strains Are Characterized by Different Conformers of PrPSc

Journal of Virology ◽

10.1128/jvi.01434-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12119-12127 ◽

Cited By ~ 31

Author(s):

Alana M. Thackray ◽

Lee Hopkins ◽

Michael A. Klein ◽

Raymond Bujdoso

Keyword(s):

Biological Diversity ◽

Guanidine Hydrochloride ◽

Serial Passage ◽

Biological Properties ◽

Proteinase K ◽

Specific Information ◽

Strain Typing ◽

Prion Strain ◽

Prion Strains ◽

Scrapie Strains

ABSTRACT The agent responsible for prion disease may exist in different forms, commonly referred to as strains, with each carrying the specific information that determines its own distinct biological properties, such as incubation period and lesion profile. Biological strain typing of ovine scrapie isolates by serial passage in conventional mice has shown some diversity in ovine prion strains. However, this biological diversity remains poorly supported by biochemical prion strain typing. The protein-only hypothesis predicts that variation between different prion strains in the same host is manifest in different conformations adopted by PrPSc. Here we have investigated the molecular properties of PrPSc associated with two principal Prnp a mouse-adapted ovine scrapie strains, namely, RML and ME7, in order to establish biochemical prion strain typing strategies that may subsequently be used to discriminate field cases of mouse-passaged ovine scrapie isolates. We used a conformation-dependent immunoassay and a conformational stability assay, together with Western blot analysis, to demonstrate that RML and ME7 PrPSc proteins show distinct biochemical and physicochemical properties. Although RML and ME7 PrPSc proteins showed similar resistance to proteolytic digestion, they differed in their glycoform profiles and levels of proteinase K (PK)-sensitive and PK-resistant isoforms. In addition, the PK-resistant core (PrP27-30) of ME7 was conformationally more stable following exposure to guanidine hydrochloride or Sarkosyl than was RML PrP27-30. Our data show that mouse-adapted ovine scrapie strains can be discriminated by their distinct conformers of PrPSc, which provides a basis to investigate their diversity at the molecular level.

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Molecular and Transmission Characteristics of Primary-Passaged Ovine Scrapie Isolates in Conventional and Ovine PrP Transgenic Mice

Journal of Virology ◽

10.1128/jvi.01454-08 ◽

2008 ◽

Vol 82 (22) ◽

pp. 11197-11207 ◽

Cited By ~ 37

Author(s):

Alana M. Thackray ◽

Lee Hopkins ◽

John Spiropoulos ◽

Raymond Bujdoso

Keyword(s):

Transgenic Mice ◽

Transmission Characteristics ◽

Prion Strain ◽

Prion Strains ◽

True Diversity ◽

Incubation Periods ◽

Primary Transmission ◽

Molecular Nature ◽

Sheep Scrapie ◽

Mouse Lines

ABSTRACT A more complete assessment of ovine prion strain diversity will be achieved by complementing biological strain typing in conventional and ovine PrP transgenic mice with a biochemical analysis of the resultant PrPSc. This will provide a correlation between ovine prion strain phenotype and the molecular nature of different PrP conformers associated with particular prion strains. Here, we have compared the molecular and transmission characteristics of ovine ARQ/ARQ and VRQ/VRQ scrapie isolates following primary passage in tg338 (VRQ) and tg59 (ARQ) ovine PrP transgenic mice and the conventional mouse lines C57BL/6 (Prnp a ), RIII (Prnp a ), and VM (Prnp b ). Our data show that these different genotypes of scrapie isolates display similar incubation periods of >350 days in conventional and tg59 mice. Facilitated transmission of sheep scrapie isolates occurred in tg338 mice, with incubation times reduced to 64 days for VRQ/VRQ inocula and to ≤210 days for ARQ/ARQ samples. Distinct genotype-specific lesion profiles were seen in the brains of conventional and tg59 mice with prion disease, which was accompanied by the accumulation of more conformationally stable PrPSc, following inoculation with ARQ/ARQ compared to VRQ/VRQ scrapie isolates. In contrast, the lesion profiles, quantities, and stability of PrPSc induced by the same inocula in tg338 mice were more similar than in the other mouse lines. Our data show that primary transmission of different genotypes of ovine prions is associated with the formation of different conformers of PrPSc with distinct molecular properties and provide the basis of a molecular approach to identify the true diversity of ovine prion strains.

