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1.
PLoS Pathog ; 18(11): e1010947, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36342968

RESUMO

Prion strains in a given type of mammalian host are distinguished by differences in clinical presentation, neuropathological lesions, survival time, and characteristics of the infecting prion protein (PrP) assemblies. Near-atomic structures of prions from two host species with different PrP sequences have been determined but comparisons of distinct prion strains of the same amino acid sequence are needed to identify purely conformational determinants of prion strain characteristics. Here we report a 3.2 Å resolution cryogenic electron microscopy-based structure of the 22L prion strain purified from the brains of mice engineered to express only PrP lacking glycophosphatidylinositol anchors [anchorless (a) 22L]. Comparison of this near-atomic structure to our recently determined structure of the aRML strain propagated in the same inbred mouse reveals that these two mouse prion strains have distinct conformational templates for growth via incorporation of PrP molecules of the same sequence. Both a22L and aRML are assembled as stacks of PrP molecules forming parallel in-register intermolecular ß-sheets and intervening loops, with single monomers spanning the ordered fibril core. Each monomer shares an N-terminal steric zipper, three major arches, and an overall V-shape, but the details of these and other conformational features differ markedly. Thus, variations in shared conformational motifs within a parallel in-register ß-stack fibril architecture provide a structural basis for prion strain differentiation within a single host genotype.


Assuntos
Príons , Animais , Camundongos , Microscopia Crioeletrônica , Genótipo , Proteínas Priônicas/genética , Príons/metabolismo , Conformação Proteica
2.
Nat Commun ; 13(1): 4005, 2022 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-35831291

RESUMO

Little is known about the structural basis of prion strains. Here we provide a high (3.0 Å) resolution cryo-electron microscopy-based structure of infectious brain-derived fibrils of the mouse anchorless RML scrapie strain which, like the recently determined hamster 263K strain, has a parallel in-register ß-sheet-based core. Several structural motifs are shared between these ex vivo prion strains, including an amino-proximal steric zipper and three ß-arches. However, detailed comparisons reveal variations in these shared structural topologies and other features. Unlike 263K and wildtype RML prions, the anchorless RML prions lack glycophosphatidylinositol anchors and are severely deficient in N-linked glycans. Nonetheless, the similarity of our anchorless RML structure to one reported for wildtype RML prion fibrils in an accompanying paper indicates that these post-translational modifications do not substantially alter the amyloid core conformation. This work demonstrates both common and divergent structural features of prion strains at the near-atomic level.


Assuntos
Príons , Scrapie , Amiloide , Animais , Encéfalo/metabolismo , Microscopia Crioeletrônica , Camundongos , Príons/metabolismo , Ovinos
3.
Mol Cell ; 81(21): 4540-4551.e6, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34433091

RESUMO

Within the extensive range of self-propagating pathologic protein aggregates of mammals, prions are the most clearly infectious (e.g., ∼109 lethal doses per milligram). The structures of such lethal assemblies of PrP molecules have been poorly understood. Here we report a near-atomic core structure of a brain-derived, fully infectious prion (263K strain). Cryo-electron microscopy showed amyloid fibrils assembled with parallel in-register intermolecular ß sheets. Each monomer provides one rung of the ordered fibril core, with N-linked glycans and glycolipid anchors projecting outward. Thus, single monomers form the templating surface for incoming monomers at fibril ends, where prion growth occurs. Comparison to another prion strain (aRML) revealed major differences in fibril morphology but, like 263K, an asymmetric fibril cross-section without paired protofilaments. These findings provide structural insights into prion propagation, strains, species barriers, and membrane pathogenesis. This structure also helps frame considerations of factors influencing the relative transmissibility of other pathologic amyloids.


