RESUMO
Amyloid-beta aggregation is considered one of the factors influencing the onset of the Alzheimer's disease. Early prevention of such aggregation should alleviate disease condition by applying small molecule compounds that shift the aggregation equilibrium toward the soluble form of the peptide or slow down the process. We have discovered that fluorinated benzenesulfonamides of particular structure slowed the amyloid-beta peptide aggregation process by more than three-fold. We synthesized a series of ortho-para and meta-para double-substituted fluorinated benzenesulfonamides that inhibited the aggregation process to a variable extent yielding a detailed picture of the structure-activity relationship. Analysis of compound chemical structure effect on aggregation in artificial cerebrospinal fluid showed the necessity to arrange the benzenesulfonamide, hydrophobic substituent, and benzoic acid in a particular way. The amyloid beta peptide aggregate fibril structures varied in cross-sectional height depending on the applied inhibitor indicating the formation of a complex with the compound. Application of selected inhibitors increased the survivability of cells affected by the amyloid beta peptide. Such compounds may be developed as drugs against Alzheimer's disease.
RESUMO
Protein aggregation in the form of amyloid fibrils has long been associated with the onset and development of various amyloidoses, including Alzheimer's, Parkinson's or prion diseases. Recent studies of their fibril formation process have revealed that amyloidogenic protein cross-interactions may impact aggregation pathways and kinetic parameters, as well as the structure of the resulting aggregates. Despite a growing number of reports exploring this type of interaction, they only cover just a small number of possible amyloidogenic protein pairings. One such pair is between two neurodegeneration-associated proteins: the pro-inflammatory S100A9 and prion protein, which are known to co-localize in vivo. In this study, we examined their cross-interaction in vitro and discovered that the fibrillar form of S100A9 modulated the aggregation pathway of mouse prion protein 89-230 fragment, while non-aggregated S100A9 also significantly inhibited its primary nucleation process. These results complement previous observations of the pro-inflammatory protein's role in amyloid aggregation and highlight its potential role against neurodegenerative disorders.
Assuntos
Amiloide , Calgranulina B , Proteínas Priônicas , Agregados Proteicos , Calgranulina B/metabolismo , Calgranulina B/química , Animais , Camundongos , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Amiloide/metabolismo , Amiloide/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/química , CinéticaRESUMO
Increasing evidence suggests that the calcium-binding and proinflammatory protein S100A9 is an important player in neuroinflammation-mediated Alzheimer's disease (AD). The amyloid co-aggregation of S100A9 with amyloid-ß (Aß) is an important hallmark of this pathology. Apolipoprotein E (ApoE) is also known to be one of the important genetic risk factors of AD. ApoE primarily exists in three isoforms, ApoE2 (Cys112/Cys158), ApoE3 (Cys112/Arg158), and ApoE4 (Arg112/Arg158). Even though the difference lies in just two amino acid residues, ApoE isoforms produce differential effects on the neuroinflammation and activation of the microglial state in AD. Here, we aim to understand the effect of the ApoE isoforms on the amyloid aggregation of S100A9. We found that both ApoE3 and ApoE4 suppress the aggregation of S100A9 in a concentration-dependent manner, even at sub-stoichiometric ratios compared to S100A9. These interactions lead to a reduction in the quantity and length of S100A9 fibrils. The inhibitory effect is more pronounced if ApoE isoforms are added in the lipid-free state versus lipidated ApoE. We found that, upon prolonged incubation, S100A9 and ApoE form low molecular weight complexes with stochiometric ratios of 1:1 and 2:1, which remain stable under SDS-gel conditions. These complexes self-assemble also under the native conditions; however, their interactions are transient, as revealed by glutaraldehyde cross-linking experiments and molecular dynamics (MD) simulation. MD simulation demonstrated that the lipid-binding C-terminal domain of ApoE and the second EF-hand calcium-binding motif of S100A9 are involved in these interactions. We found that amyloids of S100A9 are cytotoxic to neuroblastoma cells, and the presence of either ApoE isoforms does not change the level of their cytotoxicity. A significant inhibitory effect produced by both ApoE isoforms on S100A9 amyloid aggregation can modulate the amyloid-neuroinflammatory cascade in AD.
