RESUMO
The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid-liquid phase separation (LLPS) under cellular conditions and that phase-separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho-tau isolated from human Alzheimer brain. Droplet-like tau can also be observed in neurons and other cells. We found that tau droplets become gel-like in minutes, and over days start to spontaneously form thioflavin-S-positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases.
Assuntos
Doença de Alzheimer/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , Agregação Patológica de Proteínas/metabolismo , Proteínas tau/química , Proteínas tau/isolamento & purificação , Proteínas tau/metabolismo , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Benzotiazóis/metabolismo , Fenômenos Biofísicos , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Feminino , Células HEK293 , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Extração Líquido-Líquido , Camundongos , Camundongos Transgênicos , Peso Molecular , Neuroblastoma/metabolismo , Doenças Neurodegenerativas/metabolismo , Emaranhados Neurofibrilares/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Células Sf9RESUMO
Expansions of polyglutamine (polyQ) tracts in nine different proteins cause a family of neurodegenerative disorders called polyQ diseases. Because polyQ tracts are potential therapeutic targets for these pathologies there is great interest in characterizing the conformations that they adopt and in understanding how their aggregation behavior is influenced by the sequences flanking them. We used solution NMR to study at single-residue resolution a 156-residue proteolytic fragment of the androgen receptor that contains a polyQ tract associated with the disease spinobulbar muscular atrophy, also known as Kennedy disease. Our findings indicate that a Leu-rich region preceding the polyQ tract causes it to become α-helical and appears to protect the protein against aggregation, which represents a new, to our knowledge, mechanism by which sequence context can minimize the deleterious properties of these repetitive regions. Our results have implications for drug discovery for polyQ diseases because they suggest that the residues flanking these repetitive sequences may represent viable therapeutic targets.
Assuntos
Peptídeos/genética , Peptídeos/metabolismo , Sequência de Aminoácidos , Atrofia Bulboespinal Ligada ao X/genética , Atrofia Bulboespinal Ligada ao X/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Dicroísmo Circular , Difusão Dinâmica da Luz , Escherichia coli , Humanos , Cinética , Ressonância Magnética Nuclear Biomolecular , Multimerização Proteica/genética , Estrutura Secundária de Proteína/genética , Espectroscopia de Prótons por Ressonância Magnética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismoRESUMO
It has been suggested that aberrant activation of glycogen synthase kinase-3-beta (GSK-3ß) can trigger abnormal tau hyperphosphorylation and aggregation, which ultimately leads to neuronal/synaptic damage and impaired cognition in Alzheimer disease (AD). We examined if isoform-selective partial reduction of GSK-3ß can decrease pathological tau changes, including hyperphosphorylation, aggregation, and spreading, in mice with localized human wild-type tau (hTau) expression in the brain. We used adeno-associated viruses (AAVs) to express hTau locally in the entorhinal cortex of wild-type and GSK-3ß hemi-knockout (GSK-3ß-HK) mice. GSK-3ß-HK mice had significantly less accumulation of hyperphosphorylated tau in synapses and showed a significant decrease of tau protein spread between neurons. In primary neuronal cultures from GSK-3ß-HK mice, the aggregation of exogenous FTD-mutant tau was also significantly reduced. These results show that a partial decrease of GSK-3ß significantly represses tau-initiated neurodegenerative changes in the brain, and therefore is a promising therapeutic target for AD and other tauopathies.
RESUMO
Increased oxidative stress is a widely accepted factor in the development and progression of Alzheimer's disease. Here, we introduce chitosan, an antioxidant oligosaccharide, as a protective agent against H(2)O(2)/FeSO(4)-induced cell death in the NT2 neural cell line. Chitosan not only protects the neurons against cell death, as measured by MTT and caspase-3 activity, but also decreases amyloid beta formation. NT2 neurons can be used to elucidate the relationship between oxidative stress and Abeta formation. We induced Abeta formation through oxidative stress in NT2 neurons and studied the effect of chitosan. We demonstrate that chitosan can be neuroprotective by suppressing Abeta formation. We further show that chitosan exerts its protective effect by up-regulation of HO-1, gamma-GCS, Hsp-70, and Nrf2, while it inhibits activation of caspase-3 and NF-kappaB. Chitosan or chitosan derivatives have potential value as neuroprotective agents, particularly with regard to oxidative stress.
