RESUMO
The presence of amyloid fibrils of α-synuclein is closely associated with Parkinson's disease and related synucleinopathies. It is still very challenging, however, to systematically discover small molecules that prevent the formation of these aberrant aggregates. Here, we describe a structure-based approach to identify small molecules that specifically inhibit the surface-catalyzed secondary nucleation step in the aggregation of α-synuclein by binding to the surface of the amyloid fibrils. The resulting small molecules are screened using a range of kinetic and thermodynamic assays for their ability to bind α-synuclein fibrils and prevent the further generation of α-synuclein oligomers. This study demonstrates that the combination of structure-based and kinetic-based drug discovery methods can lead to the identification of small molecules that selectively inhibit the autocatalytic proliferation of α-synuclein aggregates.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Amiloide/metabolismo , Doença de Parkinson/metabolismo , Cinética , Proliferação de Células , Agregados ProteicosRESUMO
To function effectively proteins must avoid aberrant aggregation, and hence they are expected to be expressed at concentrations safely below their solubility limits. By analyzing proteome-wide mass spectrometry data of Caenorhabditis elegans, however, we show that the levels of about three-quarters of the nearly 4,000 proteins analyzed in adult animals are close to their intrinsic solubility limits, indeed exceeding them by about 10% on average. We next asked how aging and functional self-assembly influence these solubility limits. We found that despite the fact that the total quantity of proteins within the cellular environment remains approximately constant during aging, protein aggregation sharply increases between days 6 and 12 of adulthood, after the worms have reproduced, as individual proteins lose their stoichiometric balances and the cellular machinery that maintains solubility undergoes functional decline. These findings reveal that these proteins are highly prone to undergoing concentration-dependent phase separation, which on aging is rationalized in a decrease of their effective solubilities, in particular for proteins associated with translation, growth, reproduction, and the chaperone system.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteoma/química , Proteoma/metabolismo , Envelhecimento/fisiologia , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Homeostase , Espectrometria de Massas , Chaperonas Moleculares/metabolismo , Agregados Proteicos/fisiologia , Dobramento de Proteína , SolubilidadeRESUMO
Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Anticorpos/imunologia , Agregados Proteicos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Animais , Anticorpos/química , Anticorpos/metabolismo , Especificidade de Anticorpos , Caenorhabditis elegans , Modelos Animais de Doenças , Epitopos , Hipocampo/metabolismo , Camundongos , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio ÚnicoRESUMO
A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. Here, we analysed toxic oligomers of α-synuclein and its pathological variants in order to identify structural features that could be related to toxicity and found a novel structural polymorphism within G51D oligomers. These G51D oligomers can adopt a variety of ß-sheet-rich structures with differing degrees of α-helical content, and the helical structural content of these oligomers correlates with the level of induced cellular dysfunction in SH-SY5Y cells. This structure-function relationship observed in α-synuclein oligomers thus presents the α-helical structure as another potential structural determinant that may be linked with cellular toxicity in amyloid-related proteins.
Assuntos
Mutação , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Multimerização Proteica , alfa-Sinucleína/química , alfa-Sinucleína/genética , Humanos , Doenças Neurodegenerativas , Agregados Proteicos , Ligação Proteica , Multimerização Proteica/genética , Análise Espectral , alfa-Sinucleína/metabolismoRESUMO
Increasing evidence indicates that peripheral immune cells play a prominent role in neurodegeneration connected to protein misfolding, which are associated with formation of aberrant aggregates, including soluble protein misfolded oligomers. The precise links, however, between the physicochemical features of diverse oligomers and their effects on the immune system, particularly on adaptive immunity, remain currently unexplored, due partly to the transient and heterogeneous nature of the oligomers themselves. To overcome these limitations, we took advantage of two stable and well-characterized types of model oligomers (A and B), formed by HypF-N bacterial protein, type B oligomers displaying lower solvent-exposed hydrophobicity. Exposure to oligomers of human peripheral blood mononuclear cells (PBMCs) revealed differential effects, with type B, but not type A, oligomers leading to a reduction in CD4+ cells. Type A oligomers promoted enhanced differentiation towards CD4+ CD25High FoxP3+ Tregs and displayed a higher suppressive effect on lymphocyte proliferation than Tregs treated with oligomers B or untreated cells. Moreover, our results reveal Th1 and Th17 lymphocyte differentiation mediated by type A oligomers and a differential balance of TGF-ß, IL-6, IL-23, IFN-γ and IL-10 mediators. These results indicate that type B oligomers recapitulate some of the biological responses associated with Parkinson's disease in peripheral immunocompetent cells, while type A oligomers resemble responses associated with Alzheimer's disease. We anticipate that further studies characterizing the differential effects of protein misfolded oligomers on the peripheral immune system may lead to the development of blood-based diagnostics, which could report on the type and properties of oligomers present in patients.
