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Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation.
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Quinasa de Punto de Control 2 , Síndromes Neoplásicos Hereditarios , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides , Humanos , Quinasa de Punto de Control 2/química , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Síndromes Neoplásicos Hereditarios/genética , Estudios Prospectivos , Cáncer Papilar Tiroideo/genética , Neoplasias de la Tiroides/genética , Dominios Proteicos , Masculino , Femenino , Persona de Mediana EdadRESUMEN
Cutaneous melanoma (CM) is the most aggressive skin cancer, showing globally increasing incidence. Hereditary CM accounts for a significant percentage (5-15 %) of all CM cases. However, most familial cases remain without a known genetic cause. Even though, BRD9 has been associated to CM as a susceptibility gene. The molecular events following BRD9 mutagenesis are still not completely understood. In this study, we disclosed BRD9 as a key regulator in cysteine metabolism and associated altered BRD9 to increased cell proliferation, migration and invasiveness, as well as to altered melanin levels, inducing higher susceptibility to melanomagenesis. It is evident that BRD9 WT and mutated BRD9 (c.183G>C) have a different impact on cysteine metabolism, respectively by inhibiting and activating MPST expression in the metastatic A375 cell line. The effect of the mutated BRD9 variant was more evident in A375 cells than in the less invasive WM115 line. Our data point out novel molecular and metabolic mechanisms dependent on BRD9 status that potentially account for the increased risk of developing CM and enhancing CM aggressiveness. Moreover, our findings emphasize the role of cysteine metabolism remodeling in melanoma progression and open new queues to follow to explore the role of BRD9 as a melanoma susceptibility or cancer-related gene.
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Melanoma , Neoplasias Cutáneas , Humanos , Melanoma/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Cisteína , Proliferación Celular , Proteínas que Contienen Bromodominio , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Glycosyltransferases (GTs) are enzymes that catalyze the formation of glycosidic bonds and hundreds of GTs have been identified so far in humans. Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) has been associated with central nervous system diseases and cancer. However, evidence on its enzymatic properties, including its substrates, has been scarcely described. In this paper, we have produced and purified recombinant secretory GLT8D1. The enzyme was found to be N-glycosylated. Differential scanning fluorimetry was employed to analyze the stabilization of GLT8D1 by Mn2+ and nucleotides, revealing UDP as the most stabilizing nucleotide scaffold. GLT8D1 displayed glycosyltransferase activity from UDP-galactose onto N-acetylgalactosamine but with a low efficiency. Modeling of the structure revealed similarities with other GT-A fold enzymes in CAZy family GT8 and glycosyltransferases in other families with galactosyl-, glucosyl-, and xylosyltransferase activities, each with retaining catalytic mechanisms. Our study provides novel structural and functional insights into the properties of GLT8D1 with implications in pathological processes.
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Galactosiltransferasas , Glicosiltransferasas , Humanos , Galactosiltransferasas/metabolismo , Glicosiltransferasas/metabolismo , Catálisis , Uridina DifosfatoRESUMEN
INTRODUCTION: Pyruvate Dehydrogenase Complex (PDC) is a pivotal gatekeeper between cytosolic glycolysis and mitochondrial oxidative phosphorylation, playing important role in aerobic energy metabolism. Most PDC deficiency, cases being caused by mutations in PDHA1 encoding the α subunit of the rate-limiting E1 enzyme, which is characterized by abnormal phenotypes caused by energy deprivation at peripheral/central nervous systems and muscular tissues. This study aims to evaluate the potential therapeutic effect of arginine and thiamine in ameliorating mitochondrial function in patient-derived cultured cells. MATERIALS AND METHODS: PDC-deficient cell lines, carrying three different PDHA1 variants, were cultured in the absence and presence of arginine and/or thiamine at therapeutical levels, 4 mM and 100 µM, respectively. Mitochondrial bioenergetics profile was evaluated using the Seahorse extracellular flux analyzer. RESULTS: In physiological conditions, control cells presented standard values for all parameters evaluating the mitochondrial function, no differences being observed after supplementation of culture medium with therapeutic levels of arginine and/or thiamine. However, PDC-PDHA1 deficient cell lines consumed less oxygen than the control cells, but arginine and thiamine supplementation increased the basal respiration for values similar or higher than the control cell line. Moreover, arginine and thiamine treatment highlighted an inefficient oxidative phosphorylation carried out by PDC-deficient cell lines. Finally, this treatment showed an increased oxygen consumption by enzymes other than those in the respiratory chain, thus proving the dependence of these mutant cell lines on cytosolic sources for ATP production, namely glycolysis. CONCLUSIONS: This study showed that arginine and thiamine, at therapeutical levels, increase the basal oxygen consumption rate of PDC-deficient cell lines, as well as their ATP-linked respiration. This parameter measures the capacity of the cell to meet its energetic demands and, therefore, its increase reveals a higher electron flow through the respiratory chain, which is coupled to elevated oxidative phosphorylation, thus indicating an overall increased robustness in mitochondrial- related bioenergetics.
