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1.
Inorg Chem ; 59(1): 900-912, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31869218

RESUMO

The combination between dyshomeostatic levels of catecholamine neurotransmitters and redox-active metals such as copper and iron exacerbates the oxidative stress condition that typically affects neurodegenerative diseases. We report a comparative study of the oxidative reactivity of copper complexes with amyloid-ß (Aß40) and the prion peptide fragment 76-114 (PrP76-114), containing the high-affinity binding site, toward dopamine and 4-methylcatechol, in aqueous buffer and in sodium dodecyl sulfate micelles, as a model membrane environment. The competitive oxidative and covalent modifications undergone by the peptides were also evaluated. The high binding affinity of Cu/peptide to micelles and lipid membranes leads to a strong reduction (Aß40) and quenching (PrP76-114) of the oxidative efficiency of the binary complexes and to a stabilization and redox silencing of the ternary complex CuII/Aß40/PrP76-114, which is highly reactive in solution. The results improve our understanding of the pathological and protective effects associated with these complexes, depending on the physiological environment.


Assuntos
Peptídeos beta-Amiloides/química , Cobre/química , Dopamina/química , Príons/química , Dodecilsulfato de Sódio/química , Sítios de Ligação , Humanos , Micelas , Conformação Molecular , Solubilidade
2.
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
3.
Life Sci Alliance ; 2(4)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31266883

RESUMO

Prions of lower eukaryotes are self-templating protein aggregates that replicate by converting homotypic proteins into stable, tightly packed beta-sheet-rich protein assemblies. Propagation is mediated by prion domains, low-complexity regions enriched in polar and devoid of charged amino acid residues. In mammals, compositionally similar domains modulate the assembly of dynamic stress granules (SGs) that associate via multivalent weak interactions. Dysregulation of SGs composed of proteins with prion-like domains has been proposed to underlie the formation of pathological inclusions in several neurodegenerative diseases. The events that drive prion-like domains into transient or solid assemblies are not well understood. We studied the interactors of the prototype prion domain NM of Saccharomyces cerevisiae Sup35 in its soluble or fibril-induced prion conformation in the mammalian cytosol. We show that the interactomes of soluble and prionized NM overlap with that of SGs. Prion induction by exogenous seeds does not cause SG assembly, demonstrating that colocalization of aberrant protein inclusions with SG components does not necessarily reveal SGs as initial sites of protein misfolding.


Assuntos
Asparagina , Grânulos Citoplasmáticos/metabolismo , Glutamina , Fatores de Terminação de Peptídeos/química , Príons/química , Proteínas de Saccharomyces cerevisiae/química , Animais , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ontologia Genética , Camundongos , Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Domínios Proteicos , Proteólise , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
PLoS Pathog ; 15(7): e1007864, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31295325

RESUMO

Prions are unusual protein assemblies that propagate their conformationally-encoded information in absence of nucleic acids. The first prion identified, the scrapie isoform (PrPSc) of the cellular prion protein (PrPC), caused epidemic and epizootic episodes [1]. Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-ß-Sheet (PIRIBS) [2] or ß-solenoid conformations [3]. Similar folding models have also been proposed for PrPSc, although none of these have been confirmed experimentally. Recent cryo-electron microscopy (cryo-EM) and X-ray fiber-diffraction studies provided evidence that PrPSc is structured as a 4-rung ß-solenoid (4RßS) [4, 5]. Here, we combined different experimental data and computational techniques to build the first physically-plausible, atomic resolution model of mouse PrPSc, based on the 4RßS architecture. The stability of this new PrPSc model, as assessed by Molecular Dynamics (MD) simulations, was found to be comparable to that of the prion forming domain of Het-s, a naturally-occurring ß-solenoid. Importantly, the 4RßS arrangement allowed the first simulation of the sequence of events underlying PrPC conversion into PrPSc. This study provides the most updated, experimentally-driven and physically-coherent model of PrPSc, together with an unprecedented reconstruction of the mechanism underlying the self-catalytic propagation of prions.