Assuntos
Leucócitos Mononucleares/metabolismo , Deficiências na Proteostase/metabolismo , Adulto , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Ativação Linfocitária/fisiologia , Pessoa de Meia-Idade , Dobramento de Proteína , Linfócitos T Reguladores/metabolismo , Células Th1/metabolismo , Células Th17/metabolismoRESUMO
The aggregation of the 42-residue form of the amyloid-ß peptide (Aß42) is a pivotal event in Alzheimer's disease (AD). The use of chemical kinetics has recently enabled highly accurate quantifications of the effects of small molecules on specific microscopic steps in Aß42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Aß42 aggregation that uses as a read-out the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Aß42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery.
Assuntos
Peptídeos beta-Amiloides/química , Descoberta de Drogas , Fragmentos de Peptídeos/química , Doença de Alzheimer , Peptídeos beta-Amiloides/metabolismo , Animais , Caenorhabditis elegans , Líquido Cefalorraquidiano/química , Humanos , Fragmentos de Peptídeos/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
The aberrant aggregation of proteins is implicated in the onset and pathogenesis of a wide range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Mounting evidence indicates that misfolded protein oligomers produced as intermediates in the aggregation process are potent neurotoxic agents in these diseases. Because of the transient and heterogeneous nature of these elusive aggregates, however, it has proven challenging to develop therapeutics that can effectively target them. Here, we review approaches aimed at reducing oligomer toxicity, including (1) modulating the oligomer populations (e.g., by altering the kinetics of aggregation by inhibiting, enhancing, or redirecting the process), (2) modulating the oligomer properties (e.g., through the size-hydrophobicity-toxicity relationship), (3) modulating the oligomer interactions (e.g., by protecting cell membranes by displacing oligomers), and (4) reducing oligomer toxicity by potentiating the protein homeostasis system. We analyze examples of these complementary approaches, which may lead to the development of compounds capable of preventing or treating neurodegenerative disorders associated with protein aggregation.
Assuntos
Agregação Patológica de Proteínas/terapia , Multimerização Proteica , Deficiências na Proteostase/terapia , Animais , Humanos , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologiaRESUMO
Alzheimer's disease is associated with the aggregation of the amyloid-ß peptide (Aß), resulting in the deposition of amyloid plaques in brain tissue. Recent scrutiny of the mechanisms by which Aß aggregates induce neuronal dysfunction has highlighted the importance of the Aß oligomers of this protein fragment. Because of the transient and heterogeneous nature of these oligomers, however, it has been challenging to investigate the detailed mechanisms by which these species exert cytotoxicity. To address this problem, we demonstrate here the use of rationally designed single-domain antibodies (DesAbs) to characterize the structure-toxicity relationship of Aß oligomers. For this purpose, we use Zn2+-stabilized oligomers of the 40-residue form of Aß (Aß40) as models of brain Aß oligomers and two single-domain antibodies (DesAb18-24 and DesAb34-40), designed to bind to epitopes at residues 18-24 and 34-40 of Aß40, respectively. We found that the DesAbs induce a change in structure of the Zn2+-stabilized Aß40 oligomers, generating a simultaneous increase in their size and solvent-exposed hydrophobicity. We then observed that these increments in both the size and hydrophobicity of the oligomers neutralize each other in terms of their effects on cytotoxicity, as predicted by a recently proposed general structure-toxicity relationship, and observed experimentally. These results illustrate the use of the DesAbs as research tools to investigate the biophysical and cytotoxicity properties of Aß oligomers.
