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
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 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
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.
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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.
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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.
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Amiloide/química , Proteínas Priônicas/química , Multimerização Proteica , Temperatura , Conformação ProteicaRESUMO
Among the twelve catalytically active carbonic anhydrase isozymes present in the human body, the CAIX is highly overexpressed in various solid tumors. The enzyme acidifies the tumor microenvironment enabling invasion and metastatic processes. Therefore, many attempts have been made to design chemical compounds that would exhibit high affinity and selective binding to CAIX over the remaining eleven catalytically active CA isozymes to limit undesired side effects. It has been postulated that such drugs may have anticancer properties and could be used in tumor treatment. Here we have designed a series of compounds, methyl 5-sulfamoyl-benzoates, which bear a primary sulfonamide group, a well-known marker of CA inhibitors, and determined their affinities for all twelve CA isozymes. Variations of substituents on the benzenesulfonamide ring led to compound 4b, which exhibited an extremely high observed binding affinity to CAIX; the Kd was 0.12 nM. The intrinsic dissociation constant, where the binding-linked protonation reactions have been subtracted, reached 0.08 pM. The compound also exhibited more than 100-fold selectivity over the remaining CA isozymes. The X-ray crystallographic structure of compound 3b bound to CAIX showed the structural position, while several structures of compounds bound to other CA isozymes showed structural reasons for compound selectivity towards CAIX. Since this series of compounds possess physicochemical properties suitable for drugs, they may be developed for anticancer therapeutic purposes.
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Benzoatos/farmacologia , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Sulfonamidas/farmacologia , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X/métodos , Humanos , Isoenzimas/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Ligação Proteica/fisiologia , Relação Estrutura-Atividade , Termodinâmica , Microambiente Tumoral/efeitos dos fármacos , BenzenossulfonamidasRESUMO
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.
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Benzotiazóis , Insulina , Amiloide/metabolismo , Corantes Fluorescentes , Insulina/química , Microscopia de Força Atômica , Ligação ProteicaRESUMO
OBJECTIVES: The paper presents the results of an initial clinical study, which were obtained using the strain elastography imaging method based on radio frequency ultrasound signal analysis. METHODS: The technique employs endogenous motion of the liver induced by beating heart and vascular pulsatility as an excitation source of tissue microdisplacement. The potential for fibrotic tissue characterization was demonstrated using a clinical data set of radio frequency ultrasound signals (23 healthy controls, 21 subjects with hepatitis, and 16 subjects with liver cirrhosis). Parametric maps, which represent the tissue strain, were derived from the gradient of the integrated spectral coefficient parameter, and correlations with the stage of liver disease were evaluated. Average endogenous strain derived from the gradient of the integrated spectral coefficient parameter and variability (standard deviation) of the strain were evaluated in the rectangular regions of interest (sizes, 1 × 1 and 2 × 2 cm) defined by the observer. The assessment of strain was performed in different frequency subbands of endogenous motion (0-10 Hz and 10-20 Hz). RESULTS: The best distinction between the groups was observed for the average strain derived from the gradient of the integrated spectral coefficient parameter: the controls, 13.30 ± 6.62; hepatitis, 7.12 ± 7.45; cirrhosis, 3.95 ± 2.44 µm/cm (region of interest, 1 × 1 cm; frequency subband 0-10 Hz), and 10.48 ± 6.02, 8.27 ± 5.41, 3.89 ± 2.07 µm/cm, respectively (2 × 2 cm, 0-10 Hz). CONCLUSION: The investigated strain parameters showed statistically significant differences (P < .001) for the different stages of liver fibrosis in most of the cases and proved this method to be feasible.
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Técnicas de Imagem por Elasticidade/métodos , Interpretação de Imagem Assistida por Computador/métodos , Cirrose Hepática/diagnóstico por imagem , Processamento de Sinais Assistido por Computador , Adulto , Estudos de Viabilidade , Feminino , Humanos , Fígado/diagnóstico por imagem , Masculino , Movimento (Física)RESUMO
OBJECTIVES: Transcranial ultrasonography (US) is a relatively new neuroimaging modality proposed for early diagnostics of Parkinson disease (PD). The main limitation of transcranial US image-based diagnostics is a high degree of subjectivity caused by low quality of the transcranial images. The article presents a developed image analysis system and evaluates the potential of automated image analysis on transcranial US. METHODS: The system consists of algorithms for the segmentation and assessment of informative brain regions (midbrain and substantia nigra) and a decision support subsystem, which is equipped with 64 classification algorithms. Transcranial US images of 191 participants (118 patients with a clinical PD diagnosis and 73 healthy control participants) were analyzed. RESULTS: The diagnostic sensitivity and specificity achieved by the proposed system were 85% and 75%, respectively. CONCLUSIONS: Digital transcranial US image analysis is challenging, and the application of a such system as the sole instrument for decisions in clinical practice remains inconclusive. However, the proposed system could be used as a supplementary tool for automated assessment of US parameters for decision support in PD diagnostics and to reduce observer variability.