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Host Determinants of Prion Strain Diversity Independent of Prion Protein Genotype

Journal of Virology ◽

10.1128/jvi.01586-15 ◽

2015 ◽

Vol 89 (20) ◽

pp. 10427-10441 ◽

Cited By ~ 23

Author(s):

Jenna Crowell ◽

Andrew Hughson ◽

Byron Caughey ◽

Richard A. Bessen

Keyword(s):

Transgenic Mice ◽

Prion Protein ◽

Protein Gene ◽

Phenotypic Diversity ◽

Prion Diseases ◽

Conformational Stability ◽

Epigenetic Mechanism ◽

Prion Strain ◽

Disease Phenotypes ◽

Prion Strains

ABSTRACTPhenotypic diversity in prion diseases can be specified by prion strains in which biological traits are propagated through an epigenetic mechanism mediated by distinct PrPScconformations. We investigated the role of host-dependent factors on phenotypic diversity of chronic wasting disease (CWD) in different host species that express the same prion protein gene (Prnp). Two CWD strains that have distinct biological, biochemical, and pathological features were identified in transgenic mice that express the Syrian golden hamster (SGH)Prnp. The CKY strain of CWD had a shorter incubation period than the WST strain of CWD, but after transmission to SGH, the incubation period of CKY CWD was ∼150 days longer than WST CWD. Limited proteinase K digestion revealed strain-specific PrPScpolypeptide patterns that were maintained in both hosts, but the solubility and conformational stability of PrPScdiffered for the CWD strains in a host-dependent manner. WST CWD produced PrPScamyloid plaques in the brain of the SGH that were partially insoluble and stable at a high concentration of protein denaturant. However, in transgenic mice, PrPScfrom WST CWD did not assemble into plaques, was highly soluble, and had low conformational stability. Similar studies using the HY and DY strains of transmissible mink encephalopathy resulted in minor differences in prion biological and PrPScproperties between transgenic mice and SGH. These findings indicate that host-specific pathways that are independent ofPrnpcan alter the PrPScconformation of certain prion strains, leading to changes in the biophysical properties of PrPSc, neuropathology, and clinical prion disease.IMPORTANCEPrions are misfolded pathogenic proteins that cause neurodegeneration in humans and animals. Transmissible prion diseases exhibit a spectrum of disease phenotypes and the basis of this diversity is encoded in the structure of the pathogenic prion protein and propagated by an epigenetic mechanism. In the present study, we investigated prion diversity in two hosts species that express the same prion protein gene. While prior reports have demonstrated that prion strain properties are stable upon infection of the same host species and prion protein genotype, our findings indicate that certain prion strains can undergo dramatic changes in biological properties that are not dependent on the prion protein. Therefore, host factors independent of the prion protein can affect prion diversity. Understanding how host pathways can modify prion disease phenotypes may provide clues on how to alter prion formation and lead to treatments for prion, and other, human neurodegenerative diseases of protein misfolding.

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Analysis of Conformational Stability of Abnormal Prion Protein Aggregates across the Spectrum of Creutzfeldt-Jakob Disease Prions

Journal of Virology ◽

10.1128/jvi.00144-16 ◽

2016 ◽

Vol 90 (14) ◽

pp. 6244-6254 ◽

Cited By ~ 24

Author(s):

Maura Cescatti ◽

Daniela Saverioni ◽

Sabina Capellari ◽

Fabrizio Tagliavini ◽

Tetsuyuki Kitamoto ◽

...