Assuntos
Peptídeos beta-Amiloides/imunologia , Anticorpos/imunologia , Anticorpos/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Formação de Anticorpos/imunologia , Encéfalo/metabolismo , Desenho de Fármacos , Humanos , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/metabolismo , Agregados Proteicos/fisiologia , Engenharia de Proteínas/métodos , Relação Estrutura-AtividadeRESUMO
The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Adesão Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Multimerização Proteica , Animais , Proteínas de Bactérias/toxicidade , Células CHO , Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cricetulus , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Especificidade por SubstratoRESUMO
Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 µm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules.
Assuntos
Carboxil e Carbamoil Transferases/química , Clusterina/química , Proteínas de Escherichia coli/química , Cadeia B de alfa-Cristalina/química , Animais , Carboxil e Carbamoil Transferases/metabolismo , Linhagem Celular Tumoral , Clusterina/genética , Clusterina/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Camundongos , Pré-Albumina/química , Pré-Albumina/genética , Pré-Albumina/metabolismo , Agregados Proteicos , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismoRESUMO
Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species.
Assuntos
Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Multimerização Proteica , Linhagem Celular Tumoral , Humanos , Chaperonas Moleculares/genéticaRESUMO
Although human transthyretin (TTR) is associated with systemic amyloidoses, an anti-amyloidogenic effect that prevents Aß fibril formation in vitro and in animal models has been observed. Here we studied the ability of three different types of TTR, namely human tetramers (hTTR), mouse tetramers (muTTR) and an engineered monomer of the human protein (M-TTR), to suppress the toxicity of oligomers formed by two different amyloidogenic peptides/proteins (HypF-N and Aß42). muTTR is the most stable homotetramer, hTTR can dissociate into partially unfolded monomers, whereas M-TTR maintains a monomeric state. Preformed toxic HypF-N and Aß42 oligomers were incubated in the presence of each TTR then added to cell culture media. hTTR, and to a greater extent M-TTR, were found to protect human neuroblastoma cells and rat primary neurons against oligomer-induced toxicity, whereas muTTR had no protective effect. The thioflavin T assay and site-directed labeling experiments using pyrene ruled out disaggregation and structural reorganization within the discrete oligomers following incubation with TTRs, while confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and hTTR, particularly M-TTR. Moreover, atomic force microscopy (AFM), light scattering and turbidimetry analyses indicated that larger assemblies of oligomers are formed in the presence of M-TTR and, to a lesser extent, with hTTR. Overall, the data suggest a generic capacity of TTR to efficiently neutralize the toxicity of oligomers formed by misfolded proteins and reveal that such neutralization occurs through a mechanism of TTR-mediated assembly of protein oligomers into larger species, with an efficiency that correlates inversely with TTR tetramer stability.
Assuntos
Peptídeos beta-Amiloides/efeitos adversos , Proteínas Amiloidogênicas/efeitos adversos , Carboxil e Carbamoil Transferases/efeitos adversos , Proteínas de Escherichia coli/efeitos adversos , Neuroblastoma/tratamento farmacológico , Neurônios/efeitos dos fármacos , Pré-Albumina/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cultivadas , Humanos , Técnicas In Vitro , Camundongos , Microscopia de Força Atômica , Modelos Moleculares , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/metabolismo , Neurônios/patologia , Conformação Proteica , Multimerização Proteica , RatosRESUMO
Aß oligomers are being investigated as cytotoxic agents in Alzheimer's disease (AD). Because of their transient nature and conformational heterogeneity, the relationship between the structure and activity of these oligomers is still poorly understood. Hence, methods for stabilizing Aß oligomeric species relevant to AD are needed to uncover the structural determinants of their cytotoxicity. Here, we build on the observation that metal ions and metabolites have been shown to interact with Aß, influencing its aggregation and stabilizing its oligomeric species. We thus developed a method that uses zinc ions, Zn(II), to stabilize oligomers produced by the 42-residue form of Aß (Aß42), which is dysregulated in AD. These Aß42-Zn(II) oligomers are small in size, spanning the 10-30 nm range, stable at physiological temperature, and with a broad toxic profile in human neuroblastoma cells. These oligomers offer a tool to study the mechanisms of toxicity of Aß oligomers in cellular and animal AD models.