Assuntos
Encéfalo/metabolismo , Microscopia Crioeletrônica/métodos , Polissacarídeos/química , Príons/química , Príons/ultraestrutura , Amiloide/química , Animais , Glicolipídeos/química , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Camundongos , Fenótipo , Proteínas Priônicas/química , Ligação Proteica , Estrutura Secundária de Proteína , Termodinâmica
4.
Ann Clin Transl Neurol ; 7(6): 932-944, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32538552

RESUMO

OBJECTIVE: The detection of prion seeding activity in CSF and olfactory mucosal brushings using real-time quaking-induced conversion assays allows highly accurate clinical diagnosis of sporadic Creutzfeldt-Jakob disease. To gauge transmission risks associated with these biospecimens and their testing, we have bioassayed prion infectivity levels in patients' brain tissue, nasal brushings, and CSF, and assessed the pathogenicity of amplified products of real-time quaking-induced conversion assays seeded with Creutzfeldt-Jakob disease prions. METHODS: We obtained olfactory mucosal brushings and CSF from patients with a final diagnosis of sporadic Creutzfeldt-Jakob disease subtype MM1 (n = 3). Samples were inoculated intracerebrally into Tg66 transgenic mice that overexpress the homologous human 129M prion protein. The mice were evaluated for clinical, neuropathological, and biochemical evidence of prion infection. RESULTS: Patients' brain tissue at 102 to 105 fold dilutions affected 47/48 Tg66 mice. In contrast, maximum acutely tolerable doses of insoluble pellets from their olfactory mucosa brushings caused evidence of prion disease in only 4/28 inoculated mice, and no effects were seen with 10-fold dilutions. No clinical prion disease was observed in mice inoculated with antemortem CSF samples or prion-seeded real-time quaking-induced conversion assay products. INTERPRETATION: Pellets from patients' olfactory mucosa brushings had ≥10,000-fold lower infectivity per unit volume than brain tissue, while CSF lacked detectable infectivity. Nonetheless, the results suggest that appropriate precautions may be warranted in surgical interventions involving the olfactory areas. The lack of pathogenic infectivity in the real-time quaking-induced conversion assay products provides evidence that the assay does not replicate biohazardous prions in vitro.


Assuntos
Química Encefálica , Encéfalo , Síndrome de Creutzfeldt-Jakob/líquido cefalorraquidiano , Síndrome de Creutzfeldt-Jakob/transmissão , Mucosa Olfatória/química , Proteínas Priônicas/análise , Proteínas Priônicas/líquido cefalorraquidiano , Animais , Autopsia , Humanos , Camundongos , Camundongos Transgênicos , Punção Espinal
5.
Mol Cell Proteomics ; 18(12): 2388-2400, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31558565

RESUMO

Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a multi-species targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified relative to an isotopically labeled recombinant protein standard for human samples, or isotopically labeled synthetic peptides for nonhuman species. Analytical validation experiments showed process replicate coefficients of variation below 15%, good dilution linearity and recovery, and suitable performance for both CSF and brain homogenate and across humans as well as preclinical species of interest. In n = 55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared with individuals with nonprion diagnoses. Thus, lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not an artifact of ELISA-based measurement. As a result, dose-finding studies for PrP lowering drugs may need to be conducted in presymptomatic at-risk individuals rather than in symptomatic patients. We provide a targeted mass spectrometry-based method suitable for preclinical quantification of CSF PrP as a tool for drug development.


Assuntos
Espectrometria de Massas/métodos , Proteínas Priônicas/líquido cefalorraquidiano , Animais , Desenvolvimento de Medicamentos , Ensaio de Imunoadsorção Enzimática , Humanos , Macaca fascicularis , Camundongos , Doenças Priônicas/líquido cefalorraquidiano , Doenças Priônicas/tratamento farmacológico , Ratos
6.
JCI Insight ; 52019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31361599

RESUMO

Prion disease is a fatal, incurable neurodegenerative disease of humans and other mammals caused by conversion of cellular prion protein (PrP; PrPC) into a self-propagating neurotoxic conformer (prions; PrPSc). Strong genetic proofs of concept support lowering PrP expression as a therapeutic strategy. Antisense oligonucleotides (ASOs) can provide a practical route to lowering one target mRNA in the brain, but their development for prion disease has been hindered by three unresolved questions from prior work: uncertainty about mechanism of action, unclear potential for efficacy against established prion infection, and poor tolerability of drug delivery by osmotic pumps. Here we test antisense oligonucleotides (ASOs) delivered by bolus intracerebroventricular injection to intracerebrally prion-infected wild-type mice. Prophylactic treatments given every 2-3 months extended survival times 61-98%, and a single injection at 120 days post-infection, near the onset of clinical signs, extended survival 55% (87 days). In contrast, a non-targeting control ASO was ineffective. Thus, PrP lowering is the mechanism of action of ASOs effective against prion disease in vivo, and infrequent, or even single, bolus injections of ASOs can slow prion neuropathogenesis and markedly extend survival, even when initiated near clinical signs. These findings should empower development of PrP-lowering therapy for prion disease.