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Medium chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is associated with ACADM gene mutations, leading to an impaired function and/or structure of MCAD. Importantly, after import into the mitochondria, MCAD must incorporate a molecule of flavin adenine dinucleotide (FAD) per subunit and assemble into tetramers. However, the effect of MCAD amino acid substitutions on FAD incorporation has not been investigated. Herein, the commonest MCAD variant (p.K304E) and 11 additional rare variants (p.Y48C, p.R55G, p.A88P, p.Y133C, p.A140T, p.D143V, p.G224R, p.L238F, p.V264I, p.Y372N, and p.G377V) were functionally and structurally characterized. Half of the studied variants presented a FAD content <65 % compared to the wild-type. Most of them were recovered as tetramers, except the p.Y372N (mainly as dimers). No correlation was found between the levels of tetramers and FAD content. However, a correlation between FAD content and the cofactor's affinity, proteolytic stability, thermostability, and thermal inactivation was established. We showed that the studied amino acid changes in MCAD may alter the substrate chain-length dependence and the interaction with electron-transferring-flavoprotein (ETF) necessary for a proper functioning electron transfer thus adding additional layers of complexity to the pathological effect of ACADM missense mutations. Although the majority of the variant MCADs presented an impaired capacity to retain FAD during their synthesis, some of them were structurally rescued by cofactor supplementation, suggesting that in the mitochondrial environment the levels and activity of those variants may be dependent of FAD's availability thus contributing for the heterogeneity of the MCADD phenotype found in patients presenting the same genotype.
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Flavina-Adenina Dinucleótido , Mutación Missense , Humanos , Acil-CoA Deshidrogenasa/genética , Acil-CoA Deshidrogenasa/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , MutaciónRESUMEN
The structural maintenance of therapeutic proteins during formulation and/or storage is a critical aspect, particularly for multi-domain and/or multimeric proteins which usually exhibit intrinsic structural dynamics leading to aggregation with concomitant loss-of-function. Protein freeze-drying is a widely used technique to preserve protein structure and function during storage. To minimize chemical/physical stresses occurring during this process, protein stabilizers are usually included, their effect being strongly dependent on the target protein. Therefore, they should be screened for on a time-consuming case-by-case basis. Herein, differential scanning fluorimetry (DSF) and isothermal denaturation fluorimetry (ITDF) were employed to screen, among different classes of freeze-drying additives, for the most effective stabilizer of the model protein human phenylalanine hydroxylase (hPAH). Correlation studies among retrieved DSF and ITDF parameters with recovered enzyme amount and activity indicated ITDF as the most appropriate screening method. Biochemical and biophysical characterization of hPAH freeze-dried with ITDF-selected stabilizers and a long-term storage study (12 months, 5 ± 3 °C) showed that the selected compounds prevented protein aggregation and preserved hPAH structural and functional properties throughout time storage. Our results provide a solid basis towards the choice of ITDF as a high-throughput screening step for the identification of protein freeze-drying protectors.
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Fenilalanina Hidroxilasa , Humanos , Proteínas/química , Liofilización/métodos , Fluorometría , Excipientes/química , Desnaturalización ProteicaRESUMEN
Lung cancer is a lethal disease with no truly efficient therapeutic management despite the progresses, and metabolic profiling can be a way of stratifying patients who may benefit from new therapies. The present study is dedicated to profiling cysteine metabolic pathways in NSCLC cell lines and tumor samples. This was carried out by analyzing hydrogen sulfide (H2S) and ATP levels, examining mRNA and protein expression patterns of cysteine catabolic enzymes and transporters, and conducting metabolomics analysis using nuclear magnetic resonance (NMR) spectroscopy. Selenium-chrysin (SeChry) was tested as a therapeutic alternative with the aim of having an effect on cysteine catabolism and showed promising results. NSCLC cell lines presented different cysteine metabolic patterns, with A549 and H292 presenting a higher reliance on cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) to maintain H2S levels, while the PC-9 cell line presented an adaptive behavior based on the use of mercaptopyruvate sulfurtransferase (MST) and cysteine dioxygenase (CDO1), both contributing to the role of cysteine as a pyruvate source. The analyses of human lung tumor samples corroborated this variability in profiles, meaning that the expression of certain genes may be informative in defining prognosis and new targets. Heterogeneity points out individual profiles, and the identification of new targets among metabolic players is a step forward in cancer management toward personalized medicine.