Keyword(s):

Prion Protein ◽

Molecular Basis ◽

Phenotypic Variability ◽

Guanidine Hydrochloride ◽

Conformational Stability ◽

Protein Aggregates ◽

Replication Efficiency ◽

Prion Strains ◽

Jakob Disease ◽

Increasing Temperature

ABSTRACTThe wide phenotypic variability of prion diseases is thought to depend on the interaction of a host genotype with prion strains that have self-perpetuating biological properties enciphered in distinct conformations of the misfolded prion protein PrPSc. This concept is largely based on indirect approaches studying the effect of proteases or denaturing agents on the physicochemical properties of PrPScaggregates. Furthermore, most data come from studies on rodent-adapted prion strains, making current understanding of the molecular basis of strains and phenotypic variability in naturally occurring diseases, especially in humans, more limited. To fill this gap, we studied the effects of guanidine hydrochloride (GdnHCl) and heating on PrPScaggregates extracted from 60 sporadic Creutzfeldt-Jakob disease (CJD) and 6 variant CJD brains. While denaturation curves obtained after exposure of PrPScto increasing GdnHCl concentrations showed similar profiles among the 7 CJD types analyzed, PrPScexposure to increasing temperature revealed significantly different and type-specific responses. In particular, MM1 and VV2, the most prevalent and fast-replicating CJD types, showed stable and highly resistant PrPScaggregates, whereas VV1, a rare and slowly propagating type, revealed unstable aggregates that easily dissolved at low temperature. Taken together, our results indicate that the molecular interactions mediating the aggregation state of PrPSc, possibly enciphering strain diversity, are differently targeted by GdnHCl, temperature, and proteases. Furthermore, the detected positive correlation between the thermostability of PrPScaggregates and disease transmission efficiency makes inconsistent the proposed hypothesis that a decrease in conformational stability of prions results in an increase in their replication efficiency.IMPORTANCEPrion strains are defined as infectious isolates propagating distinctive phenotypic traits after transmission to syngeneic hosts. Although the molecular basis of prion strains is not fully understood, it is largely accepted that variations in prion protein conformation drive the molecular changes leading to the different phenotypes. In this study, we exposed abnormal prion protein aggregates encompassing the spectrum of human prion strains to both guanidine hydrochloride and thermal unfolding. Remarkably, while exposure to increasing temperature revealed significant strain-specific differences in the denaturation profile of the protein, treatment with guanidine hydrochloride did not. The findings suggest that thermal and chemical denaturation perturb the structure of prion protein aggregates differently. Moreover, since the most thermostable prion protein types were those associated with the most prevalent phenotypes and most rapidly and efficiently transmitting strains, the results suggest a direct correlation between strain replication efficiency and the thermostability of prion protein aggregates.

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Two distinct conformers of PrPD type 1 of sporadic Creutzfeldt–Jakob disease with codon 129VV genotype faithfully propagate in vivo

Acta Neuropathologica Communications ◽

10.1186/s40478-021-01132-7 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Ignazio Cali ◽

Juan Carlos Espinosa ◽

Satish K. Nemani ◽

Alba Marin-Moreno ◽

Manuel V. Camacho ◽

...

Keyword(s):

