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1.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809196

RESUMO

Deposition of amyloid ß (Aß) fibrils in the brain is a key pathologic hallmark of Alzheimer's disease. A class of polyphenolic biflavonoids is known to have anti-amyloidogenic effects by inhibiting aggregation of Aß and promoting disaggregation of Aß fibrils. In the present study, we further sought to investigate the structural basis of the Aß disaggregating activity of biflavonoids and their interactions at the atomic level. A thioflavin T (ThT) fluorescence assay revealed that amentoflavone-type biflavonoids promote disaggregation of Aß fibrils with varying potency due to specific structural differences. The computational analysis herein provides the first atomistic details for the mechanism of Aß disaggregation by biflavonoids. Molecular docking analysis showed that biflavonoids preferentially bind to the aromatic-rich, partially ordered N-termini of Aß fibril via the π-π interactions. Moreover, docking scores correlate well with the ThT EC50 values. Molecular dynamic simulations revealed that biflavonoids decrease the content of ß-sheet in Aß fibril in a structure-dependent manner. Hydrogen bond analysis further supported that the substitution of hydroxyl groups capable of hydrogen bond formation at two positions on the biflavonoid scaffold leads to significantly disaggregation of Aß fibrils. Taken together, our data indicate that biflavonoids promote disaggregation of Aß fibrils due to their ability to disrupt the fibril structure, suggesting biflavonoids as a lead class of compounds to develop a therapeutic agent for Alzheimer's disease.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Benzotiazóis/farmacologia , Biflavonoides/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , Doença de Alzheimer/patologia , Amiloide/antagonistas & inibidores , Amiloide/efeitos dos fármacos , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/ultraestrutura , Biflavonoides/química , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Humanos , Ligação de Hidrogênio/efeitos dos fármacos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia
2.
Molecules ; 26(4)2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670093

RESUMO

The misfolding and aggregation of polypeptide chains into ß-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence indicates that the oligomeric intermediates populated in the early stages of amyloid formation rather than the mature fibrils are responsible for the cytotoxicity and pathology and are potentially therapeutic targets. However, due to the low-populated, transient, and heterogeneous nature of amyloid oligomers, they are hard to characterize by conventional bulk methods. The development of single molecule approaches provides a powerful toolkit for investigating these oligomeric intermediates as well as the complex process of amyloid aggregation at molecular resolution. In this review, we present an overview of recent progress in characterizing the oligomerization of amyloid proteins by single molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), single-molecule photobleaching and super-resolution optical imaging. We discuss how these techniques have been applied to investigate the different aspects of amyloid oligomers and facilitate understanding of the mechanism of amyloid aggregation.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Proteínas Amiloidogênicas/química , Agregação Patológica de Proteínas/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/ultraestrutura , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/ultraestrutura , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Conformação Proteica em Folha beta/genética , Imagem Individual de Molécula , Espectrometria de Fluorescência
3.
Int J Mol Sci ; 22(4)2021 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-33668611

RESUMO

The maintenance of proteome homeostasis, or proteostasis, is crucial for preserving cellular functions and for cellular adaptation to environmental challenges and changes in physiological conditions. The capacity of cells to maintain proteostasis requires precise control and coordination of protein synthesis, folding, conformational maintenance, and clearance. Thus, protein degradation by the ubiquitin-proteasome system (UPS) or the autophagy-lysosomal system plays an essential role in cellular functions. However, failure of the UPS or the autophagic process can lead to the development of various diseases (aging-associated diseases, cancer), thus both these pathways have become attractive targets in the treatment of protein conformational diseases, such as alpha 1-antitrypsin deficiency (AATD). The Z alpha 1-antitrypsin (Z-AAT) misfolded variant of the serine protease alpha 1-antitrypsin (AAT) is caused by a structural change that predisposes it to protein aggregation and dramatic accumulation in the form of inclusion bodies within liver hepatocytes. This can lead to clinically significant liver disease requiring liver transplantation in childhood or adulthood. Treatment of mice with autophagy enhancers was found to reduce hepatic Z-AAT aggregate levels and protect them from AATD hepatotoxicity. To date, liver transplantation is the only curative therapeutic option for patients with AATD-mediated liver disease. Therefore, the development and discovery of new therapeutic approaches to delay or overcome disease progression is a top priority. Herein, we review AATD-mediated liver disease and the overall process of autophagy. We highlight the role of this system in the regulation of Z-variant degradation and its implication in AATD-medicated liver disease, including some open questions that remain challenges in the field and require further elucidation. Finally, we discuss how manipulation of autophagy could provide multiple routes of therapeutic benefit in AATD-mediated liver disease.