Assuntos
Peptídeos beta-Amiloides/efeitos adversos , Quitosana/farmacologia , Citoproteção/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/fisiologia , NF-kappa B/fisiologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Citotoxinas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/fisiologia , Multimerização Proteica/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Actinomycetes isolated from Iran soil habitats were tested for the capacity to produce compounds which can protect neurons from cell death generated by oxidative stress in NT2 neurons. Confirmation of our initial hit was accomplished via the determination of amyloid beta level using the enzyme-linked immunosorbent assay test. The most interesting amyloid beta formation inhibitor discovered in our study was a secondary metabolite which was produced by strain HM45. This bioactive strain was identified as a strain of Streptomyces antibioticus DSM 40234 using polyphasic approach. The strain HM45 was deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen as S. antibioticus DSM 41955 and University of Tehran Microorganisms Sollection as S. antibioticus UTMC 00105. This work is the first report on efficiency of an actinomycete metabolite in prohibition of neurons death caused by amyloid beta formation.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Fragmentos de Peptídeos/metabolismo , Streptomyces antibioticus/metabolismo , Morte Celular , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Genes Bacterianos , Genes de RNAr , Humanos , Irã (Geográfico) , Neurônios/citologia , Filogenia , Microbiologia do Solo , Streptomyces antibioticus/classificação , Streptomyces antibioticus/genética , Streptomyces antibioticus/isolamento & purificaçãoRESUMO
Polyglutamine (polyQ) tracts are regions of low sequence complexity frequently found in transcription factors. Tract length often correlates with transcriptional activity and expansion beyond specific thresholds in certain human proteins is the cause of polyQ disorders. To study the structural basis of the association between tract length, transcriptional activity and disease, we addressed how the conformation of the polyQ tract of the androgen receptor, associated with spinobulbar muscular atrophy (SBMA), depends on its length. Here we report that this sequence folds into a helical structure stabilized by unconventional hydrogen bonds between glutamine side chains and main chain carbonyl groups, and that its helicity directly correlates with tract length. These unusual hydrogen bonds are bifurcate with the conventional hydrogen bonds stabilizing α-helices. Our findings suggest a plausible rationale for the association between polyQ tract length and androgen receptor transcriptional activity and have implications for establishing the mechanistic basis of SBMA.
Assuntos
Atrofia Bulboespinal Ligada ao X/genética , Peptídeos/química , Conformação Proteica em alfa-Hélice/genética , Receptores Androgênicos/química , Fatores de Transcrição/química , Atrofia Bulboespinal Ligada ao X/patologia , Dicroísmo Circular , Glutamina/química , Humanos , Hidrogênio/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Mutação , Agregados Proteicos/genética , Receptores Androgênicos/genética , Fatores de Transcrição/genéticaRESUMO
Molecular chaperones such as Hsp40 and Hsp70 hold the androgen receptor (AR) in an inactive conformation. They are released in the presence of androgens, enabling transactivation and causing the receptor to become aggregation-prone. Here we show that these molecular chaperones recognize a region of the AR N-terminal domain (NTD), including a FQNLF motif, that interacts with the AR ligand-binding domain (LBD) upon activation. This suggests that competition between molecular chaperones and the LBD for the FQNLF motif regulates AR activation. We also show that, while the free NTD oligomerizes, binding to Hsp70 increases its solubility. Stabilizing the NTD-Hsp70 interaction with small molecules reduces AR aggregation and promotes its degradation in cellular and mouse models of the neuromuscular disorder spinal bulbar muscular atrophy. These results help resolve the mechanisms by which molecular chaperones regulate the balance between AR aggregation, activation and quality control.