Assuntos
Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Doenças Priônicas/tratamento farmacológico , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Descoberta de Drogas , Feminino , Terapia Genética , Camundongos , Camundongos Endogâmicos C57BL , Doenças Priônicas/patologia , Taxa de Sobrevida
7.
Nat Commun ; 10(1): 640, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718499

RESUMO

The original version of this Article contained errors in the author affiliations. Affiliation 2 incorrectly read 'Department of Neurology, The First Hospital of Jilin University, Changchun 130021 Jilin Province, China.'Affiliation 5 incorrectly read 'Department of Otolaryngology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061 Shanxi Province, China'Affiliation 9 incorrectly read 'State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.'This has now been corrected in both the PDF and HTML versions of the Article.

8.
Nat Commun ; 10(1): 247, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651538

RESUMO

A definitive pre-mortem diagnosis of prion disease depends on brain biopsy for prion detection currently and no validated alternative preclinical diagnostic tests have been reported to date. To determine the feasibility of using skin for preclinical diagnosis, here we report ultrasensitive serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays of skin samples from hamsters and humanized transgenic mice (Tg40h) at different time points after intracerebral inoculation with 263K and sCJDMM1 prions, respectively. sPMCA detects skin PrPSc as early as 2 weeks post inoculation (wpi) in hamsters and 4 wpi in Tg40h mice; RT-QuIC assay reveals earliest skin prion-seeding activity at 3 wpi in hamsters and 20 wpi in Tg40h mice. Unlike 263K-inoculated animals, mock-inoculated animals show detectable skin/brain PrPSc only after long cohabitation periods with scrapie-infected animals. Our study provides the proof-of-concept evidence that skin prions could be a biomarker for preclinical diagnosis of prion disease.


Assuntos
Bioensaio/métodos , Proteínas PrPSc/análise , Scrapie/diagnóstico , Pele/patologia , Animais , Anticorpos Monoclonais/imunologia , Biomarcadores/análise , Encéfalo/patologia , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Humanos , Mesocricetus , Camundongos , Camundongos Transgênicos , Proteínas PrPSc/imunologia , Proteínas PrPSc/patogenicidade , Scrapie/patologia
9.
PLoS Pathog ; 13(9): e1006623, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28910420

RESUMO

Mammalian prion structures and replication mechanisms are poorly understood. Most synthetic recombinant prion protein (rPrP) amyloids prepared without cofactors are non-infectious or much less infectious than bona fide tissue-derived PrPSc. This effect has been associated with differences in folding of the aggregates, manifested in part by reduced solvent exclusion and protease-resistance in rPrP amyloids, especially within residues ~90-160. Substitution of 4 lysines within residues 101-110 of rPrP (central lysine cluster) with alanines (K4A) or asparagines (K4N) allows formation of aggregates with extended proteinase K (PK) resistant cores reminiscent of PrPSc, particularly when seeded with PrPSc. Here we have compared the infectivity of rPrP aggregates made with K4N, K4A or wild-type (WT) rPrP, after seeding with scrapie brain homogenate (ScBH) or normal brain homogenate (NBH). None of these preparations caused clinical disease on first passage into rodents. However, the ScBH-seeded fibrils (only) led to a subclinical pathogenesis as indicated by increases in prion seeding activity, neuropathology, and abnormal PrP in the brain. Seeding activities usually accumulated to much higher levels in animals inoculated with ScBH-seeded fibrils made with the K4N, rather than WT, rPrP molecules. Brain homogenates from subclinical animals induced clinical disease on second passage into "hamsterized" Tg7 mice, with shorter incubation times in animals inoculated with ScBH-seeded K4N rPrP fibrils. On second passage from animals inoculated with ScBH-seeded WT fibrils, we detected an additional PK resistant PrP fragment that was similar to that of bona fide PrPSc. Together these data indicate that both the central lysine cluster and scrapie seeding of rPrP aggregates influence the induction of PrP misfolding, neuropathology and clinical manifestations upon passage in vivo. We confirm that some rPrP aggregates can initiate further aggregation without typical pathogenesis in vivo. We also provide evidence that there is little, if any, biohazard associated with routine RT-QuIC assays.