Assuntos
Doença de Alzheimer , Apolipoproteína E4 , Apolipoproteínas E , Calgranulina B , Agregados Proteicos , Humanos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide , Peptídeos beta-Amiloides/metabolismo , Apolipoproteína E3 , Apolipoproteína E4/genética , Apolipoproteínas E/genética , Doenças Neuroinflamatórias , Isoformas de Proteínas/metabolismo , Calgranulina B/metabolismoRESUMO
The microtubule-associated protein tau forms disease-specific filamentous aggregates in several different neurodegenerative diseases. In order to understand how tau undergoes misfolding into a specific filament type and to control this process for drug development purposes, it is crucial to study in vitro tau aggregation methods and investigate the structures of the obtained filaments at the atomic level. Here, we used the tau fragment dGAE, which aggregates spontaneously, to seed the formation of full-length tau filaments. The structures of dGAE and full-length tau filaments were investigated by magic-angle spinning (MAS) solid-state NMR, showing that dGAE allows propagation of a chronic traumatic encephalopathy (CTE)-like fold to the full-length tau. The obtained filaments efficiently seeded tau aggregation in HEK293T cells. This work demonstrates that in vitro preparation of disease-specific types of full-length tau filaments is feasible.
RESUMO
Protein aggregation into amyloid fibrils is associated with several amyloidoses, including neurodegenerative Alzheimer's and Parkinson's diseases. Despite years of research and numerous studies, the process is still not fully understood, which significantly impedes the search for cures of amyloid-related disorders. Recently, there has been an increase in reports of amyloidogenic protein cross-interactions during the fibril formation process, which further complicates the already intricate process of amyloid aggregation. One of these reports displayed an interaction involving Tau and prion proteins, which prompted a need for further investigation into the matter. In this work, we generated five populations of conformationally distinct prion protein amyloid fibrils and examined their interaction with Tau proteins. We observed that there was a conformation-specific association between Tau monomers and prion protein fibrils, which increased the aggregate self-association and amyloidophilic dye binding capacity. We also determined that the interaction did not induce the formation of Tau protein amyloid aggregates, but rather caused their electrostatic adsorption to the prion protein fibril surface.
Assuntos
Amiloidose , Príons , Humanos , Amiloide/metabolismo , Proteínas Priônicas/metabolismo , Proteínas tau/metabolismo , Amiloidose/metabolismo , Proteínas Amiloidogênicas , Agregados ProteicosRESUMO
The main pathological hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-ß into amyloid fibrils, leading to a neurodegeneration cascade. The current medications are far from sufficient to prevent the onset of the disease, hence requiring more research to find new alternative drugs for curing AD. In vitro inhibition experiments are one of the primary tools in testing whether a molecule may be potent to impede the aggregation of amyloid-beta peptide (Aß42). However, kinetic experiments in vitro do not match the mechanism found when aggregating Aß42 in cerebrospinal fluid. The different aggregation mechanisms and the composition of the reaction mixtures may also impact the characteristics of the inhibitor molecules. For this reason, altering the reaction mixture to resemble components found in cerebrospinal fluid (CSF) is critical to partially compensate for the mismatch between the inhibition experiments in vivo and in vitro. In this study, we used an artificial cerebrospinal fluid that contained the major components found in CSF and performed Aß42 aggregation inhibition studies using oxidized epigallocatechin-3-gallate (EGCG) and fluorinated benzenesulfonamide VR16-09. This led to a discovery of a complete turnaround of their inhibitory characteristics, rendering EGCG ineffective while significantly improving the efficacy of VR16-09. HSA was the main contributor in the mixture that significantly increased the anti-amyloid characteristics of VR16-09.