Assuntos
Androgênios/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Receptores Androgênicos/metabolismo , Animais , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Ligantes , Masculino , Camundongos , Camundongos Transgênicos , Ressonância Magnética Nuclear Biomolecular , Agregados Proteicos , Domínios Proteicos , Multimerização Proteica , Receptores Androgênicos/química , Receptores Androgênicos/genética , SolubilidadeRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
A novel artificial chaperone system using a combination of detergents and alginate was developed to refold three enzymes with totally different structures. Upon dilution of denatured protein in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to gradually remove the detergent molecules. In that respect, we used alginate, a linear copolymer of D-mannuronic acid and L-guluronic acid, to initiate and complete the refolding process. The results indicated that the extent of refolding assistance for the proteins was different due to detergent structure and also the length of hydrophobic portion of each detergent. These observed differences were attributed to the strong electrostatic and hydrophobic interactions among the capturing and stripping agents used in this investigation. Based on this newly developed method, it is expected that the protein refolding operation can be achieved easily, cheaply and efficiently.
Assuntos
Alginatos/química , Detergentes/química , Chaperonas Moleculares/química , Dobramento de Proteína , Fosfatase Alcalina/química , Anidrases Carbônicas/química , Etilenoglicol/química , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Sais/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , alfa-Amilases/químicaRESUMO
Tau is the major constituent of neurofibrillary tangles in Alzheimer's disease (AD), but the mechanism underlying tau-associated neural damage remains unclear. Here, we show that tau can directly interact with nucleoporins of the nuclear pore complex (NPC) and affect their structural and functional integrity. Pathological tau impairs nuclear import and export in tau-overexpressing transgenic mice and in human AD brain tissue. Furthermore, the nucleoporin Nup98 accumulates in the cell bodies of some tangle-bearing neurons and can facilitate tau aggregation in vitro. These data support the hypothesis that tau can directly interact with NPC components, leading to their mislocalization and consequent disruption of NPC function. This raises the possibility that NPC dysfunction contributes to tau-induced neurotoxicity in AD and tauopathies.
Assuntos
Doença de Alzheimer/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas tau/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Núcleo Celular/patologia , Citoplasma/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos TransgênicosRESUMO
Two different artificial chaperone systems were evaluated in this work using either detergents or CDs as the stripping agents. Upon dilution of urea-denatured alpha-amylase to a non-denaturing urea concentration in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to remove the detergent molecules. Our results by fluorescence, UV, turbidity measurement, circular dichroism, surface tension and activity assay indicated that the extent of refolding assistance was different due to different inter- and intra- molecular interactions in the two different systems. However, the high activity recovery in the presence of detergents, as the stripping agent, suggests that they can constitute suitable replacement for the more expensive and common stripping agent of cyclodextrins.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Ciclodextrinas/química , Chaperonas Moleculares/química , Dobramento de Proteína , alfa-Amilases/química , Detergentes/química , Desnaturação Proteica , Espectrofotometria , Ureia/químicaRESUMO
The spread of neurofibrillary tangle (NFT) pathology through the human brain is a hallmark of Alzheimer's disease (AD), which is thought to be caused by the propagation of "seeding" competent soluble misfolded tau. "TauC3", a C-terminally truncated form of tau that is generated by caspase-3 cleavage at D421, has previously been observed in NFTs and has been implicated in tau toxicity. Here we show that TauC3 is found in the seeding competent high molecular weight (HMW) protein fraction of human AD brain. Using a specific TauC3 antibody, we were able to substantially block the HMW tau seeding activity of human AD brain extracts in an in vitro tau seeding FRET assay. We propose that TauC3 could contribute to the templated tau misfolding that leads to NFT spread in AD brains.