Assuntos
Encéfalo/metabolismo , Lisina/metabolismo , Proteínas Priônicas/metabolismo , Scrapie/metabolismo , Amiloide/química , Animais , Encéfalo/patologia , Endopeptidase K/metabolismo , Camundongos Transgênicos , Proteínas PrPSc/metabolismo , Agregados Proteicos/fisiologia , Proteínas Recombinantes/metabolismo
10.
Methods Mol Biol ; 1658: 51-66, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28861782

RESUMO

Western immunoblotting is a workhorse technique used in the prion field to analyze disease-associated forms of the prion protein, termed PrPSc. The biochemical stability of PrPSc aggregates combined with the increased resistance of prion infectivity to inactivation by various treatments that inactivate most other pathogens complicates the use of Western immunoblotting as a means to characterize PrPSc samples. In this chapter, we describe a method for Western immunoblot analysis of PrPSc with an emphasis on precautions to address the biochemical and biosafety considerations associated with this procedure.


Assuntos
Amiloide/química , Western Blotting/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas PrPSc/isolamento & purificação , Agregados Proteicos , Animais , Western Blotting/instrumentação , Expressão Gênica , Humanos , Luminescência , Medições Luminescentes/métodos , Proteínas PrPSc/química , Proteínas PrPSc/imunologia , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Hidróxido de Sódio/química
11.
Methods Mol Biol ; 1658: 185-203, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28861791

RESUMO

In coping with prion diseases, it is important to have tests that are practical enough for routine applications in medicine, agriculture, wildlife biology, and research, yet sensitive enough to detect minimal amounts of infectivity. Real-time quaking-induced conversion (RT-QuIC) assays have evolved to the point where they fulfill these criteria in applications to various human and animal prion diseases. For example, RT-QuIC assays of cerebrospinal fluid and nasal brushings allow for highly sensitive (77-97%) and specific (99-100%) identification of human sCJD patients. Recent improvements have markedly enhanced sensitivity and reduced the assay time required for many samples to a matter of hours rather than days. By combining analyses of cerebrospinal fluid and nasal brushings, diagnostic sensitivities and specificities of nearly 100% can be achieved. RT-QuIC assays are based on prion-seeded amyloid fibril formation by recombinant prion protein (rPrPSen) in multiwell plates using a Thioflavin T fluorescence readout. Here we describe our current RT-QuIC methodologies as well as technical considerations in executing, troubleshooting, and adapting the assay to new strains of prions and sample types.


Assuntos
Amiloide/análise , Bioensaio , Proteínas PrPC/química , Proteínas PrPSc/química , Doenças Priônicas/diagnóstico , Amiloide/biossíntese , Amiloide/química , Animais , Benzotiazóis , Encéfalo/metabolismo , Encéfalo/patologia , Química Encefálica , Corantes Fluorescentes/química , Expressão Gênica , Humanos , Cavidade Nasal/química , Proteínas PrPC/líquido cefalorraquidiano , Proteínas PrPC/genética , Proteínas PrPSc/líquido cefalorraquidiano , Proteínas PrPSc/genética , Doenças Priônicas/líquido cefalorraquidiano , Doenças Priônicas/genética , Doenças Priônicas/patologia , Conformação Proteica em Folha beta , Dobramento de Proteína , Proteínas Recombinantes/líquido cefalorraquidiano , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sensibilidade e Especificidade , Tiazóis/química
12.
J Virol ; 91(21)2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28835493

