Estudo de moléculas derivadas da lagarta lonomia obliqua sobre o metabolismo oxidativo de condrócitos humanos
Data
2023-10-05
Tipo
Dissertação de mestrado
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Resumo
Objetivos: Estudar a atividade do peptídeo D3, derivado de uma hemolina presente em veneno da lagarta Lonomia obliqua, no metabolismo oxidativo de condrócitos humanos, investigando seus possíveis alvos moleculares por meio de diferentes estratégias de proteômica de condrócito (NHAC-kN) sob estresse oxidativo. Métodos: Para análise proteômica de condrócitos e do secretoma foram feitos tratamentos temporais na presença e ausência do peróxido de hidrogênio e do peptídeo D3. Para identificação dos possíveis alvos do D3, utilizou-se a metodologia da quimioproteômica com marcação por tags isobáricas. Após tratamento, as proteínas extraídas foram reduzidas, alquiladas e digeridas overnight e os peptídeos tripsínicos coletados. Por fim, todas as amostras foram acidificadas, dessalinizadas e secas e ressuspendidas para análise LC-MS/MS em espectrômetro de massas Thermo Scientific Q Exactive Plus. Os dados obtidos foram analisados no software MaxQuant para identificação e quantificação de proteínas. Resultados do proteoma e do secretoma das células tiveram a significância estatística determinada pelo teste T-Student bicaudal utilizando o software Perseus, com False Discovery Rate obtidos por permutação. Resultados: A proteômica e a análise do secretoma identificaram que os principais processos modulados pelo peróxido de hidrogênio foram: resposta a proteínas não enoveladas, remodelagem da matriz extracelular e secreção. O tratamento das células estimuladas com o peróxido seguido do D3 apresentou uma melhora na manutenção da matriz extracelular saudável e indícios de menor atividade secretória relacionada à degeneração da matriz, além da diminuição do estresse do retículo endoplasmático e Golgi. A análise de quimioproteômica identificou 11 possíveis alvos, sendo que a paratimosina e a dissulfeto isomerase foram identificadas em todas as abordagens. Conclusões: Pode-se afirmar, com base nos dados analisados que o peptídeo D3 tem efeito positivo em condrócitos humanos estimulados com H2O2, favorecendo manutenção da resposta secretória, manutenção das proteínas da matriz e menor estresse oxidativo das organelas secretórias.
Objectives: To evaluate the activity of the peptide D3 derived from hemolymph present in Lonomia obliqua caterpillar venom, in the oxidative metabolism of human chondrocytes by investigating the proteome and secretome of chondrocytes under oxidative stress in the presence of D3 and by applying chemoproteomics to identify putative molecular targets. Methods: For proteome and secretome analysis, treatments were performed with and without hydrogen peroxide and D3. To identify possible D3 targets, chemoproteomic assays were performed and peptides were labelled with tandem mass tags. After treatment, the extracted proteins were denatured, alkylated followed by an overnight trypsin digestion, and tryptic peptides were collected. Afterwards, all samples were acidified, desalted, dried and resuspended for LC-MS/MS analysis in a Thermo Scientific Q Exactive Plus mass spectrometer. The obtained data was analyzed using MaxQuant software for identification and quantification of proteins. The statistical significance of all results for proteomics and secretome was determined by two-tailed T-Student test using Perseus software. Results: Proteome and secretome analyzes showed that the main modulated processes were related to unfolded protein response, secretion of extracellular matrix proteins. Treatment of stimulated cells with peroxide and D3 showed an improvement in the maintenance of a healthy extracellular matrix and evidence of lower secretory activity related to inflammation and matrix degeneration. In addition, less proteins related to endoplasmic reticulum and Golgi stress were identified. Chemoproteomic analysis identified 11 possible targets, with parathymosin and disulfide isomerase were identified in all methodological approaches. Conclusions: Based on the analyzed data, the D3 peptide has presented effect on human chondrocytes stimulated with H2O2, favoring maintenance of the secretory response, maintenance of matrix proteins and possibly lower oxidative stress of the secretory organelles.
Objectives: To evaluate the activity of the peptide D3 derived from hemolymph present in Lonomia obliqua caterpillar venom, in the oxidative metabolism of human chondrocytes by investigating the proteome and secretome of chondrocytes under oxidative stress in the presence of D3 and by applying chemoproteomics to identify putative molecular targets. Methods: For proteome and secretome analysis, treatments were performed with and without hydrogen peroxide and D3. To identify possible D3 targets, chemoproteomic assays were performed and peptides were labelled with tandem mass tags. After treatment, the extracted proteins were denatured, alkylated followed by an overnight trypsin digestion, and tryptic peptides were collected. Afterwards, all samples were acidified, desalted, dried and resuspended for LC-MS/MS analysis in a Thermo Scientific Q Exactive Plus mass spectrometer. The obtained data was analyzed using MaxQuant software for identification and quantification of proteins. The statistical significance of all results for proteomics and secretome was determined by two-tailed T-Student test using Perseus software. Results: Proteome and secretome analyzes showed that the main modulated processes were related to unfolded protein response, secretion of extracellular matrix proteins. Treatment of stimulated cells with peroxide and D3 showed an improvement in the maintenance of a healthy extracellular matrix and evidence of lower secretory activity related to inflammation and matrix degeneration. In addition, less proteins related to endoplasmic reticulum and Golgi stress were identified. Chemoproteomic analysis identified 11 possible targets, with parathymosin and disulfide isomerase were identified in all methodological approaches. Conclusions: Based on the analyzed data, the D3 peptide has presented effect on human chondrocytes stimulated with H2O2, favoring maintenance of the secretory response, maintenance of matrix proteins and possibly lower oxidative stress of the secretory organelles.