Assuntos
Doença de Alzheimer , Catequina , Humanos , Fragmentos de Peptídeos/química , Peptídeos beta-Amiloides/química , Doença de Alzheimer/patologia , Amiloide , Catequina/químicaRESUMO
The calcium-binding protein S100A9 is recognized as an important component of the brain neuroinflammatory response to the onset and development of neurodegenerative disease. S100A9 is intrinsically amyloidogenic and in vivo co-aggregates with amyloid-ß peptide and α-synuclein in Alzheimer's and Parkinson's diseases, respectively. It is widely accepted that calcium dyshomeostasis plays an important role in the onset and development of these diseases, and studies have shown that elevated levels of calcium limit the potential for S100A9 to adopt a fibrillar structure. The exact mechanism by which calcium exerts its influence on the aggregation process remains unclear. Here we demonstrate that despite S100A9 exhibiting α-helical secondary structure in the absence of calcium, the protein exhibits significant plasticity with interconversion between different conformational states occurring on the micro- to milli-second timescale. This plasticity allows the population of conformational states that favour the onset of fibril formation. Magic-angle spinning solid-state NMR studies of the resulting S100A9 fibrils reveal that the S100A9 adopts a single structurally well-defined rigid fibrillar core surrounded by a shell of approximately 15-20 mobile residues, a structure that persists even when fibrils are produced in the presence of calcium ions. These studies highlight how the dysregulation of metal ion concentrations can influence the conformational equilibria of this important neuroinflammatory protein to influence the rate and nature of the amyloid deposits formed.
Assuntos
Cálcio , Doenças Neurodegenerativas , Humanos , Amiloide , Ressonância Magnética Nuclear Biomolecular , Cálcio da Dieta , Calgranulina BRESUMO
The assembly of amyloidogenic proteins into highly-structured fibrillar aggregates is related to the onset and progression of several amyloidoses, including neurodegenerative Alzheimer's or Parkinson's diseases. Despite years of research and a general understanding of the process of such aggregate formation, there are currently still very few drugs and treatment modalities available. One of the factors that is relatively insufficiently understood is the cross-interaction between different amyloid-forming proteins. In recent years, it has been shown that several of these proteins or their aggregates can alter each other's fibrillization properties, however, there are still many unknowns in the amyloid interactome. In this work, we examine the interaction between amyloid disease-related prion protein and superoxide dismutase-1. We show that not only does superoxide dismutase-1 increase the lag time of prion protein fibril formation, but it also changes the conformation of the resulting aggregates.
Assuntos
Fragmentos de Peptídeos/metabolismo , Proteínas Priônicas/metabolismo , Agregados Proteicos/efeitos dos fármacos , Superóxido Dismutase-1/metabolismo , Animais , Ligação de Hidrogênio , Camundongos , Fragmentos de Peptídeos/química , Proteínas Priônicas/química , Conformação Proteica em Folha beta/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacosRESUMO
Amyloid fibril formation is associated with several amyloidoses, including neurodegenerative Alzheimer's or Parkinson's diseases. The process of such fibrillar structure formation is still not fully understood, with new mechanistic insights appearing on a regular basis. This, in turn, has limited the development of potential anti-amyloid compounds, with only a handful of effective cures or treatment modalities available. One of the multiple amyloid aggregation factors that requires further examination is the ability of proteins to form multiple, structurally distinct aggregates, based on the environmental conditions. In this work, we examine how the initial folding state affects the fibrilization of lysozyme-an amyloidogenic protein, often used in protein aggregation studies. We show that there is a correlation between the initial state of the protein and the aggregate formation lag time, rate of elongation, resulting aggregate structural variability and dye-binding properties, as well as formation lag time and rate of elongation.