RESUMO

Accumulation of fibrillar protein aggregates is a hallmark of many diseases. While numerous proteins form fibrils by prion-like seeded polymerization in vitro, only some are transmissible and pathogenic in vivo To probe the structural features that confer transmissibility to prion protein (PrP) fibrils, we have analyzed synthetic PrP amyloids with or without the human prion disease-associated P102L mutation. The formation of infectious prions from PrP molecules in vitro has required cofactors and/or unphysiological denaturing conditions. Here, we demonstrate that, under physiologically compatible conditions without cofactors, the P102L mutation in recombinant hamster PrP promoted prion formation when seeded by minute amounts of scrapie prions in vitro Surprisingly, combination of the P102L mutation with charge-neutralizing substitutions of four nearby lysines promoted spontaneous prion formation. When inoculated into hamsters, both of these types of synthetic prions initiated substantial accumulation of prion seeding activity and protease-resistant PrP without transmissible spongiform encephalopathy (TSE) clinical signs or notable glial activation. Our evidence suggests that PrP's centrally located proline and lysine residues act as conformational switches in the in vitro formation of transmissible PrP amyloids.IMPORTANCE Many diseases involve the damaging accumulation of specific misfolded proteins in thread-like aggregates. These threads (fibrils) are capable of growing on the ends by seeding the refolding and incorporation of the normal form of the given protein. In many cases such aggregates can be infectious and propagate like prions when transmitted from one individual host to another. Some transmitted aggregates can cause fatal disease, as with human iatrogenic prion diseases, while other aggregates appear to be relatively innocuous. The factors that distinguish infectious and pathogenic protein aggregates from more innocuous ones are poorly understood. Here we have compared the combined effects of prion seeding and mutations of prion protein (PrP) on the structure and transmission properties of synthetic PrP aggregates. Our results highlight the influence of specific sequence features in the normally unstructured region of PrP that influence the infectious and neuropathogenic properties of PrP-derived aggregates.


Assuntos
Encéfalo/metabolismo , Lisina/genética , Mutação , Doenças Priônicas/transmissão , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Amiloide/química , Amiloide/metabolismo , Animais , Cricetinae , Técnicas In Vitro , Lisina/metabolismo , Doenças Priônicas/metabolismo , Prolina/genética , Prolina/metabolismo
13.
Prog Mol Biol Transl Sci ; 150: 375-388, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28838670

RESUMO

Among the most sensitive, specific and practical of methods for detecting prions are the real-time quaking-induced conversion (RT-QuIC) assays. These assays exploit the fundamental self-propagating activity of prions to amplify the presence of prion seeds by as much as a trillion-fold. The reactions can detect most of the known mammalian prion diseases, often with sensitivities greater than those of animal bioassays. RT-QuIC assays are performed in multiwell plates with fluorescence detection and have now reached the sensitivity and practicality required for routine prion disease diagnostics. Some key strains of prions within particular host species, e.g., humans, cattle, and sheep, can be discriminated by comparison of RT-QuIC responses with different recombinant prion protein substrates. The most thoroughly validated diagnostic application of RT-QuIC is in the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) using cerebrospinal fluid. Diagnostic sensitivities as high as 96% can be achieved in less than 24h with specificities of 98%-100%. The ability, if needed, to also test nasal swab samples can increase the RT-QuIC sensitivity for sCJD to virtually 100%. In addition to diagnostic applications, RT-QuIC has also been used in the testing of prion disinfectants and potential therapeutics. Mechanistically related assays are also now being developed for other protein misfolding diseases.


Assuntos
Amiloide/metabolismo , Bioensaio/métodos , Desinfetantes/uso terapêutico , Doenças Priônicas/diagnóstico , Príons/metabolismo , Animais , Humanos , Deficiências na Proteostase/diagnóstico
14.
J Virol ; 90(10): 4905-4913, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-26937029

RESUMO

UNLABELLED: Understanding the structure of PrP(Sc) and its strain variation has been one of the major challenges in prion disease biology. To study the strain-dependent conformations of PrP(Sc), we purified proteinase-resistant PrP(Sc) (PrP(RES)) from mouse brains with three different murine-adapted scrapie strains (Chandler, 22L, and Me7) and systematically tested the accessibility of epitopes of a wide range of anti-PrP and anti-PrP(Sc) specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). We found that epitopes of most anti-PrP antibodies were hidden in the folded structure of PrP(RES), even though these epitopes are revealed with guanidine denaturation. However, reactivities to a PrP(Sc)-specific conformational C-terminal antibody showed significant differences among the three different prion strains. Our results provide evidence for strain-dependent conformational variation near the C termini of molecules within PrP(Sc) multimers. IMPORTANCE: It has long been apparent that prion strains can have different conformations near the N terminus of the PrP(Sc) protease-resistant core. Here, we show that a C-terminal conformational PrP(Sc)-specific antibody reacts differently to three murine-adapted scrapie strains. These results suggest, in turn, that conformational differences in the C terminus of PrP(Sc) also contribute to the phenotypic distinction between prion strains.