Proteinase K ◽

Disease Phenotypes ◽

Prion Strains ◽

Electrophoretic Profile ◽

Incubation Periods ◽

Jakob Disease ◽

Prp Gene ◽

Codon 129

AbstractCurrent classifications of sporadic Creutzfeldt–Jakob disease (sCJD) identify five subtypes associated with different disease phenotypes. Most of these histopathological phenotypes (histotypes) co-distribute with distinct pairings of methionine (M)/valine (V) genotypes at codon 129 of the prion protein (PrP) gene and the type (1 or 2) of the disease-associated PrP (PrPD). Types 1 and 2 are defined by the molecular mass (~ 21 kDa and ~ 19 kDa, respectively) of the unglycosylated isoform of the proteinase K-resistant PrPD (resPrPD). We recently reported that the sCJDVV1 subtype (129VV homozygosity paired with PrPD type 1, T1) shows an electrophoretic profile where the resPrPD unglycosylated isoform is characterized by either one of two single bands of ~ 20 kDa (T120) and ~ 21 kDa (T121), or a doublet of ~ 21–20 kDa (T121−20). We also showed that T120 and T121 in sCJDVV have different conformational features but are associated with indistinguishable histotypes. The presence of three distinct molecular profiles of T1 is unique and raises the issue as to whether T120 and T121 represent distinct prion strains. To answer this question, brain homogenates from sCJDVV cases harboring each of the three resPrPD profiles, were inoculated to transgenic (Tg) mice expressing the human PrP-129M or PrP-129V genotypes. We found that T120 and T121 were faithfully replicated in Tg129V mice. Electrophoretic profile and incubation period of mice challenged with T121−20 resembled those of mice inoculated with T121 and T120, respectively. As in sCJDVV1, Tg129V mice challenged with T121 and T120 generated virtually undistinguishable histotypes. In Tg129M mice, T121 was not replicated while T120 and T121−20 generated a ~ 21–20 kDa doublet after lengthier incubation periods. On second passage, Tg129M mice incubation periods and regional PrP accumulation significantly differed in T120 and T121−20 challenged mice. Combined, these data indicate that T121 and T120 resPrPD represent distinct human prion strains associated with partially overlapping histotypes.

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Prions from Sporadic Creutzfeldt-Jakob Disease Patients Propagate as Strain Mixtures

mBio ◽

10.1128/mbio.00393-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 2

Author(s):

Hervé Cassard ◽

Alvina Huor ◽

Juan-Carlos Espinosa ◽

Jean-Yves Douet ◽

Severine Lugan ◽

...

Keyword(s):

Relative Proportion ◽

Significant Proportion ◽

Proteinase K ◽

Single Patient ◽

Prion Strain ◽

Prion Strains ◽

Jakob Disease ◽

The Brain

ABSTRACT Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently classified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K-digested abnormal prion protein (PrPres) isoform identified by Western blotting (type 1 or type 2). Converging evidence led to the view that MM/MV1, VV/MV2, and VV1 and MM2 sCJD cases are caused by distinct prion strains. However, in a significant proportion of sCJD patients, both type 1 and type 2 PrPres were reported to accumulate in the brain, which raised questions about the diversity of sCJD prion strains and the coexistence of two prion strains in the same patient. In this study, a panel of sCJD brain isolates (n = 29) that displayed either a single or mixed type 1/type 2 PrPres were transmitted into human-PrP-expressing mice (tgHu). These bioassays demonstrated that two distinct prion strains (M1CJD and V2CJD) were associated with the development of sCJD in MM1/MV1 and VV2/MV2 patients. However, in about 35% of the investigated VV and MV cases, transmission results were consistent with the presence of both M1CJD and V2CJD strains, including in patients who displayed a “pure” type 1 or type 2 PrPres. The use of a highly sensitive prion in vitro amplification technique that specifically probes the V2CJD strain revealed the presence of the V2CJD prion in more than 80% of the investigated isolates, including isolates that propagated as a pure M1CJD strain in tgHu. These results demonstrate that at least two sCJD prion strains can be present in a single patient. IMPORTANCE sCJD occurrence is currently assumed to result from spontaneous and stochastic formation of a misfolded PrP nucleus in the brains of affected patients. This original nucleus then recruits and converts nascent PrPC into PrPSc, leading to the propagation of prions in the patient’s brain. Our study demonstrates the coexistence of two prion strains in the brains of a majority of the 23 sCJD patients investigated. The relative proportion of these sCJD strains varied both between patients and between brain areas in a single patient. These findings strongly support the view that the replication of an sCJD prion strain in the brain of a patient can result in the propagation of different prion strain subpopulations. Beyond its conceptual importance for our understanding of prion strain properties and evolution, the sCJD strain mixture phenomenon and its frequency among patients have important implications for the development of therapeutic strategies for prion diseases.