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Abstract Introduction : The scientific production of the areas of physical therapy and occupational therapy presented important growth and international visibility. Objective : To evaluate technical and scientific indicators, in addition to the training of human resources, of scholarship researchers of scientific productivity of CNPq (PQ), in the area of physical therapy and occupational therapy. Methods : A cross-sectional, census and quantitative study was conducted from the Lattes curricula of CNPq researchers with active scholarships in December 2021. The information was extracted through the Lattes Platform of CNPq, including three dimensions of variables for analysis: a) researcher profile; b) scientific production; c) training of human resources. Results : The evaluation included 73 PQ CNPq, in the areas of physical therapy and occupational therapy. The majority were female (n = 42; 57.5%), distributed in 26 different institutions in the country, with the Southeast region being the most prevalent. The largest portion of the PQ completed their doctorate between 11 and 20 years ago (n = 41; 56.2%) and did postdoctoral work (n = 55; 75.3%) abroad (n = 32; 58.2%). The group published 9,486 scientific articles throughout its career, averaging 129.94 scientific articles per PQ. The group of PQ supervised a total of 2,561 undergraduate students, 1,748 master's and 800 doctoral students. The mean H index of PQ was 19.74 (CI 95% = 18.40-21.11; SD = 5.71; median = 19). Conclusion : The PQ scholarship researchers presented significant scientific production and training of human resources, which confirms the important development of the areas in the last ten years. However, a strong nucleation of this production was observed.
Resumo Introducão : A produção científica das áreas de fisioterapia e terapia ocupacional apresentou importante crescimento e visibilidade internacional. Objetivo : Avaliar indicadores técni-cos e científicos, além da formação de recursos humanos, de pesquisadores bolsistas de produtividade científica do CNPq (PQ), nas área de fisioterapia e terapia ocupacional. Métodos : Realizou-se estudo transversal, censitário e quantitativo, a partir dos currículos Lattes dos pesquisadores do CNPq, nas áreas mencionadas, com bolsas ativas em dezembro de 2021. As informações foram extraídas da Plataforma Lattes do CNPq, incluindo três dimensões de variáveis para análise: a) perfil do pesquisador; b) produção científica; c) formação de recursos humanos. Resultados : Foram incluídos na avaliação 73 PQ do CNPq, nas áreas de fisioterapia e terapia ocupacional. A maioria foi do sexo feminino (n = 42; 57,5%), distribuída em 26 instituições distintas no país, sendo a região Sudeste a mais prevalente. A maior parcela dos PQ finalizou o doutorado entre 11 e 20 anos atrás (n = 41; 56,2%) e realizou pós-doutorado (n = 55; 75,3%) no exterior (n = 32; 58,2%). O grupo publicou 9.486 artigos científicos durante toda a carreira, com média de 129,94 artigos científicos por PQ. O conjunto dos PQ orientou um total de 2.561 estudantes de iniciação científica, 1.748 de mestrado e 800 de doutorado. O índice H médio dos PQ foi de 19,74 (IC 95% = 18,40-21,11; DP = 5,71; mediana = 19). Conclusão : Os bolsistas PQ apresentaram significativa produção científica e formação de recursos humanos, o que confirma o importante desenvolvimento das áreas nos últimos dez anos. Observou-se, entretanto, forte nucleação dessa produção.
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Entomopathogenic nematodes are used as biological control agents against a broad range of insect pests. We ascribed the pathogenicity of these organisms to the excretory/secretory products (ESP) released by the infective nematode. Our group characterized different virulence factors produced by Steinernema carpocapsae that underlie its success as an insect pathogen. A novel ShK-like peptide (ScK1) from this nematode that presents high sequence similarity with the ShK peptide from a sea anemone was successfully produced recombinantly in Escherichia coli. The secondary structure of ScK1 appeared redox-sensitive, exhibiting a far-UV circular dichroism spectrum consistent with an alpha-helical secondary structure. Thermal denaturation of the ScK1 allowed estimating the melting temperature to 59.2 ± 0.1 °C. The results from toxicity assays using Drosophila melanogaster as a model show that injection of this peptide can kill insects in a dose-dependent manner with an LD50 of 16.9 µM per adult within 24 h. Oral administration of the fusion protein significantly reduced the locomotor activity of insects after 48 h (p < 0.05, Tukey's test). These data show that this nematode expresses insecticidal peptides with potential as next-generation insecticides.
