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Transthyretin (TTR) is an homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy. Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study, we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure. Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of subdenaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.
Assuntos
Cardiomiopatias , Pré-Albumina , Multimerização Proteica , Termodinâmica , Pré-Albumina/genética , Pré-Albumina/química , Pré-Albumina/metabolismo , Humanos , Cardiomiopatias/metabolismo , Cardiomiopatias/genética , Tiroxina/metabolismo , Tiroxina/química , Mutação de Sentido Incorreto , Amiloide/metabolismo , Amiloide/química , Amiloide/genética , Substituição de Aminoácidos , Ureia/química , Ureia/metabolismoRESUMO
The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1ß, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.
Assuntos
Armadilhas Extracelulares , Infecção por Zika virus , Zika virus , Animais , Camundongos , Doenças Neuroinflamatórias , Nervo IsquiáticoRESUMO
Biomolecular condensates, membrane-less entities arising from liquid-liquid phase separation, hold dichotomous roles in health and disease. Alongside their physiological functions, these condensates can transition to a solid phase, producing amyloid-like structures implicated in degenerative diseases and cancer. This review thoroughly examines the dual nature of biomolecular condensates, spotlighting their role in cancer, particularly concerning the p53 tumor suppressor. Given that over half of the malignant tumors possess mutations in the TP53 gene, this topic carries profound implications for future cancer treatment strategies. Notably, p53 not only misfolds but also forms biomolecular condensates and aggregates analogous to other protein-based amyloids, thus significantly influencing cancer progression through loss-of-function, negative dominance, and gain-of-function pathways. The exact molecular mechanisms underpinning the gain-of-function in mutant p53 remain elusive. However, cofactors like nucleic acids and glycosaminoglycans are known to be critical players in this intersection between diseases. Importantly, we reveal that molecules capable of inhibiting mutant p53 aggregation can curtail tumor proliferation and migration. Hence, targeting phase transitions to solid-like amorphous and amyloid-like states of mutant p53 offers a promising direction for innovative cancer diagnostics and therapeutics.
Assuntos
Neoplasias , Ácidos Nucleicos , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Agregados Proteicos , Neoplasias/metabolismo , Amiloide/químicaRESUMO
The aggregation of epitopes that are also able to bind major histocompatibility complex (MHC) alleles raises questions around the potential connection between the formation of epitope aggregates and their affinities to MHC receptors. We first performed a general bioinformatic assessment over a public dataset of MHC class II epitopes, finding that higher experimental binding correlates with higher aggregation-propensity predictors. We then focused on the case of P10, an epitope used as a vaccine candidate against Paracoccidioides brasiliensis that aggregates into amyloid fibrils. We used a computational protocol to design variants of the P10 epitope to study the connection between the binding stabilities towards human MHC class II alleles and their aggregation propensities. The binding of the designed variants was tested experimentally, as well as their aggregation capacity. High-affinity MHC class II binders in vitro were more disposed to aggregate forming amyloid fibrils capable of binding Thioflavin T and congo red, while low affinity MHC class II binders remained soluble or formed rare amorphous aggregates. This study shows a possible connection between the aggregation propensity of an epitope and its affinity for the MHC class II cleft.
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Resumo O artigo apresenta resultados de pesquisas sobre percepção pública da ciência na pandemia no Brasil, realizadas no Centro de Estudos SoU_Ciência, sediado na Universidade Federal de São Paulo (Unifesp). Com o intuito de responder à pergunta: "A pandemia da COVID-19 alterou a percepção da sociedade brasileira sobre ciência, cientistas e universidades?", realizamos estudos quantitativos e qualitativos entre agosto/2021 e julho/2022. Em levantamentos quantitativos nacionais de opinião pública, coletamos dados exclusivos de série histórica de enquetes sobre o tema no Brasil, e em grupos focais, aprofundamos estudos sobre percepção e posicionamento político de diferentes segmentos sociais. Em meio ao quadro de crescimento do negacionismo científico, retrocessos políticos e sociais, desmonte de políticas públicas, especificamente científicas e tecnológicas, decorrentes do impeachment de 2016 e da eleição de Bolsonaro em 2018, as pesquisas indicam, aparentemente contrariando a tendência política obscurantista, uma expressiva ampliação do interesse público pela ciência na pandemia no país. Este trabalho analisa a emergência de uma "onda pró-ciência" na opinião pública no Brasil, os fatores que propiciaram seu surgimento na pandemia e suas perspectivas na atualidade.