Assuntos
Anticorpos/imunologia , Epitopos/imunologia , Proteínas PrPSc/química , Proteínas PrPSc/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Camundongos , Fenótipo , Proteínas PrPSc/isolamento & purificação , Conformação Proteica , Scrapie
15.
J Virol ; 86(21): 11763-78, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22915801

RESUMO

Mammalian prions are thought to consist of misfolded aggregates (protease-resistant isoform of the prion protein [PrP(res)]) of the cellular prion protein (PrP(C)). Transmissible spongiform encephalopathy (TSE) can be induced in animals inoculated with recombinant PrP (rPrP) amyloid fibrils lacking mammalian posttranslational modifications, but this induction is inefficient in hamsters or transgenic mice overexpressing glycosylphosphatidylinositol (GPI)-anchored PrP(C). Here we show that TSE can be initiated by inoculation of misfolded rPrP into mice that express wild-type (wt) levels of PrP(C) and that synthetic prion strain propagation and selection can be affected by GPI anchoring of the host's PrP(C). To create prions de novo, we fibrillized mouse rPrP in the absence of molecular cofactors, generating fibrils with a PrP(res)-like protease-resistant banding profile. These fibrils induced the formation of PrP(res) deposits in transgenic mice coexpressing wt and GPI-anchorless PrP(C) (wt/GPI(-)) at a combined level comparable to that of PrP(C) expression in wt mice. Secondary passage into mice expressing wt, GPI(-), or wt plus GPI(-) PrP(C) induced TSE disease with novel clinical, histopathological, and biochemical phenotypes. Contrary to laboratory-adapted mouse scrapie strains, the synthetic prion agents exhibited a preference for conversion of GPI(-) PrP(C) and, in one case, caused disease only in GPI(-) mice. Our data show that novel TSE agents can be generated de novo solely from purified mouse rPrP after amplification in mice coexpressing normal levels of wt and anchorless PrP(C). These observations provide insight into the minimal elements required to create prions in vitro and suggest that the PrP(C) GPI anchor can modulate the propagation of synthetic TSE strains.


Assuntos
Príons/genética , Príons/isolamento & purificação , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Príons/patogenicidade , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
16.
J Clin Microbiol ; 50(4): 1464-6, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22238438

RESUMO

To assess prospects for early diagnosis of prion disease based on prion seeding activity in cerebrospinal fluid (CSF), we measured the activity over time in scrapie-infected hamsters by real-time quaking-induced conversion (RT-QuIC). After intracerebral inoculation, activity appeared in CSF within 1 day and plateaued weeks before the onset of clinical signs. However, after intratongue inoculation, activity first appeared in CSF with the onset of clinical signs, well after higher-level accumulation of seeding activity in brain.


Assuntos
Amiloide/líquido cefalorraquidiano , Príons/líquido cefalorraquidiano , Scrapie/líquido cefalorraquidiano , Amiloide/química , Animais , Encéfalo/patologia , Cricetinae , Cinética , Limite de Detecção , Príons/química , Scrapie/diagnóstico , Língua/patologia
17.
Virology ; 423(2): 205-13, 2012 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-22222213

RESUMO

Roles of complement factors in prion infection of the central nervous system remain unclear. In this study, we assessed the strain-dependent reactivity of complement factors in prion infections of Neuro2a (N2a) cells and mouse brains. N2a cells persistently infected with either Chandler or 22L scrapie strains were cultured in the presence of normal mouse serum (NMS), followed by staining with phosphatidylserine binding protein and early apoptosis marker Annexin V. The proportion of Annexin V positive cells was increased both in Chandler- and 22L-infected cells. Preincubation of NMS with anti-C1q, C3 and/or C9 antibodies reduced Annexin V positive cells in Chandler-infected cells, while only anti-C3 antibodies were effective on 22L-infected cells. The immunohistochemistry showed that deposition of C1q and C3 was different between Chandler- and 22L-infected mouse brains. These results indicate that the reactivity of complement factors differs between prion strains both in vitro and in vivo.