Assuntos
Amiloidose , Fármacos Dermatológicos , Amiloide/metabolismo , Proteínas Amiloidogênicas , Antivirais , Humanos , Muramidase/química , Agregados Proteicos , Dobramento de ProteínaRESUMO
S100A9 is a pro-inflammatory protein that co-aggregates with other proteins in amyloid fibril plaques. S100A9 can influence the aggregation kinetics and amyloid fibril structure of alpha-synuclein (α-syn), which is involved in Parkinson's disease. Currently, there are limited data regarding their cross-interaction and how it influences the aggregation process. In this work, we analyzed this interaction using solution 19F and 2D 15N-1H HSQC NMR spectroscopy and studied the aggregation properties of these two proteins. Here, we show that α-syn interacts with S100A9 at specific regions, which are also essential in the first step of aggregation. We also demonstrate that the 4-fluorophenylalanine label in alpha-synuclein is a sensitive probe to study interaction and aggregation using 19F NMR spectroscopy.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Amiloide/metabolismo , Calgranulina B , Humanos , Espectroscopia de Ressonância Magnética/métodos , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Protein aggregate formation is linked with multiple amyloidoses, including Alzheimer's and Parkinson's diseases. Currently, the understanding of such fibrillar structure formation and propagation is still not sufficient, the outcome of which is a lack of potent, anti-amyloid drugs. The environmental conditions used during in vitro protein aggregation assays play an important role in determining both the aggregation kinetic parameters, as well as resulting fibril structure. In the case of alpha-synuclein, ionic strength has been shown as a crucial factor in its amyloid aggregation. In this work, we examine a large sample size of alpha-synuclein aggregation reactions under thirty different ionic strength and protein concentration combinations and determine the resulting fibril structural variations using their dye-binding properties, secondary structure and morphology. We show that both ionic strength and protein concentration determine the structural variability of alpha-synuclein amyloid fibrils and that sometimes even identical conditions can result in up to four distinct types of aggregates.
Assuntos
Amiloide/química , Agregados Proteicos , Agregação Patológica de Proteínas , alfa-Sinucleína/química , Amiloide/metabolismo , Técnicas In Vitro/métodos , Cinética , Concentração Osmolar , Doença de Parkinson/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , alfa-Sinucleína/metabolismoRESUMO
Protein aggregation into amyloid fibrils is linked to multiple disorders. The understanding of how natively non-harmful proteins convert to these highly cytotoxic amyloid aggregates is still not sufficient, with new ideas and hypotheses being presented each year. Recently it has been shown that more than one type of protein aggregates may co-exist in the affected tissue of patients suffering from amyloid-related disorders, sparking the idea that amyloid aggregates formed by one protein may induce another protein's fibrillization. In this work, we examine the effect that lysozyme fibrils have on insulin amyloid aggregation. We show that not only do lysozyme fibrils affect insulin nucleation, but they also alter the mechanism of its aggregation.
Assuntos
Amiloide/metabolismo , Insulina/metabolismo , Muramidase/metabolismo , Agregação Patológica de Proteínas/metabolismo , Amiloide/ultraestrutura , Animais , Galinhas , Humanos , Agregados Proteicos , Proteínas Recombinantes/metabolismoRESUMO
Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases-a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability may depend on the state of the initial prion protein molecules.
Assuntos
Amiloide/química , Proteínas Priônicas/química , Multimerização Proteica , Temperatura , Conformação ProteicaRESUMO
The formation of amyloid fibril plaques in the brain creates inflammation and neuron death. This process is observed in neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Alpha-synuclein is the main protein found in neuronal inclusions of patients who have suffered from Parkinson's disease. S100A9 is a calcium-binding, pro-inflammation protein, which is also found in such amyloid plaques. To understand the influence of S100A9 on the aggregation of α-synuclein, we analyzed their co-aggregation kinetics and the resulting amyloid fibril structure by Fourier-transform infrared spectroscopy and atomic force microscopy. We found that the presence of S100A9 alters the aggregation kinetics of α-synuclein and stabilizes the formation of a particular amyloid fibril structure. We also show that the solution's ionic strength influences the interplay between S100A9 and α-synuclein, stabilizing a different structure of α-synuclein fibrils.