Assuntos
Autofagia , Hepatócitos , Hepatopatias , Transplante de Fígado , Fígado , Agregação Patológica de Proteínas , alfa 1-Antitripsina , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Fígado/metabolismo , Fígado/patologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Hepatopatias/cirurgia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Agregação Patológica de Proteínas/cirurgia , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/patologia
4.
Int J Mol Sci ; 22(2)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477953

RESUMO

Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 are responsible for the main spectral changes, ascribable mainly to the cell envelope modifications. A structural alteration of the membrane detected through electron microscopy analysis in the strain expressing the pathological form supports the spectroscopic results.


Assuntos
Amiloide/genética , Proteínas Amiloidogênicas/genética , Ataxina-3/genética , Doença de Machado-Joseph/genética , Membrana Celular/genética , Proliferação de Células/genética , Escherichia coli/genética , Regulação da Expressão Gênica/genética , Humanos , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Proteínas do Tecido Nervoso/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia
5.
PLoS One ; 16(1): e0241161, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33497415

RESUMO

Alpha-synuclein pre-formed fibrils (PFFs) represent a promising model system for the study of cellular processes underlying cell-to-cell transmission of alpha-synuclein proteopathic aggregates. However, the ability to differentiate the fate of internalized PFFs from those which remain in the extracellular environment remains limited due to the propensity for PFFs to adhere to the cell surface. Removal of PFFs requires repeated washing and/or specific quenching of extracellular fluorescent PFF signals. In this paper we present a new method for analyzing the fate of internalized alpha-synuclein. We inserted a tobacco etch virus (TEV) protease cleavage site between alpha-synuclein and green fluorescent protein and subjected cells to brief treatment with TEV protease after incubation with tagged PFFs. As the TEV protease is highly specific, non-toxic, and active under physiological conditions, protection from TEV cleavage can be used to distinguish internalized PFFs from those which remain attached to the cell surface. Using this experimental paradigm, downstream intracellular events can be analyzed via live or fixed cell microscopy as well as by Western blotting. We suggest that this method will be useful for understanding the fate of PFFs after endocytosis under various experimental manipulations.


Assuntos
Bioensaio , Neurônios/metabolismo , Agregação Patológica de Proteínas/metabolismo , alfa-Sinucleína/metabolismo , Animais , Linhagem Celular , Endopeptidases/genética , Endopeptidases/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , alfa-Sinucleína/genética
6.
Nat Genet ; 52(10): 1024-1035, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32989324

RESUMO

Protein aggregation is the hallmark of neurodegeneration, but the molecular mechanisms underlying late-onset Alzheimer's disease (AD) are unclear. Here we integrated transcriptomic, proteomic and epigenomic analyses of postmortem human brains to identify molecular pathways involved in AD. RNA sequencing analysis revealed upregulation of transcription- and chromatin-related genes, including the histone acetyltransferases for H3K27ac and H3K9ac. An unbiased proteomic screening singled out H3K27ac and H3K9ac as the main enrichments specific to AD. In turn, epigenomic profiling revealed gains in the histone H3 modifications H3K27ac and H3K9ac linked to transcription, chromatin and disease pathways in AD. Increasing genome-wide H3K27ac and H3K9ac in a fly model of AD exacerbated amyloid-ß42-driven neurodegeneration. Together, these findings suggest that AD involves a reconfiguration of the epigenome, wherein H3K27ac and H3K9ac affect disease pathways by dysregulating transcription- and chromatin-gene feedback loops. The identification of this process highlights potential epigenetic strategies for early-stage disease treatment.


