Browsing by Author "Marinho, Susana H."
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- Os cílios primários regulam os níveis de tiorredoxina redutase 1 e de yH2AX em resposta a níveis elevados de glucosePublication . Marques, Rira; Paiva, Mariana; Ginete, Catarina; Nolasco, Sofia; Marinho, Susana H.; Veiga, Luisa; Brito, Miguel; Soares, Helena; Carmona, BrunoA diabetes caracteriza-se por uma anormal capacidade de controlar o nível de glucose na corrente sanguínea, podendo levar a outras complicações, tais como hipertensão, doenças cardiovasculares, e retinopatia. A desregulação dos níveis de glucose na retina tem demonstrado aumentar os níveis de peróxido de hidrogénio, conduzindo a uma rutura na barreira sanguínea da retina, uma das causas de retinopatia diabética. O cílio primário é um organelo que demonstrou ter um papel no controlo do equilíbrio energético e da homeostase da glucose. Defeitos na estrutura e função dos cílios podem resultar no desenvolvimento de várias doenças, conhecidas como ciliopatias, e que incluem fenótipos como obesidade e diabetes. Neste trabalho pretendemos estudar o papel do aumento dos níveis de glucose na montagem de cílios primários em culturas de células do epitélio pigmentar da retina (RPE-1), bem como o papel dos cílios na resposta celular aos níveis elevados de glucose. Para isso, suplementámos os meios de crescimento das células RPE-1 com diferentes concentrações de glucose (5 mM, 25 mM e 5 mM de glucose + 20 mM manitol). Estas células também foram induzidas a montar cílios antes ou depois da suplementação com a glucose. Neste estudo observámos que a suplementação de glucose não afetou o número de células ciliadas, sendo que o comprimento dos cílios foi menor em células suplementadas com 25 mM de glucose. Também avaliámos os níveis nucleares de tiorredoxina redutase 1 (TXNRD1), uma das principais enzimas intervenientes na resposta ao stress oxidativo desencadeado pela hiperglicemia, e de γH2AX, um marcador celular de quebras no DNA e de senescência celular. Observámos que os níveis nucleares de TXNRD1 e de γH2AX são afetados pela adição de glucose e que a existência de cílios modula a resposta das células em resposta a níveis elevados de glucose. Estes resultados mostram que a presença de cílios primários afeta drasticamente a resposta celular às elevadas concentrações de glucose que provavelmente induzem o stress oxidativo, podendo ter um papel crucial no desenvolvimento de retinopatia diabética.
- From cilia to cancer: the two splicing variants of the human TBCCD1 genePublication . Carmona, Bruno; Justino, Gonçalo; Matos, Catarina; Pádua, Mário; Nolasco, Sofia; Marinho, Susana H.; Soares, HelenaAlmost all human genes that contain multiple exons undergo alternative splicing. