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1.
Arch Virol ; 165(3): 535-556, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32025859

RESUMO

The existence of more than 30 strains of transmissible spongiform encephalopathy (TSE) and the paucity of infectivity of purified PrPSc, as well as considerations of PrP structure, are inconsistent with the protein-only (prion) theory of TSE. Nucleic acid is a strong contender as a second component. We juxtapose two key findings: (i) PrP is a nucleic-acid-binding antimicrobial protein that is similar to retroviral Gag proteins in its ability to trigger reverse transcription. (ii) Retroelement mobilization is widely seen in TSE disease. Given further evidence that PrP also mediates nucleic acid transport into and out of the cell, a strong case is to be made that a second element - retroelement nucleic acid - bound to PrP constitutes the second component necessary to explain the multiple strains of TSE.


Assuntos
Doenças Priônicas/patologia , Proteínas Priônicas/genética , Príons/genética , Animais , Humanos , Ácidos Nucleicos , Príons/fisiologia , Ligação Proteica , Retroelementos
2.
Nat Commun ; 10(1): 4162, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519910

RESUMO

Insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, aggregates of TDP-43 occur in nearly all cases of amyotrophic lateral sclerosis (ALS). However, whether aggregates cause cellular toxicity is still not clear, even in simpler cellular systems. We reasoned that deep mutagenesis might be a powerful approach to disentangle the relationship between aggregation and toxicity. We generated >50,000 mutations in the prion-like domain (PRD) of TDP-43 and quantified their toxicity in yeast cells. Surprisingly, mutations that increase hydrophobicity and aggregation strongly decrease toxicity. In contrast, toxic variants promote the formation of dynamic liquid-like condensates. Mutations have their strongest effects in a hotspot that genetic interactions reveal to be structured in vivo, illustrating how mutagenesis can probe the in vivo structures of unstructured proteins. Our results show that aggregation of TDP-43 is not harmful but protects cells, most likely by titrating the protein away from a toxic liquid-like phase.


Assuntos
Biologia Computacional/métodos , Genômica/métodos , Biologia de Sistemas/métodos , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mutação/genética , Príons/genética , Príons/metabolismo
3.
Clin Interv Aging ; 14: 1387-1397, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31447551

RESUMO

Background: A novel prion variant, PRNP p.Tyr225Cys (c.674A>G; p.Y225C), was identified in an atypical Creutzfeldt-Jakob disease (CJD) patient. The patient had a 5-year history of progressive cognitive impairment with speech and gait disturbances. From the basic neurological examination at his first hospital visit, rigidity and myoclonic jerks in all limbs were observed without focal weakness. Electroencephalogram showed the diffuse slow continuous delta activity in the bilateral cerebral hemisphere. Magnetic resonance imaging revealed abnormalities in the brain, such as cortical signal changes and edema in the frontotemporoparietal lobes and the basal ganglia. Cerebrospinal fluid 14-3-3 protein analysis showed a weakly positive signal. Family history remained unclear, but the patient's mother and sister were diagnosed with cognitive impairment but both refused genetic testing. Methods: Targeted next generation sequencing (NGS) was performed on 50 genes, involved in different neurodegeneratives diseases, such as Alzheimer's, Parkinson's, frontotemporal dementia or prion diseases. In silico analyses and structure predictions were performed on the potential patohgenic mutations. Results: NGS and standard sequencing revealed the novel PRNP p.Tyr225Cys mutation in the patient. Structure predictions revealed that this may make the helix more flexible. In addition, the extra cysteine residue in TM-III of prion protein may result in disturbances of natural disulfide bond. Conclusion: Hence, the pathogenicity of PRNP p.Tyr225Cys was not fully confirmed at present, and its penetrance was suggested to be low. However, its possible pathogenic nature in prion diseases cannot be ignored, since Tyr/Cys exchange could disturb the helix dynamics and contribute to conformational alteration and disease progression.