Assuntos
Doença de Alzheimer/genética , Agregação Patológica de Proteínas/genética , Proteoma/genética , Transcriptoma/genética , Acetilação , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Cromatina/genética , Epigenoma/genética , Histona Acetiltransferases/genética , Código das Histonas/genética , Histonas/genética , Humanos , Fragmentos de Peptídeos/genética , Agregação Patológica de Proteínas/patologia , Transdução de Sinais/genética , Ativação Transcricional/genética
7.
PLoS One ; 15(9): e0239584, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32966331

RESUMO

Familial forms of Alzheimer's disease (AD) are caused by mutations in the presenilin genes or in the gene encoding for the amyloid precursor protein (APP). Proteolytic cleavage of APP generates the ß-amyloid peptide (Aß), which aggregates into amyloid plaques, one of the major hallmarks of AD. APP mutations within the Aß sequence, so-called intra-Aß mutations, cluster around position E693 of APP, which corresponds to position E22 in the Aß sequence. One of these mutations is the Osaka mutation, E693Δ, which has unique aggregation properties with patients showing unusually low brain amyloid levels on amyloid PET scans. Despite intense research on the pathomechanisms of different intra-Aß mutants, our knowledge is limited due to controversial findings in various studies. Here, we investigated in an ex vivo experimental system the neuro- and synaptotoxic properties of two intra-Aß mutants with different intrinsic aggregation propensities, the Osaka mutation E22Δ and the Arctic mutation E22G, and compared them to wild-type (wt) Aß. Experiments in hippocampal slice cultures from transgenic mice were complemented by treating wild-type slices with recombinantly produced Aß40 or Aß42 containing the respective intra-Aß mutations. Our analyses revealed that wt Aß and E22G Aß, both recombinant and transgenic, caused a loss of dendritic spines along with an increase in tau phosphorylation and tau-dependent neurodegeneration. In all experiments, the 42-residue variants of wt and E22G Aß showed stronger effects than the respective Aß40 isoforms. In contrast, E22Δ Aß neither reduced dendritic spine density nor resulted in increased tau phosphorylation or neuronal cell death in our ex vivo system. Our findings suggest that the previously reported major differences in the aggregation kinetics between E22G and E22Δ Aß are likely reflected in different disease pathomechanisms.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Proteínas Mutantes/genética , Mutação , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Morte Celular , Espinhas Dendríticas/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Técnicas In Vitro , Cinética , Camundongos , Camundongos Transgênicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sinapses/patologia
8.
PLoS One ; 15(8): e0233247, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857759

RESUMO

Poly(glycine-alanine) (polyGA) is one of the polydipeptides expressed in Frontotemporal Dementia and/or Amyotrophic Lateral Sclerosis 1 caused by C9ORF72 mutations and accumulates as inclusion bodies in the brain of patients. Superficially these inclusions are similar to those formed by polyglutamine (polyQ)-expanded Huntingtin exon 1 (Httex1) in Huntington's disease. Both have been reported to form an amyloid-like structure suggesting they might aggregate via similar mechanisms and therefore recruit the same repertoire of endogenous proteins. When co-expressed in the same cell, polyGA101 and Httex1(Q97) inclusions adopted immiscible phases suggesting different endogenous proteins would be enriched. Proteomic analyses identified 822 proteins in the inclusions. Only 7 were specific to polyGA and 4 specific to Httex1(Q97). Quantitation demonstrated distinct enrichment patterns for the proteins not specific to each inclusion type (up to ~8-fold normalized to total mass). The proteasome, microtubules, TriC chaperones, and translational machinery were enriched in polyGA aggregates, whereas Dnaj chaperones, nuclear envelope and RNA splicing proteins were enriched in Httex1(Q97) aggregates. Both structures revealed a collection of folding and degradation machinery including proteins in the Httex1(Q97) aggregates that are risk factors for other neurodegenerative diseases involving protein aggregation when mutated, which suggests a convergence point in the pathomechanisms of these diseases.