Assuntos
Peptídeos beta-Amiloides , Fragmentos de Peptídeos , Zinco , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Humanos , Zinco/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Linhagem Celular Tumoral , Doença de Alzheimer/metabolismo , Sobrevivência Celular/efeitos dos fármacosRESUMO
Oligomeric assemblies of the amyloid ß peptide (Aß) have been investigated for over two decades as possible neurotoxic agents in Alzheimer's disease. However, due to their heterogeneous and transient nature, it is not yet fully established which of the structural features of these oligomers may generate cellular damage. Here, we study distinct oligomer species formed by Aß40 (the 40-residue form of Aß) in the presence of four different metal ions (Al3+, Cu2+, Fe2+, and Zn2+) and show that they differ in their structure and toxicity in human neuroblastoma cells. We then describe a correlation between the size of the oligomers and their neurotoxic activity, which provides a type of structure-toxicity relationship for these Aß40 oligomer species. These results provide insight into the possible role of metal ions in Alzheimer's disease by the stabilization of Aß oligomers.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/química , Metais , Íons , Fragmentos de Peptídeos/químicaRESUMO
The aberrant assembly of peptides and proteins into fibrillar aggregates proceeds through oligomeric intermediates that are thought to be the primary pathogenic species in many protein deposition diseases. We describe two types of oligomers formed by the HypF-N protein that are morphologically and tinctorially similar, as detected with atomic force microscopy and thioflavin T assays, though one is benign when added to cell cultures whereas the other is toxic. Structural investigation at a residue-specific level using site-directed labeling with pyrene indicated differences in the packing of the hydrophobic interactions between adjacent protein molecules in the oligomers. The lower degree of hydrophobic packing was found to correlate with a higher ability to penetrate the cell membrane and cause an influx of Ca(2+) ions. Our findings suggest that structural flexibility and hydrophobic exposure are primary determinants of the ability of oligomeric assemblies to cause cellular dysfunction and its consequences, such as neurodegeneration.
Assuntos
Carboxil e Carbamoil Transferases/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Multimerização Proteica , Carboxil e Carbamoil Transferases/metabolismo , Carboxil e Carbamoil Transferases/ultraestrutura , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Ligação Proteica , Conformação ProteicaRESUMO
Peptides and proteins can convert from their soluble forms into highly ordered fibrillar aggregates, giving rise to pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. It is increasingly recognized that protein oligomers forming early in the process of fibril aggregation represent the pathogenic species in protein deposition diseases. The N-terminal domain of the HypF protein from Escherichia coli (HypF-N) has previously been shown to form, under distinct conditions, two types of HypF-N oligomers with indistinguishable morphologies but distinct structural features at the molecular level. Only the oligomer type exposing hydrophobic surfaces and possessing sufficient structural plasticity is toxic (type A), whereas the other type is benign to cultured cells (type B). Here we show that only type A oligomers are able to induce a Ca(2+) influx from the cell medium to the cytosol, to penetrate the plasma membrane, to increase intracellular reactive oxygen species production, lipid peroxidation and release of intracellular calcein, resulting in the activation of the apoptotic pathway. Remarkably, these oligomers can also induce a loss of cholinergic neurons when injected into rat brains. By contrast, markers of cellular stress and viability were unaffected in cultured and rat neuronal cells exposed to type B oligomers. The analysis of the time scales of such effects indicates that the difference of toxicity between the two oligomer types involve the early events of the toxicity cascade, shedding new light on the mechanism of action of protein oligomers and on the molecular targets for the therapeutic intervention against protein deposition diseases.
Assuntos
Cálcio/metabolismo , Carboxil e Carbamoil Transferases/química , Carboxil e Carbamoil Transferases/farmacologia , Neurônios Colinérgicos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Neurônios Colinérgicos/química , Modelos Animais de Doenças , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Terapia de Alvo Molecular , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Age-related changes in cellular metabolism can affect brain homeostasis, creating conditions that are permissive to the onset and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Although the roles of metabolites have been extensively studied with regard to cellular signaling pathways, their effects on protein aggregation remain relatively unexplored. By computationally analysing the Human Metabolome Database, we identified two endogenous metabolites, carnosine and kynurenic acid, that inhibit the aggregation of the amyloid beta peptide (Aß) and rescue a C. elegans model of Alzheimer's disease. We found that these metabolites act by triggering a cytosolic unfolded protein response through the transcription factor HSF-1 and downstream chaperones HSP40/J-proteins DNJ-12 and DNJ-19. These results help rationalise previous observations regarding the possible anti-ageing benefits of these metabolites by providing a mechanism for their action. Taken together, our findings provide a link between metabolite homeostasis and protein homeostasis, which could inspire preventative interventions against neurodegenerative disorders.