Assuntos
Sistemas de Apoio a Decisões Clínicas , Processamento de Imagem Assistida por Computador/métodos , Doença de Parkinson/diagnóstico por imagem , Ultrassonografia Doppler Transcraniana/métodos , Encéfalo/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Essential tremor (ET) and Parkinson's disease (PD) are considered distinct disorders. The aim of the study was to look for a link or any distinguishing features by transcranial sonography (TCS), together with the clinical examination findings in a group of patients with overlapping phenotype of ET and PD (ET-PD). METHODS: A prospective observational case-control study was carried out from the 3rd January 2011 until 30th January 2013 at the Hospital of Lithuanian University of Health Sciences. The final study group consisted of 15 patients with ET-PD, 116 patients with ET-only and 141 patients with PD-only. The control group included 101 subjects. Clinical diagnosis was of a diagnostic standard. RESULTS: The main ultrasonographic findings in the ET-PD group were similar to those of the PD-only: hyperechogenicity of the substantia nigra (66.7%, p < 0.001) and nuclei raphe interruptions/absence (38.5%, p < 0.001). The single distinguishing TCS finding in ET-PD group was a lentiform nucleus hyperechogenicity (26.7%), however this was only significant when compared to controls (p = 0.006). An asymmetrical onset of symptoms (73.3%) in ET-PD group was characteristic to PD-only. The ET-PD patients had the longest disease duration (median 6 years, p < 0.001), the most frequent rate of positive family history (53.3%, p = 0.005), rather low prevalence of cogwheel rigidity (26.7%, p < 0.001), and higher mean Hoehn & Yahr scores compared to PD-only (2.6 ± 0.8 vs. 1.8 ± 0.8, p = 0.012). CONCLUSIONS: The main TCS findings of the present study in patients with overlapping ET-PD phenotype were similar to the PD-only group. The highest positive family history rate among ET-PD patients indicates a strong hereditary predisposition and needs genetic underpinnings. Some ET patients, who look like they may be developing co-morbid PD clinically, may have an alternative diagnosis for Parkinsonism, which could be delineated by TCS examination.
Assuntos
Encéfalo/patologia , Tremor Essencial/diagnóstico por imagem , Doença de Parkinson/diagnóstico por imagem , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Ultrassonografia Doppler TranscranianaRESUMO
Protein fibril formation and accumulation are associated with dozens of amyloidoses, including the widespread and yet-incurable Alzheimer's and Parkinson's diseases. Currently, there are still several aspects of amyloid aggregation that are not fully understood, which negatively contributes to the development of disease-altering drugs and treatments. One factor which requires a more in-depth analysis is the effect of the environment on both the initial state of amyloidogenic proteins and their aggregation process and resulting fibril characteristics. In this work, we examine how lysozyme's folding state influences its amyloid formation kinetics and resulting aggregate structural characteristics under several different pH conditions, ranging from acidic to neutral. We demonstrate that both the initial state of the protein and the solution's pH value have a significant combined effect on the variability of the resulting aggregate secondary structures, as well as their stabilities, interactions with amyloid-specific dye molecules, and self-replication properties.
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Amiloide , Dobramento de Proteína , Amiloide/química , Muramidase/química , Estrutura Secundária de Proteína , Concentração de Íons de HidrogênioRESUMO
The 14-3-3 proteins are a highly conserved adaptor protein family with multi-layer functions, abundantly expressed in the brain. The 14-3-3 proteins modulate phosphorylation, regulate enzymatic activity and can act as chaperones. Most importantly, they play an important role in various neurodegenerative disorders due to their vast interaction partners. Particularly, the 14-3-3ζ isoform is known to co-localize in aggregation tangles in both Alzheimer's and Parkinson's diseases as a result of protein-protein interactions. These abnormal clumps consist of amyloid fibrils, insoluble aggregates, mainly formed by the amyloid-ß, tau and α-synuclein proteins. However, the molecular basis of if and how 14-3-3ζ can aggregate into amyloid fibrils is unknown. In this study, we describe the formation of amyloid fibrils by 14-3-3ζ using a comprehensive approach that combines bioinformatic tools, amyloid-specific dye binding, secondary structure analysis and atomic force microscopy. The results presented herein characterize the amyloidogenic properties of 14-3-3ζ and imply that the well-folded protein undergoes aggregation to ß-sheet-rich amyloid fibrils.
