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The crosstalk between inflammatory conditions and glycosylation in rare diseases of glycosylation
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A glicosilação está envolvida em vários processos biológicos, e defeitos nesta via estão associados a patologias, como doenças congénitas da glicosilação (CDG). Fosfomanomutase 2 (PMM2)-CDG, o subtipo mais frequente, resulta de mutações recessivas no gene PMM2, que codifica uma enzima essencial para a biossíntese de N-glicanos. Redução da atividade da PMM2 provoca hipoglicosilação de glicoproteínas, originando envolvimento multissistémico, incluindo disfunção imunitária. Atualmente, não existem terapias eficazes. Contudo, chaperones farmacológicas (CFs), capazes de estabilizar proteínas misfolded e aumentar atividade enzimática, demonstraram eficácia in vitro na restauração da N-glicosilação.
Esta tese explorou dois aspetos: (i) o potencial terapêutico de três CFs previamente identificadas por ferramentas quimioinformáticas e (ii) o impacto da N-glicosilação defeituosa na desregulação imunitária e suscetibilidade a infeções. Fibroblastos de doentes com PMM2-CDG e controlos foram incubados com as CFs em diferentes períodos. Staining com Concanavalina A revelou aumento nos níveis de N-glicanos em fibroblastos de doentes após 72h de tratamento com todas as CFs, embora este efeito não se mantivesse às 120h. Análise da expressão da enzima PMM2 e da proteína altamente N-glicosilada, ICAM-1, não mostrou diferenças significativas entre doentes e controlos, sugerindo que estas CFs podem melhorar a N-glicosilação geral, mas poderão ser insuficientes para restaurar a expressão ou a glicosilação adequada de glicoproteínas específicas.
Para avaliar a função imunitária, amostras de sangue total de doentes e controlos foram estimuladas (PMA+Ionomicina). A imunofenotipagem revelou aumento da expressão do marcador de ativação CD69 e redução do imune-checkpoint PD-L1, enquanto o perfil de citocinas mostrou secreção aumentada de IL-2 e IL-6 em amostras estimuladas de PMM2-CDG, sugerindo que células sanguíneas dos doentes são responsivas, mas podem apresentar atividade desregulada ou exacerbada.
Compreender o impacto da glicosilação defeituosa na fisiopatologia e na resposta imunitária é crucial para desenvolver estratégias terapêuticas eficazes para PMM2-CDG e melhorar a qualidade de vida dos doentes.
Glycosylation is involved in several biological processes, and defects in this pathway are associated with pathologies, such as congenital disorders of glycosylation (CDG). Phosphomannomutase 2 (PMM2)-CDG, the most frequent subtype, results from recessive mutations in the PMM2 gene, which encodes an enzyme essential for N-glycan biosynthesis. Reduced PMM2 activity causes hypoglycosylation of glycoproteins, leading to multisystemic involvement, including immunological dysfunction. Currently, there are no effective therapies. However, pharmacological chaperones (PCs), capable of stabilizing misfolded proteins and enhancing enzyme activity, have shown in vitro efficacy in restoring N-glycosylation. This thesis explored two aspects: (i) the therapeutic potential of three PCs previously identified by chemoinformatic tools, and (ii) the impact of defective N-glycosylation on immune dysregulation and susceptibility to infections. Fibroblasts from PMM2-CDG patients and controls were incubated with the PC candidates at different time points. Concanavalin A staining revealed increased N-glycans levels in patient fibroblasts after 72h of treatment with all PCs, although this effect was not sustained at 120h. Analysis of the expression of the enzyme PMM2 and the highly N-glycosylated protein, ICAM-1, showed no significant differences between patients and controls, suggesting that these PCs may improve general N-glycosylation, but may be insufficient to restore the expression or proper glycosylation of specific glycoproteins. To assess immune function, whole blood samples from patients and controls were stimulated with PMA and Ionomycin. Immunophenotyping revealed increased expression of the activation marker CD69 and reduced expression of the immune-checkpoint PD-L1, while cytokine profiling showed elevated IL-2 and IL-6 secretion in stimulated PMM2-CDG samples, indicating that patient blood cells are responsive but may exhibit dysregulated or heightened activity. Understanding the impact of defective glycosylation on pathophysiology and immune response is crucial for developing effective therapeutic strategies for PMM2-CDG and improving patients’ quality of life.
Glycosylation is involved in several biological processes, and defects in this pathway are associated with pathologies, such as congenital disorders of glycosylation (CDG). Phosphomannomutase 2 (PMM2)-CDG, the most frequent subtype, results from recessive mutations in the PMM2 gene, which encodes an enzyme essential for N-glycan biosynthesis. Reduced PMM2 activity causes hypoglycosylation of glycoproteins, leading to multisystemic involvement, including immunological dysfunction. Currently, there are no effective therapies. However, pharmacological chaperones (PCs), capable of stabilizing misfolded proteins and enhancing enzyme activity, have shown in vitro efficacy in restoring N-glycosylation. This thesis explored two aspects: (i) the therapeutic potential of three PCs previously identified by chemoinformatic tools, and (ii) the impact of defective N-glycosylation on immune dysregulation and susceptibility to infections. Fibroblasts from PMM2-CDG patients and controls were incubated with the PC candidates at different time points. Concanavalin A staining revealed increased N-glycans levels in patient fibroblasts after 72h of treatment with all PCs, although this effect was not sustained at 120h. Analysis of the expression of the enzyme PMM2 and the highly N-glycosylated protein, ICAM-1, showed no significant differences between patients and controls, suggesting that these PCs may improve general N-glycosylation, but may be insufficient to restore the expression or proper glycosylation of specific glycoproteins. To assess immune function, whole blood samples from patients and controls were stimulated with PMA and Ionomycin. Immunophenotyping revealed increased expression of the activation marker CD69 and reduced expression of the immune-checkpoint PD-L1, while cytokine profiling showed elevated IL-2 and IL-6 secretion in stimulated PMM2-CDG samples, indicating that patient blood cells are responsive but may exhibit dysregulated or heightened activity. Understanding the impact of defective glycosylation on pathophysiology and immune response is crucial for developing effective therapeutic strategies for PMM2-CDG and improving patients’ quality of life.
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Phosphomannomutase 2 - Congenital Disorders of Glycosylation N-Glycosylation Immune system Pharmacological Chaperones Drug Repurposing