Assuntos
Corpos de Inclusão/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Linhagem Celular , Éxons , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Peptídeos/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Proteínas/genética , Proteólise , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Risco , Solubilidade
9.
PLoS Pathog ; 16(6): e1008611, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511263

RESUMO

Human infection with avian influenza A (H5N1) and (H7N9) viruses causes severe respiratory diseases. PB1-F2 protein is a critical virulence factor that suppresses early type I interferon response, but the mechanism of its action in relation to high pathogenicity is not well understood. Here we show that PB1-F2 protein of H7N9 virus is a particularly potent suppressor of antiviral signaling through formation of protein aggregates on mitochondria and inhibition of TRIM31-MAVS interaction, leading to prevention of K63-polyubiquitination and aggregation of MAVS. Unaggregated MAVS accumulated on fragmented mitochondria is prone to degradation by both proteasomal and lysosomal pathways. These properties are proprietary to PB1-F2 of H7N9 virus but not shared by its counterpart in WSN virus. A recombinant virus deficient of PB1-F2 of H7N9 induces more interferon ß in infected cells. Our findings reveal a subtype-specific mechanism for destabilization of MAVS and suppression of interferon response by PB1-F2 of H7N9 virus.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Subtipo H7N9 do Vírus da Influenza A/metabolismo , Influenza Humana/metabolismo , Agregação Patológica de Proteínas/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cães , Células HEK293 , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/patologia , Interferon beta/genética , Interferon beta/metabolismo , Células Madin Darby de Rim Canino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Agregação Patológica de Proteínas/genética , Células THP-1 , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/genética
10.
Nat Med ; 26(8): 1256-1263, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32572268

RESUMO

Alzheimer's disease (AD) causes unrelenting, progressive cognitive impairments, but its course is heterogeneous, with a broad range of rates of cognitive decline1. The spread of tau aggregates (neurofibrillary tangles) across the cerebral cortex parallels symptom severity2,3. We hypothesized that the kinetics of tau spread may vary if the properties of the propagating tau proteins vary across individuals. We carried out biochemical, biophysical, MS and both cell- and animal-based-bioactivity assays to characterize tau in 32 patients with AD. We found striking patient-to-patient heterogeneity in the hyperphosphorylated species of soluble, oligomeric, seed-competent tau. Tau seeding activity correlates with the aggressiveness of the clinical disease, and some post-translational modification (PTM) sites appear to be associated with both enhanced seeding activity and worse clinical outcomes, whereas others are not. These data suggest that different individuals with 'typical' AD may have distinct biochemical features of tau. These data are consistent with the possibility that individuals with AD, much like people with cancer, may have multiple molecular drivers of an otherwise common phenotype, and emphasize the potential for personalized therapeutic approaches for slowing clinical progression of AD.


Assuntos
Doença de Alzheimer/genética , Disfunção Cognitiva/genética , Agregação Patológica de Proteínas/genética , Proteínas tau/genética , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Disfunção Cognitiva/patologia , Feminino , Heterogeneidade Genética , Humanos , Masculino , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/genética , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Fosforilação , Agregação Patológica de Proteínas/patologia , Índice de Gravidade de Doença
11.
Genes Dev ; 34(11-12): 785-805, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32381627