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Drosophila melanogaster , Nematodos , Animales , Insectos , Agentes de Control Biológico , VirulenciaRESUMEN
In this review encouraged by original data, we first provided in vivo evidence that the kidney, comparative to the liver or brain, is an organ particularly rich in cysteine. In the kidney, the total availability of cysteine was higher in cortex tissue than in the medulla and distributed in free reduced, free oxidized and protein-bound fractions (in descending order). Next, we provided a comprehensive integrated review on the evidence that supports the reliance on cysteine of the kidney beyond cysteine antioxidant properties, highlighting the relevance of cysteine and its renal metabolism in the control of cysteine excess in the body as a pivotal source of metabolites to kidney biomass and bioenergetics and a promoter of adaptive responses to stressors. This view might translate into novel perspectives on the mechanisms of kidney function and blood pressure regulation and on clinical implications of the cysteine-related thiolome as a tool in precision medicine.
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Cisteína/metabolismo , Riñón/metabolismo , Medicina de Precisión , Encéfalo/metabolismo , Humanos , Hígado/metabolismo , Especificidad de ÓrganosRESUMEN
The new Coronavirus, responsible for the COVID-19 disease, is the most discussed topic in the current days, and the forecast numbers of new cases and deaths are the most important source of data in governmental decision-making. The present work presents a prediction model with two different approaches concerning the input data, by using Artificial Neural Networks (ANN). The use of a substantial mitigation procedure adopted (mandatory use of masks) was experimented as an input to the network, in order to evaluate the improvement in the results. The ANN forecasting model was demonstrated to predict with higher accuracy within the next twenty days using the information about the mandatory use of face masks. The final results showed that the twenty days ahead forecasting was made with an error of 24,7% and 1,6% for the number of cumulative cases of infection and deaths for Brazil, and 37,9% and 33,8% for Portuguese time series, respectively.
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The 'gasotransmitters' hydrogen sulfide (H2S), nitric oxide (NO), and carbon monoxide (CO) act as second messengers in human physiology, mediating signal transduction via interaction with or chemical modification of protein targets, thereby regulating processes such as neurotransmission, blood flow, immunomodulation, or energy metabolism. Due to their broad reactivity and potential toxicity, the biosynthesis and breakdown of H2S, NO, and CO are tightly regulated. Growing evidence highlights the active role of gasotransmitters in their mutual cross-regulation. In human physiology, the transsulfuration enzymes cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are prominent H2S enzymatic sources. While CBS is known to be inhibited by NO and CO, little is known about CSE regulation by gasotransmitters. Herein, we investigated the effect of s-nitrosation on CSE catalytic activity. H2S production by recombinant human CSE was found to be inhibited by the physiological nitrosating agent s-nitrosoglutathione (GSNO), while reduced glutathione had no effect. GSNO-induced inhibition was partially reverted by ascorbate and accompanied by the disappearance of one solvent accessible protein thiol. By combining differential derivatization procedures and mass spectrometry-based analysis with functional assays, seven out of the ten protein cysteine residues, namely Cys84, Cys109, Cys137, Cys172, Cys229, Cys307, and Cys310, were identified as targets of s-nitrosation. By generating conservative Cys-to-Ser variants of the identified s-nitrosated cysteines, Cys137 was identified as most significantly contributing to the GSNO-mediated CSE inhibition. These results highlight a new mechanism of crosstalk between gasotransmitters.