Assuntos
Proteínas do Sistema Complemento/imunologia , Proteínas PrPSc/imunologia , Scrapie/imunologia , Animais , Anticorpos/imunologia , Encéfalo/imunologia , Encéfalo/patologia , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Scrapie/patologia
18.
Biochemistry ; 50(21): 4479-90, 2011 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-21539311

RESUMO

Mammalian prion diseases involve conversion of normal prion protein, PrP(C), to a pathological aggregated state (PrP(res)). The three-dimensional structure of PrP(res) is not known, but infrared (IR) spectroscopy has indicated high, strain-dependent ß-sheet content. PrP(res) molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifications by comparing wild-type PrP(res) with GPI- and glycan-deficient PrP(res) produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrP(res) revealed conformational differences between the 22L, ME7, and Chandler (RML) murine scrapie strains, most notably in bands attributed to ß-sheets. These PrP(res) spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild-type and anchorless 22L PrP(res) were nearly indistinguishable. With ME7 PrP(res), modest differences between the wild-type and anchorless spectra were detected, notably an ∼2 cm(-1) shift in an apparent ß-sheet band. Collectively, the data provide evidence that the glycans and anchor do not grossly affect the strain-specific secondary structures of PrP(res), at least relative to the differences observed between strains, but can subtly affect turns and certain ß-sheet components. Recently reported H-D exchange analyses of anchorless PrP(res) preparations strongly suggested the presence of strain-dependent, solvent-inaccessible ß-core structures throughout most of the C-terminal half of PrP(res) molecules, with no remaining α-helix. Our IR data provide evidence that similar core structures also comprise wild-type PrP(res).


Assuntos
Glicosilfosfatidilinositóis/química , Polissacarídeos/química , Proteínas PrPSc/química , Animais , Eletroforese em Gel de Poliacrilamida , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Proteínas PrPSc/isolamento & purificação , Conformação Proteica , Espectrofotometria Infravermelho
19.
Nat Struct Mol Biol ; 18(4): 504-6, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21441913

RESUMO

One of the mysteries in prion research is the structure of the infectious form of mammalian prion protein PrP(Sc). Here we used mass spectrometry analysis of hydrogen-deuterium exchange to examine brain-derived PrP(Sc). Our data indicate that, contrary to popular models, prion-protein conversion involves refolding of the entire region from residue ~80-90 to the C-terminus, which in PrP(Sc) consists of ß-strands and relatively short turns and/or loops, with no native α-helices present.


Assuntos
Química Encefálica , Príons/química , Animais , Deutério , Hidrogênio , Espectrometria de Massas
20.
J Biol Chem ; 285(19): 14083-7, 2010 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-20304915

RESUMO

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases that are associated with the conformational conversion of a normal prion protein, PrP(C), to a misfolded aggregated form, PrP(Sc). The protein-only hypothesis asserts that PrP(Sc) itself represents the infectious TSE agent. Although this model is supported by rapidly growing experimental data, unequivocal proof has been elusive. The protein misfolding cyclic amplification reactions have been recently shown to propagate prions using brain-derived or recombinant prion protein, but only in the presence of additional cofactors such as nucleic acids and lipids. Here, using a protein misfolding cyclic amplification variation, we show that prions causing transmissible spongiform encephalopathy in wild-type hamsters can be generated solely from highly purified, bacterially expressed recombinant hamster prion protein without any mammalian or synthetic cofactors (other than buffer salts and detergent). These findings provide strong support for the protein-only hypothesis of TSE diseases, as well as argue that cofactors such as nucleic acids, other polyanions, or lipids are non-obligatory for prion protein conversion to the infectious form.


Assuntos
Lipídeos , Ácidos Nucleicos , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Cricetinae , Feminino , Mesocricetus , Proteínas PrPSc/isolamento & purificação , Doenças Priônicas/transmissão , Doenças Priônicas/veterinária , Dobramento de Proteína , Proteínas Recombinantes/isolamento & purificação
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