RESUMO

Dysregulation of the DNA/RNA-binding protein FUS causes certain subtypes of ALS/FTD by largely unknown mechanisms. Recent evidence has shown that FUS toxic gain of function due either to mutations or to increased expression can disrupt critical cellular processes, including mitochondrial functions. Here, we demonstrate that in human cells overexpressing wild-type FUS or expressing mutant derivatives, the protein associates with multiple mRNAs, and these are enriched in mRNAs encoding mitochondrial respiratory chain components. Notably, this sequestration leads to reduced levels of the encoded proteins, which is sufficient to bring about disorganized mitochondrial networks, reduced aerobic respiration and increased reactive oxygen species. We further show that mutant FUS associates with mitochondria and with mRNAs encoded by the mitochondrial genome. Importantly, similar results were also observed in fibroblasts derived from ALS patients with FUS mutations. Finally, we demonstrate that FUS loss of function does not underlie the observed mitochondrial dysfunction, and also provides a mechanism for the preferential sequestration of the respiratory chain complex mRNAs by FUS that does not involve sequence-specific binding. Together, our data reveal that respiratory chain complex mRNA sequestration underlies the mitochondrial defects characteristic of ALS/FTD and contributes to the FUS toxic gain of function linked to this disease spectrum.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/fisiopatologia , Regulação da Expressão Gênica/genética , Mitocôndrias/patologia , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Linhagem Celular , Respiração Celular/genética , Células Cultivadas , Transporte de Elétrons/genética , Genoma Mitocondrial , Humanos , Mitocôndrias/genética , Mutação , Agregação Patológica de Proteínas/genética , Ligação Proteica/genética
12.
Sci Rep ; 10(1): 7721, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382058

RESUMO

Amyloids are protein aggregates observed in several diseases, for example in Alzheimer's and Parkinson's diseases. An aggregate has a very regular beta structure with a tightly packed core, which spontaneously assumes a steric zipper form. Experimental methods enable studying such peptides, however they are tedious and costly, therefore inappropriate for genomewide studies. Several bioinformatic methods have been proposed to evaluate protein propensity to form an amyloid. However, the knowledge of aggregate structures is usually not taken into account. We propose PATH (Prediction of Amyloidogenicity by THreading) - a novel structure-based method for predicting amyloidogenicity and show that involving available structures of amyloidogenic fragments enhances classification performance. Experimental aggregate structures were used in templatebased modeling to recognize the most stable representative structural class of a query peptide. Several machine learning methods were then applied on the structural models, using their energy terms. Finally, we identified the most important terms in classification of amyloidogenic peptides. The proposed method outperforms most of the currently available methods for predicting amyloidogenicity, with its area under ROC curve equal to 0.876. Furthermore, the method gave insight into significance of selected structural features and the potentially most stable structural class of a peptide fragment if subjected to crystallization.


Assuntos
Amiloide/ultraestrutura , Fragmentos de Peptídeos/ultraestrutura , Conformação Proteica em Folha beta/genética , Software , Algoritmos , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Amiloide/química , Biologia Computacional/métodos , Humanos , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fragmentos de Peptídeos/química , Agregados Proteicos/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia
13.
Phys Chem Chem Phys ; 22(17): 9225-9232, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32307496

RESUMO

The p53 protein is a tumor suppressor and the most often mutated protein in human cancers. Recent studies reported that p53 mutants, including two of the common cancer mutants (R175H and R273H), are more prone to aggregation than wild type (WT) p53 and their pathological aggregation can lead to diverse cancers. However, the underlying molecular mechanism is poorly understood. Herein, we investigated the structural and dynamic properties of R175H and R273H mutants of the p53 core domain (p53C) by performing extensive all-atom molecular dynamics simulations. We found that both R175H and R273H mutants exhibit a well preserved ß-sheet structure, but a larger hydrophobic surface area and higher loop flexibility than WT p53C. These conformational properties are consistent with the structural features of aggregation-prone molten-globule states. Our data also provide the details on how the two mutations lead to an increased flexibility of loop2. Moreover, using dynamic network analysis, we identified the allosteric path through which the R273H mutation induces an increased flexibility of the distant N-terminal region of loop2. These results provide mechanistic insights into the high aggregation propensities of R175H and R273H mutants.