Assuntos
Síndrome de Creutzfeldt-Jakob/genética , Príons/genética , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Progressão da Doença , Eletroencefalografia , Humanos , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Priônicas , República da Coreia
4.
Nucleic Acids Res ; 47(16): 8807-8820, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31299085

RESUMO

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3'-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Regulação Fúngica da Expressão Gênica , Terminação Traducional da Cadeia Peptídica , Príons/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Arginina/metabolismo , Sítios de Ligação , Clonagem Molecular , Códon/química , Códon/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Lisina/metabolismo , Modelos Moleculares , Príons/química , Príons/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
5.
Acta Vet Hung ; 67(2): 174-182, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31238729

RESUMO

Prion diseases are fatal neurodegenerative diseases characterised by the accumulation of an abnormal prion protein isoform (PrPSc), which is converted from the normal prion protein (PrPC). Prion diseases have been reported in an extensive number of species but not in horses up to now; therefore, horses are known to be a species resistant to prion diseases. The prion-like protein gene (PRND) is closely located downstream of the prion protein gene (PRNP) and the prion-like protein (Doppel) is a homologue with PrP. Previous studies have shown that an association between prion diseases and polymorphisms of the PRND gene is reported in the main hosts of prion diseases. Hence, we examined the genetic variations of the PRND gene in Thoroughbred horses. Interestingly, polymorphisms of the PRND gene were not detected. In addition, we conducted a comparative analysis of the amino acid sequences of the PRND gene to identify the differences between horses and other species. The amino acid sequence of the horse PRND gene showed the highest identity to that of sheep (83.7%), followed by that of goats, cattle and humans. To the best of our knowledge, this is the first genetic study of the PRND gene in horses.


Assuntos
Cavalos/genética , Fases de Leitura Aberta/genética , Polimorfismo Genético , Príons/genética , Sequência de Aminoácidos , Animais , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Cavalos/metabolismo , Príons/química , Príons/metabolismo , Alinhamento de Sequência
6.
J Vet Diagn Invest ; 31(4): 629-633, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31238800

RESUMO

Scrapie resistance or susceptibility in sheep is associated with single nucleotide polymorphisms (SNPs) at codons 136, 154, and 171 of the prion protein gene (PRNP). In addition, phenylalanine mutation at codon 141 has been recognized as a risk factor for atypical scrapie. In contrast, K222, D146, and S146 alleles confer genetic resistance to classical scrapie in goats. High-throughput genotyping technologies would provide significant benefits in scrapie eradication plans. The ability to resolve oligonucleotides varying in mass by less than a single nucleotide makes MALDI-TOF mass spectrometry (MS) a suitable platform for PRNP genotyping. We evaluated the commercial Myriapod scrapie kit (Diatech Pharmacogenetics), associated with a highly automated processing platform incorporating MALDI-TOF MS technology, to detect SNPs at codons 136, 154, 171, 141, and 222 of small ruminant PRNP. The Myriapod scrapie kit was accredited according to UNI CEI EN ISO/IEC 17025. We present the genotyping results of 10,960 sheep in Sicily and 1,822 goats in Sicily and Calabria (southern Italy) tested during 2017. We found a high frequency (43.9%) of the protective ARR allele in sheep and a promising 12.3% of the resistant K222 variant in goats. This efficient and high-throughput method is suitable for extensive PRNP genotyping, as requested in the European scrapie eradication plan.


Assuntos
Genótipo , Doenças das Cabras/genética , Proteínas Priônicas/genética , Scrapie/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Alelos , Animais , Predisposição Genética para Doença , Cabras , Mutação , Príons/genética , Ovinos , Sicília
7.
Mol Cell Biol ; 39(18)2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31208977