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The effects of prion protein proteolysis and disaggregation on the strain properties of hamster scrapie

Journal of General Virology ◽

10.1099/vir.0.2008/002303-0 ◽

2008 ◽

Vol 89 (10) ◽

pp. 2642-2650 ◽

Cited By ~ 15

Author(s):

Andrea M. Deleault ◽

Nathan R. Deleault ◽

Brent T. Harris ◽

Judy R. Rees ◽

Surachai Supattapone

Keyword(s):

Physical Properties ◽

Conformational Stability ◽

Proteinase K ◽

Prion Strain ◽

Biochemical Characteristics ◽

Strain Diversity ◽

Protease Digestion ◽

Scrapie Strains ◽

Strain Dependent ◽

Mammalian Prions

Native mammalian prions exist in self-propagating strains that exhibit distinctive clinical, pathological and biochemical characteristics. Prion strain diversity is associated with variations in PrPSc conformation, but it remains unknown precisely which physical properties of the PrPSc molecules are required to encipher mammalian prion strain phenotypes. In this study, we subjected prion-infected brain homogenates derived from three different hamster scrapie strains to either (i) proteinase K digestion or (ii) sonication, and inoculated the modified samples into normal hamsters. The results show that the strain-specific clinical features and neuropathological profiles of inoculated animals were not affected by either treatment. Similarly, the strain-dependent biochemical characteristics of the PrPSc molecules (including electrophoretic mobility, glycoform composition, conformational stability and susceptibility to protease digestion) in infected animals were unaffected by either proteolysis or sonication of the original inocula. These results indicate that the infectious strain properties of native prions do not appear to be altered by PrPSc disaggregation, and that maintenance of such properties does not require the N-domain (approximately residues 23–90) of the protease-resistant PrPSc molecules or protease-sensitive PrPSc molecules.

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Isoleucine Plays an Important Role for Maintaining Immune Function

Current Protein and Peptide Science ◽

10.2174/1389203720666190305163135 ◽

2019 ◽

Vol 20 (7) ◽

pp. 644-651 ◽

Cited By ~ 3

Author(s):

Changsong Gu ◽

Xiangbing Mao ◽

Daiwen Chen ◽

Bing Yu ◽

Qing Yang

Keyword(s):

Amino Acids ◽

Acid Metabolism ◽

Branched Chain Amino Acids ◽

Whole Body ◽

Immune Functions ◽

Host Defense Peptides ◽

Innate And Adaptive Immunity ◽

Physiological Functions ◽

Branched Chain ◽

The Relationship

Branched chain amino acids are the essential nutrients for humans and many animals. As functional amino acids, they play important roles in physiological functions, including immune functions. Isoleucine, as one of the branched chain amino acids, is also critical in physiological functions of the whole body, such as growth, immunity, protein metabolism, fatty acid metabolism and glucose transportation. Isoleucine can improve the immune system, including immune organs, cells and reactive substances. Recent studies have also shown that isoleucine may induce the expression of host defense peptides (i.e., β-defensins) that can regulate host innate and adaptive immunity. In addition, isoleucine administration can restore the effect of some pathogens on the health of humans and animals via increasing the expression of β-defensins. Therefore, the present review will emphatically discuss the effect of isoleucine on immunity while summarizing the relationship between branched chain amino acids and immune functions.