Abstract This article presents the results of our study on the public perception of science during the COVID-19 pandemic in Brazil, carried out at the Centro de Estudos SoU_Ciência, from Universidade Federal de São Paulo (UNIFESP). To answer the question: "Has the COVID-19 pandemic changed the perception of Brazilian society about science, scientists, and universities?", quantitative and qualitative studies were conducted between August 2021 and July 2022. In national quantitative public opinion surveys, we collected exclusive data from a historical series of polls on the subject in Brazil, and in focus groups, we deepened studies on the perception and political position of different social segments. Amid the growth of scientific denialism; political and social setbacks; and the dismantling of public policies, specifically scientific and technological, resulting from the impeachment of 2016 and the election of Bolsonaro in 2018, research indicates, apparently contrary to an obscurantist political tendency, a significant expansion of public interest in science during the pandemic in the country. This paper analyzes the emergence of a "pro-science wave" in public opinion in Brazil, the factors that led to its emergence during the pandemic, and its current prospects.
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Studies showed that JM-20, a benzodiazepine-dihydropyridine hybrid molecule, protects against rotenone and 6-hydroxydopamine neurotoxicity. However, its protective effects against cytotoxicity induced by endogenous neurotoxins involved in Parkinson's disease (PD) pathogenesis have never been investigated. In this study, we evaluated the ability of JM-20 to inhibit alpha-synuclein (aSyn) aggregation. We also evaluated the interactions of JM-20 with aSyn by molecular docking and molecular dynamics and assessed the protective effect of JM-20 against aminochrome cytotoxicity. We demonstrated that JM-20 induced the formation of heterogeneous amyloid fibrils, which were innocuous to primary cultures of mesencephalic cells. Moreover, JM-20 reduced the average size of aSyn positive inclusions in H4 cells transfected with SynT wild-type and synphilin-1-V5, but not in HEK cells transfected with synphilin-1-GFP. In silico studies showed the interaction between JM-20 and the aSyn-binding site. Additionally, we showed that JM-20 protects SH-SY5Y cells against aminochrome cytotoxicity. These results reinforce the potential of JM-20 as a neuroprotective compound for PD and suggest aSyn as a molecular target for JM-20.
Assuntos
Di-Hidropiridinas , Neuroblastoma , Doença de Parkinson , Humanos , alfa-Sinucleína , Benzodiazepinas , Simulação de Acoplamento Molecular , Doença de Parkinson/tratamento farmacológicoRESUMO
Aged and photoaged skin exhibit fine wrinkles that are signs of epidermal inflammation and degeneration. It has been shown that healthy elderly skin expresses amyloidogenic proteins, including α-Synuclein, which are known to oligomerize and trigger inflammation and neurodegeneration. However, little is known about their putative role in skin physiology and sensitivity. To unravel this possible role, we investigated the impact of oligomeric α-Synuclein (Oα-Syn) in 2D and 3D keratinocyte human models. Exogenous Oα-Syn caused degeneration of reconstructed human epidermis (RHE) by diminishing proliferation and thickness of the stratum basale. Oα-Syn also increased NF-kB nuclear translocation in keratinocytes and triggered inflammation in the RHE, by increasing expression of interleukin-1ß and tumor necrosis factor-alpha, and the release of tumor necrosis factor-alpha in a time-dependent manner. Dexamethasone and an IL-1ß inhibitor partially diminished RHE degeneration caused by Oα-Syn. These findings suggest that Oα-Syn induces epidermal inflammation and decreases keratinocyte proliferation, and therefore might contribute to epidermal degeneration observed in human skin aging.