RESUMO

Membrane type 1-matrix metalloproteinase (MT1-MMP) and tumor necrosis factor α (TNF-α)-converting enzyme (TACE) are prominent membrane-anchored metalloproteinases that regulate the turnover of extracellular matrix (ECM) components and bioactive molecules required for cancer proliferation. In this study, we describe a novel approach that would allow tissue inhibitor of metalloproteinase 1 (TIMP-1), the endogenous inhibitor of the matrix metalloproteinases (MMPs), to be translocated to the cell membrane for simultaneous MT1-MMP/TACE inhibition. We achieve this by fusing T1TACE, a designer TIMP-1 with superb affinities for MT1-MMP and TACE, to the glycosyl-phosphatidyl inositol anchor of prions to create a membrane-tethered, broad-spectrum inhibitor, named T1Pr αTACE, that colocalizes with MT1-MMP and TACE on the cell surface. Transduction of T1Pr αTACE in human fibrosarcoma cells results not only in a substantial reduction in gelatinolytic and TNF-α/heparin binding epithelial growth factor shedding activities but also in a loss of tubulogenic capability in three-dimensional matrices. In renal carcinoma, T1Pr αTACE triggers cellular senescence and disrupts MMP-mediated proteolysis of ECM components such as fibronectin and collagen I, leading to an impairment in cell motility and survival under both in vitro and in vivo conditions. Taken together, our findings may provide a new perspective in TIMP targeting that could be exploited to halt metastatic renal carcinoma progression.


Assuntos
Carcinoma de Células Renais/terapia , Glicosilfosfatidilinositóis/metabolismo , Neoplasias Renais/terapia , Príons/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Inibidor Tecidual de Metaloproteinase-1/genética , Células A549 , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Renais/metabolismo , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Transporte Proteico , Proteínas Recombinantes de Fusão/farmacologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Transdução Genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Int J Mol Sci ; 20(11)2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31146333

RESUMO

The yeast [PSI+] prion, formed by the Sup35 (eRF3) protein, has multiple structural variants differing in the strength of nonsense suppressor phenotype. Structure of [PSI+] and its variation are characterized poorly. Here, we mapped Sup35 amyloid cores of 26 [PSI+] ex vivo prions of different origin using proteinase K digestion and mass spectrometric identification of resistant peptides. In all [PSI+] variants the Sup35 amino acid residues 2-32 were fully resistant and the region up to residue 72 was partially resistant. Proteinase K-resistant structures were also found within regions 73-124, 125-153, and 154-221, but their presence differed between [PSI+] isolates. Two distinct digestion patterns were observed for region 2-72, which always correlated with the "strong" and "weak" [PSI+] nonsense suppressor phenotypes. Also, all [PSI+] with a weak pattern were eliminated by multicopy HSP104 gene and were not toxic when combined with multicopy SUP35. [PSI+] with a strong pattern showed opposite properties, being resistant to multicopy HSP104 and lethal with multicopy SUP35. Thus, Sup35 prion cores can be composed of up to four elements. [PSI+] variants can be divided into two classes reliably distinguishable basing on structure of the first element and the described assays.


Assuntos
Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Endopeptidase K/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Príons/química , Príons/genética , Domínios Proteicos , Multimerização Proteica , Proteólise , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
9.
PLoS One ; 14(5): e0216621, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31071138

RESUMO

Longitudinal studies of chronic wasting disease (CWD) in the native host have provided considerable understanding of how this prion disease continues to efficiently spread among cervid species. These studies entail great cost in animal, time and financial support. A variety of methods have emerged including transgenic mouse bioassay, western blot, enzyme-linked immunoassay (ELISA), immunohistochemistry (IHC), serial protein misfolding cyclic amplification (sPMCA) and real time quaking-induced conversion (RT-QuIC), that deepen our understanding of this and other protein misfolding disorders. To further characterize an inoculum source used for ongoing CWD studies and to determine how the readouts from each of these assays compare, we assayed a CWD-positive brain pool homogenate (CBP6) and a mouse dilutional bioassay of this homogenate using the above detection methods. We demonstrate that: (i) amplification assays enhanced detection of amyloid seeding activity in the CWD+ cervid brain pool to levels beyond mouse LD50, (ii) conventional detection methods (IHC and western blot) performed well in identifying the presence of PrPSc in terminal brain tissue yet lack sufficient detection sensitivity to identify all CWD-infected mice, and (iii) the incorporation of amplification assays enhanced detection of CWD-infected mice near the LD50. This cross-platform analysis provides a basis to calibrate the relative sensitivities of CWD detection assays.