Assuntos
Doença de Alzheimer/metabolismo , Anticorpos/metabolismo , Encéfalo/metabolismo , Proteínas tau/imunologia , Idoso de 80 Anos ou mais , Especificidade de Anticorpos , Caspase 3/metabolismo , Linhagem Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/metabolismo , Dobramento de Proteína , Proteínas tau/químicaRESUMO
The progression of many neurodegenerative diseases is assumed to be caused by misfolding of specific characteristic diseases related proteins, resulting in aggregation and fibril formation of these proteins. Protein misfolding associated age related diseases, although different in disease manifestations, share striking similarities. In all cases, one disease protein aggregates and loses its function or additionally shows a toxic gain of function. However, the clear link between these individual amyloid-like protein aggregates and cellular toxicity is often still uncertain. The similar features of protein misfolding and aggregation in this group of proteins, all involved in age related neurodegenerative diseases, results in high interest in characterization of their structural properties. We review here recent findings on structural properties of some age related disease proteins, in the context of their biological importance in disease.
Assuntos
Envelhecimento/metabolismo , Amiloide/metabolismo , Doenças Neurodegenerativas/metabolismo , Agregação Patológica de Proteínas/metabolismo , Envelhecimento/patologia , Animais , Humanos , Doenças Neurodegenerativas/patologia , Agregação Patológica de Proteínas/patologiaRESUMO
Alzheimer's disease (AD), the most relevant cause of dementia in elderly, is characterized by amyloid ß (Aß) containing plaques and neurofibrillatory tangles, synaptic and neuronal loss, along with progressive cognitive impairment in short-term memory. However, mechanistic links between protein kinase A (PKA), oxidative stress and memory loss in response to Aß remain elusive. In the present study, we examined the effects of post-training bilateral intra-hippocampal infusions of the specific protein kinase AII inhibitor, H-89, on memory deficits induced by Aß (1-42) in Aß-pretreated rats. H-89 and Aß were administered immediately after completion of training. All animals were trained for 4 consecutive days and tested 9 and 19 days after the infusions. Significant differences were observed in the time and distance of finding the hidden platform in Aß treated animals after 19 days. Interestingly, intra-hippocampal infusion of H-89 (5µM/side) significantly prevented the Aß-induced memory impairment. Furthermore, evaluation of NFκB (nuclear factor-κB), and antioxidant enzymes, such as γ-GCS (glutamylcysteine synthetase), HO-1 (hemeoxygenase-1), GSH (glutathione), and SOD (superoxide dismutase) confirmed the protective effect of H-89. Given the possible neuroprotective effects of H-89 on Aß-induced memory impairment, our results may open a new avenue for the prevention of AD by PKAII signaling pathway inhibitor.
Assuntos
Peptídeos beta-Amiloides/administração & dosagem , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/administração & dosagem , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glutationa/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Isoquinolinas/administração & dosagem , Masculino , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/genética , Memória de Curto Prazo/efeitos dos fármacos , NF-kappa B/genética , Inibidores de Proteínas Quinases/administração & dosagem , Ratos , Ratos Wistar , Sulfonamidas/administração & dosagemRESUMO
Alzheimer's disease (AD) a progressive neurodegenerative disorder of later life, is characterized by brain deposition of amyloid beta-protein (Abeta) plaques, accumulation of intracellular neurofibrillatory tangles, synaptic loss and neuronal cell death. There is significant evidence that oxidative stress is a critical event in the pathogenesis of AD. In the present study Abeta formation was induced in NT2N neurons, one of the most appropriate cell line models in AD. Our results indicate that oxidative stress resulting from the treatment of H(2)O(2)/FeSO(4) and/or 4-hydroxy-2-noenal (HNE) can be inhibited in the presence of tBHQ, a known inducer of nuclear factor-erythroid 2 related factor 2 (Nrf2) in NT2N neurons and can therefore be used to elucidate the relationship between oxidative stress, Abeta formation and Nrf2. The role of Nrf2 was confirmed using retinoic acid as an inhibitor of Nrf2. It provides the first documentation that tBHQ not only protects the neurons against cell death but also decreases amyloid beta formation. Moreover, the results indicate that oxidative stress fosters Abeta formation in NT2N neurons, creating a vicious neurodegenerative loop.