Assuntos
Proteínas PrPSc/química , Proteínas PrPSc/patogenicidade , Príons/química , Príons/patogenicidade , Animais , Microscopia Crioeletrônica , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas PrPC , Proteínas PrPSc/ultraestrutura , Príons/ultraestrutura , Conformação Proteica , Estrutura Quaternária de Proteína
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.
Nanoscale ; 11(26): 12680-12694, 2019 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-31237592

RESUMO

Amyloids have been exploited to build amazing bioactive materials. In most cases, short synthetic peptides constitute the functional components of such materials. The controlled assembly of globular proteins into active amyloid nanofibrils is still challenging, because the formation of amyloids implies a conformational conversion towards a ß-sheet-rich structure, with a concomitant loss of the native fold and the inactivation of the protein. There is, however, a remarkable exception to this rule: yeast prions. They are singular proteins able to switch between a soluble and an amyloid state. In both states, the structure of their globular domains remains essentially intact. The transit between these two conformations is encoded in prion domains (PrDs): long and disordered sequences to which the active globular domains are appended. PrDs are much larger than typical self-assembling peptides. This seriously limits their use for nanotechnological applications. We have recently shown that these domains contain soft amyloid cores (SACs) that suffice to nucleate their self-assembly reaction. Here we genetically fused a model SAC with different globular proteins. We demonstrate that this very short sequence acts as a minimalist PrD, driving the selective and slow assembly of the initially soluble fusion proteins into amyloid fibrils in which the globular proteins retain their native structure and display high activity. Overall, we provide here a novel, modular and straightforward strategy to build active protein-based nanomaterials at a preparative scale.


Assuntos
Amiloide/química , Nanofibras/química , Nanoestruturas/química , Príons/química , Fatores de Processamento de RNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Domínios Proteicos , Engenharia de Proteínas
7.
Inorg Chem ; 58(14): 8995-9003, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31247811

RESUMO

Recently, we reported on a series of aminomethylene-phosphonate (AMP) analogues, bearing one or two heterocyclic groups on the aminomethylene moiety, as promising Zn(II) chelators. Given the strong Zn(II) binding properties of these compounds, they may find useful applications in metal chelation therapy. With a goal of inhibiting the devastating oxidative damage caused by prion protein in prion diseases, we explored the most promising ligand, {bis[(1H-imidazol-4-yl)methyl]amino}methylphosphonic acid, AMP-(Im)2, 4, as an inhibitor of the oxidative reactivity associated with the Cu(II) complex of prion peptide fragment 84-114. Specifically, we first characterized the Cu(II) complex with AMP-(Im)2 by ultraviolet-visible spectroscopy and electrochemical measurements that indicated the high chemical and electrochemical stability of the complex. Potentiometric pH titration provided evidence of the formation of a stable 1:1 [Cu(II)-AMP-(Im)2]+ complex (ML), with successive binding of a second AMP-(Im)2 molecule yielding ML2 complex [Cu(II)-(AMP-(Im)2)2]+ (log K' = 15.55), and log ß' = 19.84 for ML2 complex. The CuN3O1 ML complex was demonstrated by X-ray crystallography, indicating the thermodynamically stable square pyramidal complex. Chelation of Cu(II) by 4 significantly reduced the oxidation potential of the former. CuCl2 and the 1:2 Cu:AMP-(Im)2 complex showed one-electron redox of Cu(II)/Cu(I) at 0.13 and -0.35 V, respectively. Indeed, 4 was found to be a potent antioxidant that at a 1:1:1 AMP-(Im)2:Cu(II)-PrP84-114 molar ratio almost totally inhibited the oxidation reaction of 4-methylcatechol. Circular dichroism data suggest that this antioxidant activity is due to formation of a ternary, redox inactive Cu(II)-Prp84-114-[AMP-(Im)2] complex. Future studies in prion disease animal models are warranted to assess the potential of 4 to inhibit the devastating oxidative damage caused by PrP.


Assuntos
Cobre/química , Isoxazóis/química , Príons/química , Tetrazóis/química , Modelos Moleculares , Oxirredução , Conformação Proteica
8.
ACS Chem Biol ; 14(7): 1410-1417, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31194501

RESUMO

Amyloid cascade and neuroinflammation are hallmarks of neurodegenerative diseases, and pro-inflammatory S100A9 protein is central to both of them. Here, we have shown that NCAM1 peptide constructs carrying polycationic sequences derived from Aß peptide (KKLVFF) and PrP protein (KKRPKP) significantly promote the S100A9 amyloid self-assembly in a concentration-dependent manner by making transient interactions with individual S100A9 molecules, perturbing its native structure and acting as catalysts. Since the individual molecule misfolding is a rate-limiting step in S100A9 amyloid aggregation, the effects of the NCAM1 construct on the native S100A9 are so critical for its amyloid self-assembly. S100A9 rapid self-assembly into large aggregated clumps may prevent its amyloid tissue propagation, and by modulating S100A9 aggregation as a part of the amyloid cascade, the whole process may be effectively tuned.