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Among gynecologic malignancies, ovarian cancer is the third most prevalent and the most common cause of death, especially due to diagnosis at an advanced stage together with resistance to therapy. As a solid tumor grows, cancer cells in the microenvironment are exposed to regions of hypoxia, a selective pressure prompting tumor progression and chemoresistance. We have previously shown that cysteine contributes to the adaptation to this hypoxic microenvironment, but the mechanisms by which cysteine protects ovarian cancer cells from hypoxia-induced death are still to be unveiled. Herein, we hypothesized that cysteine contribution relies on cellular metabolism reprogramming and energy production, being cysteine itself a metabolic source. Our results strongly supported a role of xCT symporter in energy production that requires cysteine metabolism instead of hydrogen sulfide (H2S) per se. Cysteine degradation depends on the action of the H2S-synthesizing enzymes cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and/or 3-mercaptopyruvate sulfurtransferase (MpST; together with cysteine aminotransferase, CAT). In normoxia, CBS and CSE inhibition had a mild impact on cysteine-sustained ATP production, pointing out the relevance of CAT + MpST pathway. However, in hypoxia, the concomitant inhibition of CBS and CSE had a stronger impact on ATP synthesis, thus also supporting a role of their hydrogen sulfide and/or cysteine persulfide-synthesizing activity in this stressful condition. However, the relative contributions of each of these enzymes (CBS/CSE/MpST) on cysteine-derived ATP synthesis under hypoxia remains unclear, due to the lack of specific inhibitors. Strikingly, NMR analysis strongly supported a role of cysteine in the whole cellular metabolism rewiring under hypoxia. Additionally, the use of cysteine to supply biosynthesis and bioenergetics was reinforced, bringing cysteine to the plateau of a main carbon sources in cancer. Collectively, this work supports that sulfur and carbon metabolism reprogramming underlies the adaptation to hypoxic microenvironment promoted by cysteine in ovarian cancer.
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The use of artificial neural networks (ANNs) is a great contribution to medical studies since the application of forecasting concepts allows for the analysis of future diseases propagation. In this context, this paper presents a study of the new coronavirus SARS-COV-2 with a focus on verifying the virus propagation associated with mitigation procedures and massive vaccination campaigns. There were two proposed methodologies in making predictions 28 days ahead for the number of new cases, deaths, and ICU patients of five European countries: Portugal, France, Italy, the United Kingdom, and Germany. A case study of the results of massive immunization in Israel was also considered. The data input of cases, deaths, and daily ICU patients was normalized to reduce discrepant numbers due to the countries' size and the cumulative vaccination values by the percentage of population immunized (with at least one dose of the vaccine). As a comparative criterion, the calculation of the mean absolute error (MAE) of all predictions presents the best methodology, targeting other possibilities of use for the method proposed. The best architecture achieved a general MAE for the 1-to-28-day ahead forecast, which is lower than 30 cases, 0.6 deaths, and 2.5 ICU patients per million people.
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Our general goal was to non-invasively evaluate kidney tubular dysfunction. We developed a strategy based on cysteine (Cys) disulfide stress mechanism that underlies kidney dysfunction. There is scarce information regarding the fate of Cys-disulfides (CysSSX), but evidence shows they might be detoxified in proximal tubular cells by the action of N-acetyltransferase 8 (NAT8). This enzyme promotes the addition of an N-acetyl moiety to cysteine-S-conjugates, forming mercapturates that are eliminated in urine. Therefore, we developed a strategy to quantify mercapturates of CysSSX in urine as surrogate of disulfide stress and NAT8 activity in kidney tubular cells. We use a reduction agent for the selective reduction of disulfide bonds. The obtained N-acetylcysteine moiety of the mercapturates from cysteine disulfides was monitored by fluorescence detection. The method was applied to urine from mice and rat as well as individuals with healthy kidney and kidney disease.
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Cisteína , Enfermedades Renales , Acetilcisteína , Animales , Disulfuros , Riñón , Ratones , RatasRESUMEN
Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.
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Fenilalanina Hidroxilasa/metabolismo , Quinolonas/química , Quinolonas/farmacología , Dominio Catalítico , Espectroscopía de Resonancia por Spin del Electrón , Fluorometría , Células HEK293 , Humanos , Enfermedades Metabólicas/metabolismo , Modelos Moleculares , Fenilalanina/metabolismo , Fenilcetonurias/metabolismo , Resonancia por Plasmón de Superficie , TripsinaRESUMEN
Pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-coenzyme A, hinging glycolysis and the tricarboxylic acid cycle. PDC deficiency, an inborn error of metabolism, has a broad phenotypic spectrum. Symptoms range from fatal lactic acidosis or progressive neuromuscular impairment in the neonatal period, to chronic neurodegeneration. Most disease-causing mutations in PDC deficiency affect the PDHA1 gene, encoding the α subunit of the PDC-E1 component. Detailed biophysical analysis of pathogenic protein variants is a challenging approach to support the design of therapies based on improving and correcting protein structure and function. Herein, we report the characterization of clinically relevant PDC-E1α variants identified in Portuguese PDC deficient patients. These variants bear amino acid substitutions in different structural regions of PDC-E1α. The structural and functional analyses of recombinant heterotetrameric (αα'ßß') PDC-E1 variants, combined with molecular dynamics (MD) simulations, show a limited impact of the amino acid changes on the conformational stability, apart from the increased propensity for aggregation of the p.R253G variant as compared to wild-type PDC-E1. However, all variants presented a functional impairment in terms of lower residual PDC-E1 enzymatic activity and ≈3-100 × lower affinity for the thiamine pyrophosphate (TPP) cofactor, in comparison with wild-type PDC-E1. MD simulations neatly showed generally decreased stability (increased flexibility) of all variants with respect to the WT heterotetramer, particularly in the TPP binding region. These results are discussed in light of disease severity of the patients bearing such mutations and highlight the difficulty of developing chaperone-based therapies for PDC deficiency.