Assuntos
Doença de Alzheimer/metabolismo , Caenorhabditis elegans/metabolismo , Carnosina/metabolismo , Modelos Animais de Doenças , Ácido Cinurênico/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Carnosina/farmacologia , Citosol/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Ácido Cinurênico/farmacologia , Agregados Proteicos , Agregação Patológica de Proteínas/prevenção & controle , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Misfolded protein oligomers are increasingly recognized as highly cytotoxic agents in a wide range of human disorders associated with protein aggregation. In this study, we assessed the possible uptake and resulting toxic effects of model protein oligomers administered to C. elegans through the culture medium. We used an automated machine-vision, high-throughput screening procedure to monitor the phenotypic changes in the worms, in combination with confocal microscopy to monitor the diffusion of the oligomers, and oxidative stress assays to detect their toxic effects. Our results suggest that the oligomers can diffuse from the intestinal lumen to other tissues, resulting in a disease phenotype. We also observed that pre-incubation of the oligomers with a molecular chaperone (αB-crystallin) or a small molecule inhibitor of protein aggregation (squalamine), reduced the oligomer absorption. These results indicate that exogenous misfolded protein oligomers can be taken up by the worms from their environment and spread across tissues, giving rise to pathological effects in regions distant from their place of absorbance.
Assuntos
Caenorhabditis elegans , Intestinos , Animais , Ensaios de Triagem em Larga Escala , FenótipoRESUMO
Alzheimer's disease, which is the most common form of dementia, is characterized by the aggregation of the amyloid ß peptide (Aß) and by an impairment of calcium homeostasis caused by excessive activation of glutamatergic receptors (excitotoxicity). Here, we studied the effects on calcium homeostasis caused by the formation of Aß oligomeric assemblies. We found that Aß oligomers cause a rapid influx of calcium ions (Ca2+) across the cell membrane by rapidly activating extrasynaptic N-methyl-d-aspartate (NMDA) receptors and, to a lower extent, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. We also observed, however, that misfolded oligomers do not interact directly with these receptors. Further experiments with lysophosphatidylcholine and arachidonic acid, which cause membrane compression and stretch, respectively, indicated that these receptors are activated through a change in membrane tension induced by the oligomers and transmitted mechanically to the receptors via the lipid bilayer. Indeed, lysophosphatidylcholine is able to neutralize the oligomer-induced activation of the NMDA receptors, whereas arachidonic acid activates the receptors similarly to the oligomers with no additive effects. An increased rotational freedom observed for a fluorescent probe embedded within the membrane in the presence of the oligomers also indicates a membrane stretch. These results reveal a mechanism of toxicity of Aß oligomers in Alzheimer's disease through the perturbation of the mechanical properties of lipid membranes sensed by NMDA and AMPA receptors.
Assuntos
Doença de Alzheimer , Receptores de N-Metil-D-Aspartato , Peptídeos beta-Amiloides/metabolismo , Cálcio/metabolismo , Homeostase , Humanos , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiônicoRESUMO
The aggregation of the human islet amyloid polypeptide (IAPP) is associated with diabetes type II. A quantitative understanding of this connection at the molecular level requires that the aggregation mechanism of IAPP is resolved in terms of the underlying microscopic steps. Here we have systematically studied recombinant IAPP, with amidated C-terminus in oxidised form with a disulphide bond between residues 3 and 7, using thioflavin T fluorescence to monitor the formation of amyloid fibrils as a function of time and IAPP concentration. We used global kinetic analyses to connect the macroscopic measurements of aggregation to the microscopic mechanisms, and show that the generation of new aggregates is dominated by the secondary nucleation of monomers on the fibril surface. We then exposed insulinoma cells to aliquots extracted from different time points of the aggregation process, finding the highest toxicity at the midpoint of the reaction, when the secondary nucleation rate reaches its maximum. These results identify IAPP oligomers as the most cytotoxic species generated during IAPP aggregation, and suggest that compounds that target secondary nucleation of IAPP could be most effective as therapeutic candidates for diabetes type II.