Assuntos
Amiloide/análise , Bioensaio/métodos , Encéfalo/metabolismo , Cervos/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Príons/análise , Doença de Emaciação Crônica/diagnóstico , Animais , Encéfalo/patologia , Camundongos , Camundongos Transgênicos , Proteínas Priônicas/genética , Príons/genética , Doença de Emaciação Crônica/transmissão
10.
Molecules ; 24(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022909

RESUMO

Fused in sarcoma (FUS) is a DNA/RNA binding protein that is involved in RNA metabolism and DNA repair. Numerous reports have demonstrated by pathological and genetic analysis that FUS is associated with a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and polyglutamine diseases. Traditionally, the fibrillar aggregation of FUS was considered to be the cause of those diseases, especially via its prion-like domains (PrLDs), which are rich in glutamine and asparagine residues. Lately, a nonfibrillar self-assembling phenomenon, liquid-liquid phase separation (LLPS), was observed in FUS, and studies of its functions, mechanism, and mutual transformation with pathogenic amyloid have been emerging. This review summarizes recent studies on FUS self-assembling, including both aggregation and LLPS as well as their relationship with the pathology of ALS, FTLD, and other neurodegenerative diseases.


Assuntos
Doenças Neurodegenerativas/genética , Agregação Patológica de Proteínas/genética , Proteína FUS de Ligação a RNA/química , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Asparagina/química , Asparagina/genética , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/patologia , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Humanos , Doenças Neurodegenerativas/patologia , Peptídeos/química , Peptídeos/genética , Príons/química , Príons/genética , Agregação Patológica de Proteínas/patologia , Domínios Proteicos/genética , Proteína FUS de Ligação a RNA/genética
11.
Methods Mol Biol ; 1958: 237-261, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945222

RESUMO

Proteins with prion-like behavior are attracting an increasing interest, since accumulating evidences indicate that they play relevant roles both in health and disease. The self-assembly of these proteins into insoluble aggregates is associated with severe neuropathological processes such as amyotrophic lateral sclerosis (ALS). However, in normal conditions, they are known to accomplish a wide range of functional roles. The conformational duality of prion-like proteins is often encoded in specific protein regions, named prion-like domains (PrLDs). PrLDs are usually long and disordered regions of low complexity. We have shown that PrLDs might contain soft-amyloid cores that contribute significantly to trigger their aggregation, as well as to support their propagation. Further exploration of the role of these sequences in the conformational conversion of prion-like proteins might provide novel insights into the mechanism of action and regulation of these polypeptides, enabling the future development of therapeutic strategies. Here, we describe a set of methodologies aimed to identify and characterize these short amyloid stretches in a protein or proteome of interest, ranging from in silico detection to in vitro and in vivo evaluation and validation.


Assuntos
Biologia Molecular/métodos , Proteínas Priônicas/química , Príons/química , Sequência de Aminoácidos/genética , Amiloide/química , Amiloide/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Humanos , Proteínas Priônicas/genética , Príons/genética , Agregados Proteicos/genética , Domínios Proteicos/genética , Proteoma/química , Proteoma/genética
12.
Biochim Biophys Acta Proteins Proteom ; 1867(10): 922-932, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30986567

RESUMO

The dynamic nature of the tau protein under physiological conditions is likely to be critical for it to perform its diverse functions inside a cell. Under some conditions, this intrinsically disordered protein assembles into pathogenic aggregates that are self-perpetuating, toxic and infectious in nature. The role of liquid-liquid phase separation in the initiation of the aggregation reaction remains to be delineated. Depending on the nature of the aggregate, its structure, and its localization, neurodegenerative disorders with diverse clinical features are manifested. The prion-like mechanism by which these aggregates propagate and spread across the brain is not well understood. Various factors (PTMs, mutations) have been strongly associated with the pathological aggregates of tau. However, little is known about how these factors modulate the pathological properties linked to aggregation. This review describes the current progress towards understanding the mechanism of propagation of tau aggregates.