Assuntos
Amiloide/química , Calgranulina B/química , Agregados Proteicos , alfa-Sinucleína/química , Humanos , Proteínas Recombinantes/químicaRESUMO
A central characteristic of Alzheimer's disease (AD) and other tauopathies is the accumulation of aggregated and misfolded Tau deposits in the brain. Tau-targeting therapies for AD have been unsuccessful in patients to date. Here we show that human polymerase δ-interacting protein 2 (PolDIP2) interacts with Tau. With a set of complementary methods, including thioflavin-T-based aggregation kinetic assays, Tau oligomer-specific dot-blot analysis, and single oligomer/fibril analysis by atomic force microscopy, we demonstrate that PolDIP2 inhibits Tau aggregation and amyloid fibril growth in vitro. The identification of PolDIP2 as a potential regulator of cellular Tau aggregation should be considered for future Tau-targeting therapeutics.
Assuntos
Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Proteínas Nucleares/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Benzotiazóis , Encéfalo/metabolismo , Humanos , Proteínas Nucleares/genética , TauopatiasRESUMO
Prion diseases are associated with conformational conversion of cellular prion protein into a misfolded pathogenic form, which resembles many properties of amyloid fibrils. The same prion protein sequence can misfold into different conformations, which are responsible for variations in prion disease phenotypes (prion strains). In this work, we use atomic force microscopy, FTIR spectroscopy and magic-angle spinning NMR to devise structural models of mouse prion protein fibrils prepared in three different denaturing conditions. We find that the fibril core region as well as the structure of its N- and C-terminal parts is almost identical between the three fibrils. In contrast, the central part differs in length of ß-strands and the arrangement of charged residues. We propose that the denaturant ionic strength plays a major role in determining the structure of fibrils obtained in a particular condition by stabilizing fibril core interior-facing glutamic acid residues.
Assuntos
Amiloide/metabolismo , Doenças Priônicas/metabolismo , Proteínas Priônicas/metabolismo , Agregação Patológica de Proteínas/metabolismo , Sequência de Aminoácidos , Amiloide/química , Animais , Isótopos de Carbono/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Microscopia de Força Atômica/métodos , Isótopos de Nitrogênio/metabolismo , Proteínas Priônicas/química , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Relação Estrutura-AtividadeRESUMO
The amyloid cascade is central for the neurodegeneration disease pathology, including Alzheimer's and Parkinson's, and remains the focus of much current research. S100A9 protein drives the amyloid-neuroinflammatory cascade in these diseases. DOPA and cyclen-based compounds were used as amyloid modifiers and inhibitors previously, and DOPA is also used as a precursor of dopamine in Parkinson's treatment. Here, by using fluorescence titration experiments we showed that five selected ligands: DOPA-D-H-DOPA, DOPA-H-H-DOPA, DOPA-D-H, DOPA-cyclen, and H-E-cyclen, bind to S100A9 with apparent Kd in the sub-micromolar range. Ligand docking and molecular dynamic simulation showed that all compounds bind to S100A9 in more than one binding site and with different ligand mobility and H-bonds involved in each site, which all together is consistent with the apparent binding determined in fluorescence experiments. By using amyloid kinetic analysis, monitored by thioflavin-T fluorescence, and AFM imaging, we found that S100A9 co-aggregation with these compounds does not hinder amyloid formation but leads to morphological changes in the amyloid fibrils, manifested in fibril thickening. Thicker fibrils were not observed upon fibrillation of S100A9 alone and may influence the amyloid tissue propagation and modulate S100A9 amyloid assembly as part of the amyloid-neuroinflammatory cascade in neurodegenerative diseases.