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Doenças Neurodegenerativas , Doença de Parkinson , Humanos , Amiloide/química , Proteínas 14-3-3/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Doença de Parkinson/metabolismo , Peptídeos beta-Amiloides/metabolismo , Doenças Neurodegenerativas/metabolismoRESUMO
Protein liquid-liquid phase separation (LLPS) is a rapidly emerging field of study on biomolecular condensate formation. In recent years, this phenomenon has been implicated in the process of amyloid fibril formation, serving as an intermediate step between the native protein transition into their aggregated state. The formation of fibrils via LLPS has been demonstrated for a number of proteins related to neurodegenerative disorders, as well as other amyloidoses. Despite the surge in amyloid-related LLPS studies, the influence of protein condensate formation on the end-point fibril characteristics is still far from fully understood. In this work, we compare alpha-synuclein aggregation under different conditions, which promote or negate its LLPS and examine the differences between the formed aggregates. We show that alpha-synuclein phase separation generates a wide variety of assemblies with distinct secondary structures and morphologies. The LLPS-induced structures also possess higher levels of toxicity to cells, indicating that biomolecular condensate formation may be a critical step in the appearance of disease-related fibril variants.
RESUMO
Amyloid fibrils are protein aggregates formed by protein assembly through cross ß structures. Inhibition of amyloid fibril formation may contribute to therapy against amyloid-related disorders like Parkinson's, Alzheimer's, and type 2 diabetes. Here we report that several fluorinated sulfonamide compounds, previously shown to inhibit human carbonic anhydrase, also inhibit the fibrillation of different proteins. Using a range of spectroscopic, microscopic and chromatographic techniques, we found that the two fluorinated sulfonamide compounds completely inhibit insulin fibrillation over a period of 16 h and moderately suppress α-synuclein and Aß fibrillation. In addition, these compounds decreased cell toxicity of insulin incubated under fibrillation-inducing conditions. We ascribe these effects to their ability to maintain insulin in the native monomeric state. Molecular dynamic simulations suggest that these compounds inhibit insulin self-association by interacting with residues at the dimer interface. This highlights the general anti-aggregative properties of aromatic sulfonamides and suggests that sulfonamide compounds which inhibit carbonic anhydrase activity may have potential as therapeutic agents against amyloid-related disorders.
Assuntos
Anidrases Carbônicas , Diabetes Mellitus Tipo 2 , Humanos , Insulina/química , Amiloide/química , Sulfonamidas/farmacologia , Inibidores da Anidrase Carbônica/farmacologiaRESUMO
BACKGROUND: Hyperechogenicity of the substantia nigra (SN+), detected by transcranial sonography (TCS), was reported as a characteristic finding in Parkinson's disease (PD), with high diagnostic accuracy values, when compared mainly to healthy controls or essential tremor (ET) group. However, some data is accumulating that the SN + could be detected in other neurodegenerative and even in non-neurodegenerative disorders too. Our aim was to estimate the diagnostic accuracy of TCS, mainly focusing on the specificity point, when applied to a range of the parkinsonian disorders, and comparing to the degenerative cognitive syndromes. METHODS: A prospective study was carried out at the Hospital of Lithuanian University of Health Sciences from January until September 2011. Initially, a TCS and clinical examination were performed on 258 patients and 76 controls. The General Electric Voluson 730 Expert ultrasound system was used. There were 12.8% of cases excluded with insufficient temporal bones, and 4.3% excluded with an unclear diagnosis. The studied sample consisted of the groups: PD (n = 71, 33.2%), ET (n = 58, 27.1%), PD and ET (n = 10, 4.7%), atypical parkinsonian syndromes (APS) (n = 3, 1.4%), hereditary neurodegenerative parkinsonism (HDP) (n = 3, 1.4%), secondary parkinsonism (SP) (n = 23, 10.8%), mild cognitive impairment (MCI) (n = 33, 15.4%), dementia (n = 13, 6.1%), and control (n = 71). RESULTS: There were 80.3% of PD patients at stages 1 & 2 according to Hoehn and Yahr. At the cut-off value of 0.20 cm² of the SN+, the sensitivity for PD was 94.3% and the specificity - 63.3% (ROC analysis, AUC 0.891), in comparison to the rest of the cohort. At the cut-off value of 0.26 cm², the sensitivity was 90% and the specificity 82.4%.The estimations for the lowest specificity for PD, in comparison to the latter subgroups (at the cut-off values of 0.20 cm² and 0.26 cm², respectively) were: 0% and 33.3% to APS, 33.3% and 66.7% to HDP, 34.8% and 69.6% to SP, 55.2% and 82.8% to ET, 75% and 91.7% to dementia. CONCLUSIONS: The high sensitivity of the test could be employed as a valuable screening tool. But TCS is more useful as a supplementary diagnostic method, due to the specificity values not being comprehensive.