Assuntos
Proteínas Intrinsicamente Desordenadas , Mutação , Príons , Agregados Proteicos , Proteínas tau , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Príons/química , Príons/genética , Príons/metabolismo , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismo
13.
Viruses ; 11(3)2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30875755

RESUMO

Increasing evidence suggests that neurodegenerative disorders share a common pathogenic feature: the presence of deposits of misfolded proteins with altered physicochemical properties in the Central Nervous System. Despite a lack of infectivity, experimental data show that the replication and propagation of neurodegenerative disease-related proteins including amyloid-ß (Aß), tau, α-synuclein and the transactive response DNA-binding protein of 43 kDa (TDP-43) share a similar pathological mechanism with prions. These observations have led to the terminology of "prion-like" to distinguish between conditions with noninfectious characteristics but similarities with the prion replication and propagation process. Prions are considered to adapt their conformation to changes in the context of the environment of replication. This process is known as either prion selection or adaptation, where a distinct conformer present in the initial prion population with higher propensity to propagate in the new environment is able to prevail over the others during the replication process. In the last years, many studies have shown that prion-like proteins share not only the prion replication paradigm but also the specific ability to aggregate in different conformations, i.e., strains, with relevant clinical, diagnostic and therapeutic implications. This review focuses on the molecular basis of the strain phenomenon in prion and prion-like proteins.


Assuntos
Doenças Neurodegenerativas/patologia , Proteínas Priônicas/genética , Príons/genética , Príons/patogenicidade , Peptídeos beta-Amiloides/genética , Animais , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Humanos , Camundongos , Dobramento de Proteína , Proteínas tau/genética
14.
Biochim Biophys Acta Proteins Proteom ; 1867(10): 933-940, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30826454

RESUMO

Several RNA-binding proteins undergo reversible liquid-liquid phase transitions, which, in pathological conditions, might evolve into transitions to solid-state phases, giving rise to amyloid structures. Amyloidogenic and prion-like proteins, such as the tumor suppressor protein p53 and the mammalian prion protein (PrP), bind RNAs specifically or nonspecifically, resulting in changes in their propensity to undergo aggregation. Mutant p53 aggregation seems to play a crucial role in cancer through loss of function, negative dominance and gain of function. PrP conversion modulated by RNA results in highly toxic aggregates. Here, we review data on the modulatory action of RNAs on the aggregation of both proteins.


Assuntos
Amiloide , Mutação , Príons , Agregados Proteicos , RNA , Proteína Supressora de Tumor p53 , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Animais , Humanos , Príons/química , Príons/genética , Príons/metabolismo , RNA/química , RNA/genética , RNA/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Viruses ; 11(3)2019 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-30857327

RESUMO

The known amyloid-based prions of Saccharomyces cerevisiae each have multiple heritable forms, called "prion variants" or "prion strains". These variants, all based on the same prion protein sequence, differ in their biological properties and their detailed amyloid structures, although each of the few examined to date have an in-register parallel folded ß sheet architecture. Here, we review the range of biological properties of yeast prion variants, factors affecting their generation and propagation, the interaction of prion variants with each other, the mutability of prions, and their segregation during mitotic growth. After early differentiation between strong and weak stable and unstable variants, the parameters distinguishing the variants has dramatically increased, only occasionally correlating with the strong/weak paradigm. A sensitivity to inter- and intraspecies barriers, anti-prion systems, and chaperone deficiencies or excesses and other factors all have dramatic selective effects on prion variants. Recent studies of anti-prion systems, which cure prions in wild strains, have revealed an enormous array of new variants, normally eliminated as they arise and so not previously studied. This work suggests that defects in the anti-prion systems, analogous to immune deficiencies, may be at the root of some human amyloidoses.


Assuntos
Variação Genética , Chaperonas Moleculares , Príons/genética , Príons/patogenicidade , Saccharomyces cerevisiae/genética , Amiloide/química , Amiloide/genética , Mutação , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/genética
16.
Genes (Basel) ; 10(3)2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871095