Assuntos
Amiloide/imunologia , Antígeno CD56/imunologia , Calgranulina B/imunologia , Agregação Patológica de Proteínas/imunologia , Sequência de Aminoácidos , Amiloide/química , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Antígeno CD56/química , Calgranulina B/química , Humanos , Inflamação/imunologia , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Príons/química , Príons/imunologia , Agregados Proteicos
9.
Nat Commun ; 10(1): 2593, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197139

RESUMO

Prion-like domains (PLDs), defined by their low sequence complexity and intrinsic disorder, are present in hundreds of human proteins. Although gain-of-function mutations in the PLDs of neuronal RNA-binding proteins have been linked to neurodegenerative disease progression, the physiological role of PLDs and their range of molecular functions are still largely unknown. Here, we show that the PLD of Drosophila Imp, a conserved component of neuronal ribonucleoprotein (RNP) granules, is essential for the developmentally-controlled localization of Imp RNP granules to axons and regulates in vivo axonal remodeling. Furthermore, we demonstrate that Imp PLD restricts, rather than promotes, granule assembly, revealing a novel modulatory function for PLDs in RNP granule homeostasis. Swapping the position of Imp PLD compromises RNP granule dynamic assembly but not transport, suggesting that these two functions are uncoupled. Together, our study uncovers a physiological function for PLDs in the spatio-temporal control of neuronal RNP assemblies.


Assuntos
Transporte Axonal/fisiologia , Grânulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/metabolismo , Domínios Proteicos/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Microscopia de Fluorescência , Modelos Animais , Príons/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
10.
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
11.
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
12.
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
13.
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
14.
Viruses ; 11(3)2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30823361

RESUMO

The abnormal protein aggregates in progressive neurodegenerative disorders, such as Alzheimer's, Parkinson's and prion diseases, adopt a generic structural form called amyloid fibrils. The precise amyloid fold can differ between patients and these differences are related to distinct neuropathological phenotypes of the diseases. A key focus in current research is the molecular mechanism governing such structural diversity, known as amyloid polymorphism. In this review, we focus on our recent work on recombinant prion protein (recPrP) and the use of pressure as a variable for perturbing protein structure. We suggest that the amyloid polymorphism is based on volumetric features. Accordingly, pressure is the thermodynamic parameter that fits best to exploit volume differences within the states of a chemical reaction, since it shifts the equilibrium constant to the state that has the smaller volume. In this context, there are analogies with the process of correct protein folding, the high pressure-induced effects of which have been studied for more than a century and which provides a valuable source of inspiration. We present a short overview of this background and review our recent results regarding the folding, misfolding, and aggregation-disaggregation of recPrP under pressure. We present preliminary experiments aimed at identifying how prion protein fibril diversity is related to the quaternary structure by using pressure and varying protein sequences. Finally, we consider outstanding questions and testable mechanistic hypotheses regarding the multiplicity of states in the amyloid fold.


Assuntos
Amiloide/genética , Polimorfismo Genético , Pressão , Proteínas Priônicas/genética , Amiloide/química , Humanos , Doenças Priônicas , Príons/química , Agregados Proteicos , Conformação Proteica , Dobramento de Proteína , Termodinâmica
15.
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
16.
Viruses ; 11(2)2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30696005

RESUMO

The mechanism of prion strain diversity remains unsolved. Investigation of inheritance and diversification of protein-based pathogenic information demands the identification of the detailed structures of abnormal isoforms of the prion protein (PrPSc); however, achieving purification is difficult without affecting infectivity. Similar prion-like properties are recognized also in other disease-associated in-register parallel ß-sheet amyloids including Tau and α-synuclein (αSyn) amyloids. Investigations into structures of those amyloids via solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy recently made remarkable advances due to their relatively small sizes and lack of post-translational modifications. Herein, we review advances regarding pathogenic amyloids, particularly Tau and αSyn, and discuss implications about strain diversity mechanisms of prion/PrPSc from the perspective that PrPSc is an in-register parallel ß-sheet amyloid. Additionally, we present our recent data of molecular dynamics simulations of αSyn amyloid, which suggest significance of compatibility between ß-sheet propensities of the substrate and local structures of the template for stability of amyloid structures. Detailed structures of αSyn and Tau amyloids are excellent models of pathogenic amyloids, including PrPSc, to elucidate strain diversity and pathogenic mechanisms.