Assuntos
Modelos Moleculares , Mutação/genética , Neoplasias/genética , Agregação Patológica de Proteínas/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica/genética , Domínios Proteicos/genética , Estrutura Terciária de Proteína
14.
Mol Cell ; 78(2): 329-345.e9, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32268122

RESUMO

Neural stem and progenitor cells (NSPCs) are critical for continued cellular replacement in the adult brain. Lifelong maintenance of a functional NSPC pool necessitates stringent mechanisms to preserve a pristine proteome. We find that the NSPC chaperone network robustly maintains misfolded protein solubility and stress resilience through high levels of the ATP-dependent chaperonin TRiC/CCT. Strikingly, NSPC differentiation rewires the cellular chaperone network, reducing TRiC/CCT levels and inducing those of the ATP-independent small heat shock proteins (sHSPs). This switches the proteostasis strategy in neural progeny cells to promote sequestration of misfolded proteins into protective inclusions. The chaperone network of NSPCs is more effective than that of differentiated cells, leading to improved management of proteotoxic stress and amyloidogenic proteins. However, NSPC proteostasis is impaired by brain aging. The less efficient chaperone network of differentiated neural progeny may contribute to their enhanced susceptibility to neurodegenerative diseases characterized by aberrant protein misfolding and aggregation.


Assuntos
Envelhecimento/genética , Chaperonas Moleculares/genética , Células-Tronco Neurais/metabolismo , Agregação Patológica de Proteínas/genética , Trifosfato de Adenosina/genética , Envelhecimento/patologia , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Diferenciação Celular/genética , Chaperoninas/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Camundongos , Chaperonas Moleculares/metabolismo , Células-Tronco Neurais/patologia , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Células-Tronco/metabolismo , Células-Tronco/patologia
15.
Sci Rep ; 10(1): 4011, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132634

RESUMO

Protein aggregation has been one of the leading triggers of various disease conditions, such as Alzheimer's, Parkinson's and other amyloidosis. TGFBI-associated corneal dystrophies are protein aggregation disorders in which the mutant TGFBIp aggregates and accumulates in the cornea, leading to a reduction in visual acuity and blindness in severe cases. Currently, the only therapy available is invasive and there is a known recurrence after surgery. In this study, we tested the inhibitory and amyloid dissociation properties of four osmolytes in an in-vitro TGFBI peptide aggregation model. The 23-amino acid long peptide (TGFBIp 611-633 with the mutation c.623 G>R) from the 4th FAS-1 domain of TGFBIp that rapidly forms amyloid fibrils was used in the study. Several biophysical methods like Thioflavin T (ThT) fluorescence, Circular Dichroism (CD), fluorescence microscopy and Transmission electron microscopy (TEM) were used to study the inhibitory and amyloid disaggregation properties of the four osmolytes (Betaine, Raffinose, Sarcosine, and Taurine). The osmolytes were effective in both inhibiting and disaggregating the amyloid fibrils derived from TGFBIp 611-633 c.623 G>R peptide. The osmolytes did not have an adverse toxic effect on cultured human corneal fibroblast cells and could potentially be a useful therapeutic strategy for patients with TGFBIp corneal dystrophies.


Assuntos
Amiloide , Córnea , Proteínas da Matriz Extracelular , Fibroblastos , Peptídeos , Agregação Patológica de Proteínas , Fator de Crescimento Transformador beta , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Linhagem Celular , Córnea/metabolismo , Córnea/patologia , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
16.
Int J Mol Sci ; 21(4)2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32098273

RESUMO

Human cells express large amounts of different proteins continuously that must fold into well-defined structures that need to remain correctly folded and assemble in order to ensure their cellular and biological functions. The integrity of this protein balance/homeostasis, also named proteostasis, is maintained by the proteostasis network (PN). This integrated biological system, which comprises about 2000 proteins (chaperones, folding enzymes, degradation components), control and coordinate protein synthesis folding and localization, conformational maintenance, and degradation. This network is particularly challenged by mutations such as those found in genetic diseases, because of the inability of an altered peptide sequence to properly engage PN components that trigger misfolding and loss of function. Thus, deletions found in the ΔF508 variant of the Cystic Fibrosis (CF) transmembrane regulator (CFTR) triggering CF or missense mutations found in the Z variant of Alpha 1-Antitrypsin deficiency (AATD), leading to lung and liver diseases, can accelerate misfolding and/or generate aggregates. Conversely to CF variants, for which three correctors are already approved (ivacaftor, lumacaftor/ivacaftor, and most recently tezacaftor/ivacaftor), there are limited therapeutic options for AATD. Therefore, a more detailed understanding of the PN components governing AAT variant biogenesis and their manipulation by pharmacological intervention could delay, or even better, avoid the onset of AATD-related pathologies.