RESUMO

Prions are infectious, self-perpetuating protein conformers. In mammals, pathological aggregation of the prion protein causes incurable neurodegenerative disorders, while in yeast Saccharomyces cerevisiae, prion formation may be neutral or even beneficial. According to the prevailing contemporary point of view, prion formation is considered to be a functional inactivation of the corresponding protein whose conformational state shifts from the functional monomeric one to the infectious aggregated one. The Swi1 protein forms the [SWI⁺] prion and belongs to the nucleosome remodeler complex SWI/SNF controlling the expression of a significant part of the yeast genome. In this work, we performed RNA sequencing of isogenic S. cerevisiae strains grown on the media containing galactose as the sole carbon source. These strains bore the [SWI⁺] prion or had its structural gene SWI1 deleted. The comparative analysis showed that [SWI⁺] affects genome expression significantly weaker as compared to the SWI1 deletion. Moreover, in contrast to [SWI⁺], the SWI1 deletion causes the general inhibition of translation-related genes expression and chromosome I disomy. At the same time, the [SWI⁺] prion exhibits a specific pattern of modulation of the metabolic pathways and some biological processes and functions, as well as the expression of several genes. Thus, the [SWI⁺] prion only partially corresponds to the loss-of-function of SWI1 and demonstrates several gain-of-function traits.


Assuntos
Proteínas Cromossômicas não Histona/genética , Príons/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcriptoma , Proteínas Cromossômicas não Histona/metabolismo , Deleção de Genes , Príons/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
17.
Prion ; 13(1): 65-76, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30777498

RESUMO

Genetic variability in the prion protein (Prnp) gene influences host susceptibility to many pathogenic prion diseases. Understanding the distribution of susceptible Prnp variants and determining factors influencing spatial genetic patterns are important components of many chronic wasting disease mitigation strategies. Here, we describe Prnp variability in white-tailed deer (Odocoileus virginianus) from the Mid-Atlantic region of the United States of America, an area with a recent history of infection and low disease incidence. This population is characterized by lower rates of polymorphism and significantly higher frequencies of the more susceptible 96GG genotype compared to previously surveyed populations. The prevalence of the most susceptible genotypes at disease-associated loci did vary among subregions, indicating that populations have innate differences in genotype-dictated susceptibility.


Assuntos
Cervos/genética , Polimorfismo Genético , Proteínas Priônicas/genética , Doença de Emaciação Crônica/genética , Animais , Predisposição Genética para Doença , Genótipo , Príons/genética
18.
J Biol Chem ; 294(13): 4911-4923, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30705093

RESUMO

Prions are infectious protein aggregates that cause several fatal neurodegenerative diseases. Prion research has been hindered by a lack of cellular paradigms for studying the replication of prions from different species. Although hamster prions have been widely used to study prion replication in animals and within in vitro amplification systems, they have proved challenging to propagate in cultured cells. Because the murine catecholaminergic cell line CAD5 is susceptible to a diverse range of mouse prion strains, we hypothesized that it might also be capable of propagating nonmouse prions. Here, using CRISPR/Cas9-mediated genome engineering, we demonstrate that CAD5 cells lacking endogenous mouse PrP expression (CAD5-PrP-/- cells) can be chronically infected with hamster prions following stable expression of hamster PrP. When exposed to the 263K, HY, or 139H hamster prion strains, these cells stably propagated high levels of protease-resistant PrP. Hamster prion replication required absence of mouse PrP, and hamster PrP inhibited the propagation of mouse prions. Cellular homogenates from 263K-infected cells exhibited prion seeding activity in the RT-QuIC assay and were infectious to naïve cells expressing hamster PrP. Interestingly, murine N2a neuroblastoma cells ablated for endogenous PrP expression were susceptible to mouse prions, but not hamster prions upon expression of cognate PrP, suggesting that CAD5 cells either possess cellular factors that enhance or lack factors that restrict the diversity of prion strains that can be propagated. We conclude that transfected CAD5-PrP-/- cells may be a useful tool for assessing the biology of prion strains and dissecting the mechanism of prion replication.