Assuntos
Amiloide/química , Proteínas Priônicas/química , Príons/genética , Animais , Humanos , Camundongos , Simulação de Dinâmica Molecular , Proteínas PrPSc/química , Doenças Priônicas , Príons/química , Conformação Proteica em Folha beta , alfa-Sinucleína/genética , Proteínas tau/genética
17.
Mol Neurobiol ; 56(1): 367-377, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29704200

RESUMO

Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrPSc) in the central nervous system. Although several small compounds that bind to normal PrP (PrPC) have been shown to inhibit structural conversion of the protein, an effective therapy for human prion disease remains to be established. In this study, we screened 1200 existing drugs approved by the US Food and Drug Administration (FDA) for anti-prion activity using surface plasmon resonance imaging (SPRi). Of these drugs, 31 showed strong binding activity to recombinant human PrP, and three of these reduced the accumulation of PrPSc in prion-infected cells. One of the active compounds, alprenolol hydrochloride, which is used clinically as a ß-adrenergic blocker for hypertension, also reduced the accumulation of PrPSc in the brains of prion-infected mice at the middle stage of the disease when the drug was administered orally with their daily water from the day after infection. Docking simulation analysis suggested that alprenolol hydrochloride fitted into the hotspot within mouse PrPC, which is known as the most fragile structure within the protein. These findings provide evidence that SPRi is useful in identifying effective drug candidates for neurodegenerative diseases caused by abnormal protein aggregation, such as prion diseases.


Assuntos
Alprenolol/farmacologia , Imagem Tridimensional , Príons/antagonistas & inibidores , Alprenolol/química , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Espectroscopia de Ressonância Magnética , Camundongos , Simulação de Acoplamento Molecular , Oxprenolol/química , Oxprenolol/farmacologia , Proteínas PrPSc/metabolismo , Príons/química , Príons/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Ressonância de Plasmônio de Superfície , Análise de Sobrevida
18.
Methods Mol Biol ; 1873: 305-316, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30341619

RESUMO

Prion (PrPC) is an endogenous protein found mainly in the nervous system, and its misfolded isoform (PrPSc) is associated with a group of neurodegenerative disorders known as transmissible spongiform encephalopathies, or simply prion diseases. The PrPSc isoform shows an intriguing ability to self-perpetuate, acting as template for PrPC misfolding and consequent aggregation. Aggregation in vitro and in vivo follows a fibrillation processes that is associated with neurodegeneration. Therefore, it is important to investigate and understand the molecular mechanisms involved in this process; such understanding also allows investigation of the action of possible candidate molecules to inhibit this process. Here, we highlight useful in vitro methodologies and analyses that were developed using PrP as a protein model but that, as other amyloid proteins also exhibit the same behavior, may be applied to understand other "prion-like" diseases such as Alzheimer's and Parkinson's disease.


Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Príons/antagonistas & inibidores , Príons/química , Encéfalo/metabolismo , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Príons/isolamento & purificação , Príons/metabolismo , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
19.
FEMS Yeast Res ; 19(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30329039

RESUMO

I retrace my path from math to medicine to biochemistry to yeast genetics, my focus on infectious diseases of yeast and finally prions. My discovery of yeast prions relied on my particular focus on the logical relations of non-chromosomal genetic elements and the chromosomal genes involved in their propagation and expression. Pursuing an understanding of yeast prions involved structural biology based on genetics, solid-state NMR, population genetics and more genetics.


Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Príons/química , Príons/metabolismo , Dobramento de Proteína , Leveduras/genética , Leveduras/metabolismo , Genética Microbiana/tendências , História do Século XX , História do Século XXI , Biologia Molecular/tendências
20.
Curr Genet ; 65(2): 387-392, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30310993

RESUMO

Protein aggregation in vivo is generally combated by extensive proteostatic defenses. Many proteostasis factors specifically recognize aggregation-prone features and re-fold or degrade the targeted protein. However, protein aggregation is not uncommon, suggesting that some proteins employ evasive strategies to aggregate in spite of the proteostasis machinery. Therefore, in addition to understanding the inherent aggregation propensity of protein sequences, it is important to understand how these sequences affect proteostatic recognition and regulation in vivo. In a recent study, we used a genetic mutagenesis and screening approach to explore the aggregation or degradation promoting effects of the canonical amino acids in the context of G-rich and Q/N-rich prion-like domains (PrLDs). Our results indicate that aggregation propensity scales are strongly influenced by the interplay between specific PrLD features and proteostatic recognition. Here, we briefly review these results and expand upon their potential implications. In addition, a preliminary exploration of the yeast proteome suggests that these proteostatic regulation heuristics may influence the compositional features of native G-rich and Q/N-rich domains in yeast. These results improve our understanding of the features affecting the aggregation and proteostatic regulation of prion-like domains in a cellular context, and suggest that the sequence space for native prion-like domains may be shaped by proteostatic constraints.


Assuntos
Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Príons/genética , Príons/metabolismo , Agregados Proteicos , Domínios e Motivos de Interação entre Proteínas , Animais , Variação Genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas Priônicas/química , Príons/química , Agregação Patológica de Proteínas , Ligação Proteica , Proteólise
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