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Fator de Necrose Tumoral alfa , alfa-Sinucleína , Idoso , Epiderme/metabolismo , Epiderme/patologia , Humanos , Inflamação/metabolismo , Queratinócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Fungal infection is an important health problem in Latin America, and in Brazil in particular. Paracoccidioides (mainly P. brasiliensis and P. lutzii) is responsible for paracoccidioidomycosis, a disease that affects mainly the lungs. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells, which makes this protein an interesting target of study. A specific stretch of 15 amino acids that spans the region 181-195 (named P10) of gp43 is an important epitope of gp43 that is being envisioned as a vaccine candidate. Here we show that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. Seeds obtained by fragmentation of P10 fibrils were able to induce the aggregation of P4, but not P23, two other peptides derived from gp43. In silico analysis revealed several regions within the P10 sequence that can form amyloid with steric zipper architecture. Besides, in-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by several proteases, which suggests that P10 could be formed under physiological conditions. Considering our data in the context of a potential vaccine development, we redesigned the sequence of P10, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.
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Amiloide/química , Antígenos de Neoplasias/química , Antígenos/química , Paracoccidioides/imunologia , Paracoccidioidomicose/prevenção & controle , Algoritmos , Animais , Antígenos de Fungos/imunologia , Dicroísmo Circular , Biologia Computacional/métodos , Simulação por Computador , Epitopos , Proteínas Fúngicas/química , Vacinas Fúngicas/imunologia , Glicoproteínas/química , Humanos , Técnicas In Vitro , Paracoccidioidomicose/imunologia , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Software , Solventes/química , Desenvolvimento de VacinasRESUMO
Despite evidence of health benefits from kefir administration, a systematic review with meta-analysis on bioactive compounds associated with these benefits is still absent in the literature. Kefir is fermented milk resulting from the metabolism of a complex microbiota in symbiosis. Recent researches have investigated the bioactive compounds responsible for the preventive and therapeutic effects attributed to kefir. However, differences in functional potential between industrial and artisanal kefir are still controversial. Firstly, we identified differences in the microbial composition among both types of kefir. Available evidence concerning the action of different bioactive compounds from kefir on health, both from in vitro and in vivo studies, was subsequently summarized to draw a primary conclusion of the dose and the intervention time for effect, the producer microorganisms, the precursor in the milk, and the action mechanism. Meta-analysis was performed to investigate the statistically significant differences (P < 0.05) between intervention and control and between both types of kefir for each health effect studied. In summary, the bioactive compounds more commonly reported were exopolysaccharides, including kefiran, bioactive peptides, and organic acids, especially lactic acid. Kefir bioactive compounds presented antimicrobial, anticancer, and immune-modulatory activities corroborated by the meta-analysis. However, clinical evidence is urgently needed to strengthen the practical applicability of these bioactive compounds. The mechanisms of their action were diverse, indicating that they can act by different signaling pathways. Still, industrial and artisanal kefir may differ regarding functional potential-OR of 8.56 (95% CI: 2.27-32.21, P ≤ .001)-according to the observed health effect, which can be associated with differences in the microbial composition between both types of kefir.
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Anti-Infecciosos/farmacologia , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Agentes de Imunomodulação/farmacologia , Kefir , Leite/química , Animais , Fermentação , HumanosRESUMO
The accumulation of protein aggregates in human tissues is a hallmark of more than 40 diseases called amyloidoses. In seven of these disorders, the aggregation is associated with neurodegenerative processes in the central nervous system such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). The aggregation occurs when certain soluble proteins lose their physiological function and become toxic amyloid species. The amyloid assembly consists of protein filament interactions, which can form fibrillar structures rich in ß-sheets. Despite the frequent incidence of these diseases among the elderly, the available treatments are limited and at best palliative, and new therapeutic approaches are needed. Among the many natural compounds that have been evaluated for their ability to prevent or delay the amyloidogenic process is epigallocatechin-3-gallate (EGCG), an abundant and potent polyphenolic molecule present in green tea that has extensive biological activity. There is evidence for EGCG's ability to inhibit the aggregation of α-synuclein, amyloid-ß, and huntingtin proteins, respectively associated with PD, AD, and HD. It prevents fibrillogenesis (in vitro and in vivo), reduces amyloid cytotoxicity, and remodels fibrils to form non-toxic amorphous species that lack seed propagation. Although it is an antioxidant, EGCG in an oxidized state can promote fibrils' remodeling through formation of Schiff bases and crosslinking the fibrils. Moreover, microparticles to drug delivery were synthesized from oxidized EGCG and loaded with a second anti-amyloidogenic molecule, obtaining a synergistic therapeutic effect. Here, we describe several pre-clinical and clinical studies involving EGCG and neurodegenerative diseases and their related mechanisms.