Assuntos
Príons/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Cricetinae , Edição de Genes , Camundongos , Príons/genética
19.
Brain ; 142(3): 760-770, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30698738

RESUMO

A common presentation of inherited prion disease is Gerstmann-Sträussler-Scheinker syndrome, typically presenting with gait ataxia and painful dysaesthesiae in the legs evolving over 2-5 years. The most frequent molecular genetic diagnosis is a P102L mutation of the prion protein gene (PRNP). There is no explanation for why this clinical syndrome is so distinct from Creutzfeldt-Jakob disease, and biomarkers of the early stages of disease have not been developed. Here we aimed, first, at determining if quantitative neurophysiological assessments could predict clinical diagnosis or disability and monitor progression and, second, to determine the neuropathological basis of the initial clinical and neurophysiological findings. We investigated subjects known to carry the P102L mutation in the longitudinal observational UK National Prion Monitoring Cohort study, with serial assessments of clinical features, peripheral nerve conduction, H and F components, threshold tracking and histamine flare and itch response and neuropathological examination in some of those who died. Twenty-three subjects were studied over a period of up to 12 years, including 65 neurophysiological assessments at the same department. Six were symptomatic throughout and six became symptomatic during the study. Neurophysiological abnormalities were restricted to the lower limbs. In symptomatic patients around the time of, or shortly after, symptom onset the H-reflex was lost. Lower limb thermal thresholds were at floor/ceiling in some at presentation, in others thresholds progressively deteriorated. Itch sensation to histamine injection was lost in most symptomatic patients. In six patients with initial assessments in the asymptomatic stage of the disease, a progressive deterioration in the ability to detect warm temperatures in the feet was observed prior to clinical diagnosis and the onset of disability. All of these six patients developed objective abnormalities of either warm or cold sensation prior to the onset of significant symptoms or clinical diagnosis. Autopsy examination in five patients (including two not followed clinically) showed prion protein in the substantia gelatinosa, spinothalamic tracts, posterior columns and nuclei and in the neuropil surrounding anterior horn cells. In conclusion, sensory symptoms and loss of reflexes in Gerstmann-Sträussler-Scheinker syndrome can be explained by neuropathological changes in the spinal cord. We conclude that the sensory symptoms and loss of lower limb reflexes in Gerstmann-Sträussler-Scheinker syndrome is due to pathology in the caudal spinal cord. Neuro-physiological measures become abnormal around the time of symptom onset, prior to diagnosis, and may be of value for improved early diagnosis and for recruitment and monitoring of progression in clinical trials.


Assuntos
Doenças Priônicas/patologia , Proteínas Priônicas/genética , Medula Espinal/patologia , Adulto , Idoso , Biomarcadores/sangue , Encéfalo/patologia , Estudos de Coortes , Síndrome de Creutzfeldt-Jakob/patologia , Feminino , Doença de Gerstmann-Straussler-Scheinker/patologia , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Mutação , Neurofisiologia , Linhagem , Proteínas Priônicas/metabolismo , Príons/genética
20.
Vet Rec ; 184(3): 97, 2019 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-30602491

RESUMO

The transmissible spongiform encephalopathy scrapie of sheep/goats and chronic wasting disease of cervids are associated with environmental reservoirs of infectivity. Preventing environmental prions acting as a source of infectivity to healthy animals is of major concern to farms that have had outbreaks of scrapie and also to the health management of wild and farmed cervids. Here, an efficient scrapie decontamination protocol was applied to a farm with high levels of environmental contamination with the scrapie agent. Post-decontamination, no prion material was detected within samples taken from the farm buildings as determined using a sensitive in vitro replication assay (sPMCA). A bioassay consisting of 25 newborn lambs of highly susceptible prion protein genotype VRQ/VRQ introduced into this decontaminated barn was carried out in addition to sampling and analysis of dust samples that were collected during the bioassay. Twenty-four of the animals examined by immunohistochemical analysis of lymphatic tissues were scrapie-positive during the bioassay, samples of dust collected within the barn were positive by month 3. The data illustrates the difficulty in decontaminating farm buildings from scrapie, and demonstrates the likely contribution of farm dust to the recontamination of these environments to levels that are capable of causing disease.


Assuntos
Descontaminação/normas , Fazendas , Príons/isolamento & purificação , Scrapie/transmissão , Animais , Animais Recém-Nascidos , Bioensaio/veterinária , Poeira , Monitoramento Ambiental , Genótipo , Príons/genética , Scrapie/epidemiologia , Ovinos , Reino Unido/epidemiologia
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