Assuntos
Agregação Patológica de Proteínas/metabolismo , Dobramento de Proteína , Deficiências na Proteostase/metabolismo , Proteostase , Deficiência de alfa 1-Antitripsina/metabolismo , Humanos , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Deficiências na Proteostase/genética , Deficiências na Proteostase/patologia , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/patologia
17.
Circ Res ; 126(7): 907-922, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32081062

RESUMO

RATIONALE: Compromised protein quality control can result in proteotoxic intracellular protein aggregates in the heart, leading to cardiac disease and heart failure. Defining the participants and understanding the underlying mechanisms of cardiac protein aggregation is critical for seeking therapeutic targets. We identified Ube2v1 (ubiquitin-conjugating enzyme E2 variant 1) in a genome-wide screen designed to identify novel effectors of the aggregation process. However, its role in the cardiomyocyte is undefined. OBJECTIVE: To assess whether Ube2v1 regulates the protein aggregation caused by cardiomyocyte expression of a mutant αB crystallin (CryABR120G) and identify how Ube2v1 exerts its effect. METHODS AND RESULTS: Neonatal rat ventricular cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB (CryABWT) or CryABR120G. Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryABR120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryABWT and CryABR120G-expressing neonatal rat ventricular cardiomyocytes. Ubiquitin proteasome system performance was analyzed using a ubiquitin proteasome system reporter protein. Ube2v1 knockdown improved ubiquitin proteasome system performance and promoted the degradation of insoluble ubiquitinated proteins in CryABR120G cardiomyocytes but did not alter autophagic flux. Lys (K) 63-linked ubiquitination modulated by Ube2v1 expression enhanced protein aggregation and contributed to Ube2v1's function in regulating protein aggregate formation. Knocking out Ube2v1 exclusively in cardiomyocytes by using AAV9 (adeno-associated virus 9) to deliver multiplexed single guide RNAs against Ube2v1 in cardiac-specific Cas9 mice alleviated CryABR120G-induced protein aggregation, improved cardiac function, and prolonged lifespan in vivo. CONCLUSIONS: Ube2v1 plays an important role in protein aggregate formation, partially by enhancing K63 ubiquitination during a proteotoxic stimulus. Inhibition of Ube2v1 decreases CryABR120G-induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.


Assuntos
Lisina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregação Patológica de Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Estudo de Associação Genômica Ampla/métodos , Humanos , Masculino , Camundongos Transgênicos , Mutação , Miócitos Cardíacos/metabolismo , Agregação Patológica de Proteínas/genética , Ratos , Fatores de Transcrição/genética , Enzimas de Conjugação de Ubiquitina/genética , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismo
18.
Nat Commun ; 11(1): 1004, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081878

RESUMO

Cytoplasmic aggregation of TDP-43 characterizes degenerating neurons in most cases of amyotrophic lateral sclerosis (ALS). Here, we develop an optogenetic TDP-43 variant (opTDP-43), whose multimerization status can be modulated in vivo through external light illumination. Using the translucent zebrafish neuromuscular system, we demonstrate that short-term light stimulation reversibly induces cytoplasmic opTDP-43 mislocalization, but not aggregation, in the spinal motor neuron, leading to an axon outgrowth defect associated with myofiber denervation. In contrast, opTDP-43 forms pathological aggregates in the cytoplasm after longer-term illumination and seeds non-optogenetic TDP-43 aggregation. Furthermore, we find that an ALS-linked mutation in the intrinsically disordered region (IDR) exacerbates the light-dependent opTDP-43 toxicity on locomotor behavior. Together, our results propose that IDR-mediated TDP-43 oligomerization triggers both acute and long-term pathologies of motor neurons, which may be relevant to the pathogenesis and progression of ALS.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Esclerose Amiotrófica Lateral/genética , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Mutação , Optogenética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Multimerização Proteica , Estabilidade Proteica , Regulação para Cima , Peixe-Zebra
19.
Mol Cell Biochem ; 466(1-2): 117-128, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32056106