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Simulación de Dinámica Molecular , Mutación Missense , Piruvato Deshidrogenasa (Lipoamida)/química , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa , Tiamina Pirofosfato/química , Sustitución de Aminoácidos , Estabilidad de Enzimas , Humanos , Piruvato Deshidrogenasa (Lipoamida)/genética , Piruvato Deshidrogenasa (Lipoamida)/metabolismo , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/enzimología , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/genética , Tiamina Pirofosfato/genética , Tiamina Pirofosfato/metabolismoRESUMEN
The present works contributes with a rheological characterization of the most commonly used polymers and solvents to formulate amorphous solid dispersions by means of spray drying process: copovidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose acetate succinate (HPMCAS) and a copolymer of methacrylic acid and methyl methacrylate (1:1 ratio and commercially known as Eudragit L100). The organic-based solutions are characterized in terms of surface tension, shear viscosity and relaxation time. HPMC and HPMCAS solutions exhibit shear thinning behaviour, i.e. decrease of shear viscosity with the increasing shear rate imposed, while the samples with Eudragit L100 can be considered Boger fluids, showing a constant viscosity but maintaining its viscoelastic character. Under uniaxial extensional flow, these polymers solutions exhibit a viscoelastic behavior with an increasing relaxation time with an increasing concentration, and in some cases showed a 'beads-on-string' effect. In contrast, copovidone showed a Newtonian fluid behavior, with the absence of elasticity. The fundamental understanding and characterization of the present work may be further applied to atomization modelling and support and expedite spray drying process development.
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Metilcelulosa , Secado por Pulverización , Derivados de la Hipromelosa , Polímeros , Reología , Soluciones , ViscosidadRESUMEN
Sulfane sulfur species comprise a variety of biologically relevant hydrogen sulfide (H2S)-derived species, including per- and poly-sulfidated low molecular weight compounds and proteins. A growing body of evidence suggests that H2S, currently recognized as a key signaling molecule in human physiology and pathophysiology, plays an important role in cancer biology by modulating cell bioenergetics and contributing to metabolic reprogramming. This is accomplished through functional modulation of target proteins via H2S binding to heme iron centers or H2S-mediated reversible per- or poly-sulfidation of specific cysteine residues. Since sulfane sulfur species are increasingly viewed not only as a major source of H2S but also as key mediators of some of the biological effects commonly attributed to H2S, the multifaceted role of these species in cancer biology is reviewed here with reference to H2S, focusing on their metabolism, signaling function, impact on cell bioenergetics and anti-tumoral properties.
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Metabolismo Energético , Sulfuro de Hidrógeno/metabolismo , Neoplasias/metabolismo , Azufre/metabolismo , HumanosRESUMEN
To enable survival in adverse conditions, cancer cells undergo global metabolic adaptations. The amino acid cysteine actively contributes to cancer metabolic remodelling on three different levels: first, in its free form, in redox control, as a component of the antioxidant glutathione or its involvement in protein s-cysteinylation, a reversible post-translational modification; second, as a substrate for the production of hydrogen sulphide (H2S), which feeds the mitochondrial electron transfer chain and mediates per-sulphidation of ATPase and glycolytic enzymes, thereby stimulating cellular bioenergetics; and, finally, as a carbon source for epigenetic regulation, biomass production and energy production. This review will provide a systematic portrayal of the role of cysteine in cancer biology as a source of carbon and sulphur atoms, the pivotal role of cysteine in different metabolic pathways and the importance of H2S as an energetic substrate and signalling molecule. The different pools of cysteine in the cell and within the body, and their putative use as prognostic cancer markers will be also addressed. Finally, we will discuss the pharmacological means and potential of targeting cysteine metabolism for the treatment of cancer.