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Monoclonal antibody studies of creatine kinase. The ART epitope: evidence for an intermediate in protein folding

Biochemical Journal ◽

10.1042/bj2570461 ◽

1989 ◽

Vol 257 (2) ◽

pp. 461-469 ◽

Cited By ~ 25

Author(s):

G E Morris

Keyword(s):

Amino Acids ◽

Monoclonal Antibody ◽

Creatine Kinase ◽

Specific Binding ◽

Trypsin Digestion ◽

Proteinase K ◽

Enzyme Linked Immunosorbent Assay ◽

Muscle Creatine Kinase ◽

Western Blots ◽

Methionine Residues

Chemical cleavage at cysteine residues with nitrothiocyanobenzoic acid shows that the last 98 amino acids of the 380-amino-acid sequence of chick muscle creatine kinase are sufficient for binding of the monoclonal antibody CK-ART. Removal of the last 30 amino acids by cleavage at methionine residues with CNBr results in loss of CK-ART binding. CK-ART binding is also lost when these C-terminal methionine residues are oxidized to sulphoxide, but binding is regained on reduction. Proteinase K ‘nicks’ native CK at a single site near the C-terminus and two fragments of 327 amino acides and 53 amino acids can be separated by subsequent SDS or urea treatment. CK-ART still binds normally to ‘nicked’ CK, which is enzymically inactive. After treatment with either urea (in a competition enzyme-linked immunosorbent assay) or SDS (on Western blots), however, CK-ART binds to neither of the two fragments, although these treatments do not affect binding to intact CK. This suggests that parts of both CK fragments contribute to the CK-ART epitope. CK-ART is both species- and isoenzyme-specific, binding only to chick M-CK. The only C-terminal regions containing chick-specific sequences are residues 300-312 and residues 368-371, the latter group being close to the essential methionine residues. We suggest that one, or possibly both, of these regions is involved in forming the conformational epitope on the surface of the CK molecule which CK-ART recognizes. Native CK is resistant to trypsin digestion. The C-terminal half of urea-treated and partly-refolded CK is also resistant to trypsin digestion, whereas the N-terminal half is readily digested. The results suggest a C-terminal region which can refold more rapidly than the rest of the CK molecule and provide evidence for an intermediate in CK refolding.

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Unfolding of Helical Poly(L-Glutamic Acid) in N,N-Dimethylformamide Probed by Pyrene Excimer Fluorescence (PEF)

Polymers ◽

10.3390/polym13111690 ◽

2021 ◽

Vol 13 (11) ◽

pp. 1690

Author(s):

Weize Yuan ◽

Remi Casier ◽

Jean Duhamel

Keyword(s):

Glutamic Acid ◽

Local Density ◽

Guanidine Hydrochloride ◽

Racemic Mixture ◽

Excimer Fluorescence ◽

Excimer Formation ◽

Blob Model ◽

Α Helix ◽

The Relationship ◽

Pyrene Excimer Formation

The denaturation undergone by α–helical poly(L-glutamic acid) (PLGA) in N,N-dimethylformamide upon addition of guanidine hydrochloride (GdHCl) was characterized by comparing the fluorescence of a series of PLGA constructs randomly labeled with the dye pyrene (Py-PLGA) to that of a series of Py-PDLGA samples prepared from a racemic mixture of D,L-glutamic acid. The process of pyrene excimer formation (PEF) was taken advantage of to probe changes in the conformation of α–helical Py-PLGA. Fluorescence Blob Model (FBM) analysis of the fluorescence decays of the Py-PLGA and Py-PDLGA constructs yielded the average number (<Nblob>) of glutamic acids located inside a blob, which represented the volume probed by an excited pyrenyl label. <Nblob> remained constant for randomly coiled Py-PDLGA but decreased from ~20 to ~10 glutamic acids for the Py-PLGA samples as GdHCl was added to the solution. The decrease in <Nblob> reflected the decrease in the local density of PLGA as the α–helix unraveled in solution. The changes in <Nblob> with GdHCl concentration was used to determine the change in Gibbs energy required to denature the PLGA α–helix in DMF. The relationship between <Nblob> and the local density of macromolecules can now be applied to characterize the conformation of macromolecules in solution.

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