Assuntos
Disfunção Cognitiva/diagnóstico por imagem , Doença de Parkinson Secundária/diagnóstico por imagem , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/diagnóstico , Transtornos Parkinsonianos/diagnóstico por imagem , Transtornos Parkinsonianos/diagnóstico , Substância Negra/diagnóstico por imagem , Idoso , Estudos de Casos e Controles , Demência/diagnóstico por imagem , Tremor Essencial/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Sensibilidade e Especificidade , UltrassonografiaRESUMO
Protein aggregation in the form of amyloid fibrils is linked with the onset and progression of more than 30 amyloidoses, including multiple neurodegenerative disorders, such as Alzheimer's or Parkinson's disease. Despite countless studies and years of research, the process of such aggregate formation is still not fully understood. One peculiar aspect of amyloids is that they appear to be capable of undergoing structural rearrangements even after the fibrils have already formed. Such a phenomenon was reported to occur in the case of alpha-synuclein and amyloid beta aggregates after a long period of incubation. In this work, we examine whether incubation at an elevated temperature can induce the restructurization of four different conformation alpha-synuclein amyloid fibrils. We show that this structural alteration occurs in a relatively brief time period, when the aggregates are incubated at 60 °C. Additionally, it appears that during this process multiple conformationally-distinct alpha-synuclein fibrils all shift towards an identical secondary structure.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/química , Amiloide/química , Peptídeos beta-Amiloides/química , Temperatura , Doença de Parkinson/metabolismoRESUMO
Amyloid-ß and α-synuclein aggregation into amyloid fibrils is linked to the onset and progression of Alzheimer's and Parkinson's diseases. While there are only a few disease-modifying drugs, it is essential to search for new, more effective ways to encounter these neurodegenerative diseases. Multiple research articles have shown that the autoxidation of flavone is a critical factor for activating the inhibitory potential against the protein aggregation. Despite this, the structure of the newly-formed inhibitors is unknown. In this research, we examined the autoxidation products of 2',3'-dihydroxyflavone that were previously shown to possess one of the most prominent inhibitory effects against amyloid-ß aggregation. Their analysis using HPLC suggested the formation of polymeric molecules that were isolated using a 3 kDa cut-off. These polymeric structures were indicated as the most potent inhibitors based on protein aggregation kinetics and AFM studies. This revelation was confirmed using MALDI-TOF and NMR. We also show that active molecules have a tendency to reduce the Amyloid-ß and α-synuclein aggregates toxicity to SH-SY5Y cells.
RESUMO
The increasing prevalence of amyloid-related disorders, such as Alzheimer's or Parkinson's disease, raises the need for effective anti-amyloid drugs. It has been shown on numerous occasions that flavones, a group of naturally occurring anti-oxidants, can impact the aggregation process of several amyloidogenic proteins and peptides, including amyloid-beta. Due to flavone autoxidation at neutral pH, it is uncertain if the effective inhibitor is the initial molecule or a product of this reaction, as many anti-amyloid assays attempt to mimic physiological conditions. In this work, we examine the aggregation-inhibiting properties of flavones before and after they are oxidized. The oxidation of flavones was monitored by measuring the UV-vis absorbance spectrum change over time. The protein aggregation kinetics were followed by measuring the amyloidophilic dye thioflavin-T (ThT) fluorescence intensity change. Atomic force microscopy was employed to image the aggregates formed with the most prominent inhibitors. We demonstrate that flavones, which undergo autoxidation, have a far greater potency at inhibiting the aggregation of both the disease-related amyloid-beta, as well as a model amyloidogenic protein-insulin. Oxidized 6,2',3'-trihydroxyflavone was the most potent inhibitor affecting both insulin (7-fold inhibition) and amyloid-beta (2-fold inhibition). We also show that this tendency to autoxidize is related to the positions of the flavone hydroxyl groups.