Assuntos
Amiloide/química , Calgranulina B/química , Di-Hidroxifenilalanina/química , Simulação de Dinâmica Molecular , Agregados Proteicos , HumanosRESUMO
Tau is a microtubule-associated protein, found at high levels in neurons, and its aggregation is associated with neurodegeneration. Recently, it was found that tau can be actively secreted from neurons, but the effects of extracellular tau on neuronal viability are unclear. In this study, we investigated whether extracellular tau2N4R can cause neurotoxicity in primary cultures of rat brain neurons and glial cells. Cell cultures were examined for neuronal loss, death, and phosphatidylserine exposure, as well as for microglial phagocytosis by fluorescence microscopy. Aggregation of tau2N4R was assessed by atomic force microscopy. We found that extracellular addition of tau induced a gradual loss of neurons over 1-2 days, without neuronal necrosis or apoptosis, but accompanied by proliferation of microglia in the neuronal-glial co-cultures. Tau addition caused exposure of the 'eat-me' signal phosphatidylserine on the surface of living neurons, and this was prevented by elimination of the microglia or by inhibition of neutral sphingomyelinase. Tau also increased the phagocytic activity of pure microglia, and this was blocked by inhibitors of neutral sphingomyelinase or protein kinase C. The neuronal loss induced by tau was prevented by inhibitors of neutral sphingomyelinase, protein kinase C or the phagocytic receptor MerTK, or by eliminating microglia from the cultures. The data suggest that extracellular tau induces primary phagocytosis of stressed neurons by activated microglia, and identifies multiple ways in which the neuronal loss induced by tau can be prevented.
Assuntos
Microglia/efeitos dos fármacos , Neurônios , Fagocitose/efeitos dos fármacos , Proteínas tau/farmacologia , Animais , Células Cultivadas , Técnicas de Cocultura , Microglia/metabolismo , Neurônios/patologia , Ratos , Proteínas tau/metabolismoRESUMO
Amyloidogenic protein aggregation into highly structured fibrils is linked to more than 30 amyloidoses, including several neurodegenerative disorders. Despite significant progress in trying to understand the process of amyloid formation, there is still no cure or effective treatment available. A number of studies involving potential anti-amyloid compounds rely on the use of a fluorescent probe-thioflavin-T-to track the appearance, growth, or disassembly of these cytotoxic aggregates. Despite the wide application of this dye molecule, its interaction with amyloid fibrils is still poorly understood. Recent reports have shown it may possess distinct binding modes and fluorescence intensities based on the conformation of the examined fibrils. In this work, we generate insulin fibrils under four different conditions and attempt to identify distinct conformations using both classic methods, such as atomic force microscopy and Fourier-transform infrared spectroscopy, as well as their ThT binding ability and fluorescence quantum yield. We show that there is a significant variance of ThT fluorescence quantum yields, excitation/emission maxima positions, and binding modes between distinct insulin fibril conformations.
Assuntos
Benzotiazóis , Insulina , Amiloide/metabolismo , Corantes Fluorescentes , Insulina/química , Microscopia de Força Atômica , Ligação ProteicaRESUMO
The formation of amyloid fibrils is linked to multiple neurodegenerative disorders, including Alzheimer's and Parkinson's disease. Despite years of research and countless studies on the topic of such aggregate formation, as well as their resulting structure, the current knowledge is still fairly limited. One of the main aspects prohibiting effective aggregation tracking is the environment's effect on amyloid-specific dyes, namely thioflavin-T (ThT). Currently, there are only a few studies hinting at ionic strength being one of the factors that modulate the dye's binding affinity and fluorescence intensity. In this work we explore this effect under a range of ionic strength conditions, using insulin, lysozyme, mouse prion protein, and α-synuclein fibrils. We show that ionic strength is an extremely important factor affecting both the binding affinity, as well as the fluorescence intensity of ThT.