RESUMO

Aberrant structural formations of Cu/Zn superoxide dismutase enzyme (SOD1) are the probable mechanism by which circumscribed mutations in the SOD1 gene cause familial amyotrophic lateral sclerosis (ALS1). SOD1 forms aberrant structures which can proceed by nucleation to insoluble aggregates. Here, the SOD1 aggregation reaction was investigated predominantly by time-course studies on ALS1 variants G85R, G37R, D101G, and D101N in human embryonic kidney cells (HEK293FT), with analysis by detergent ultracentrifugation extractions and high-resolution PAGE methodologies. Nucleation was found to be pseudo-zeroth order and dependent on time and concentration at constant 37.0 °C and pH 7.4. The predominant subsets of the total SOD1 expression set which comprised the nucleation phase were both soluble and insoluble inactive monomers, trimers, and hexamers with reduced intra-disulfide bonds. Superoxide exposure via paraquat initiated the formation of SOD1 trimers in untransfected SH-SY5Y cells and increased the aggregation propensity of G85R in HEK293FT. These data show the kinetic formation of aberrant SOD1 subsets implicated in ALS1 and indicate that superoxide substrate may initiate its radical polymerization. In an instance of the utility of methodological reductionism in molecular theory: though many ALS1 variants retain their global enzymatic activity, the SOD1 subsets most implicated in causing ALS1 do not retain their specific activity.


Assuntos
Mutação de Sentido Incorreto , Agregação Patológica de Proteínas/metabolismo , Superóxido Dismutase-1/metabolismo , Substituição de Aminoácidos , Esclerose Amiotrófica Lateral/genética , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Agregação Patológica de Proteínas/genética , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genética
20.
Curr Protoc Neurosci ; 91(1): e88, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32049438

RESUMO

Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by motor symptoms such as tremor, slowness of movement, rigidity, and postural instability, as well as non-motor features like sleep disturbances, loss of ability to smell, depression, constipation, and pain. Motor symptoms are caused by depletion of dopamine in the striatum due to the progressive loss of dopamine neurons in the substantia nigra pars compacta. Approximately 10% of PD cases are familial arising from genetic mutations in α-synuclein, LRRK2, DJ-1, PINK1, parkin, and several other proteins. The majority of PD cases are, however, idiopathic, i.e., having no clear etiology. PD is characterized by progressive accumulation of insoluble inclusions, known as Lewy bodies, mostly composed of α-synuclein and membrane components. The cause of PD is currently attributed to cellular proteostasis deregulation and mitochondrial dysfunction, which are likely interdependent. In addition, neuroinflammation is present in brains of PD patients, but whether it is the cause or consequence of neurodegeneration remains to be studied. Rodents do not develop PD or PD-like motor symptoms spontaneously; however, neurotoxins, genetic mutations, viral vector-mediated transgene expression and, recently, injections of misfolded α-synuclein have been successfully utilized to model certain aspects of the disease. Here, we critically review the advantages and drawbacks of rodent PD models and discuss approaches to advance pre-clinical PD research towards successful disease-modifying therapy. © 2020 The Authors.


Assuntos
Neurotoxinas/toxicidade , Transtornos Parkinsonianos , Animais , Corpo Estriado/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/ultraestrutura , Avaliação Pré-Clínica de Medicamentos/métodos , Previsões , Estudo de Associação Genômica Ampla , Técnicas Histológicas , Humanos , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson/genética , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/patologia , Praguicidas/toxicidade , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Ratos , Substância Negra/efeitos dos fármacos , Sinucleinopatias/genética , Sinucleinopatias/patologia , alfa-Sinucleína/biossíntese , alfa-Sinucleína/genética
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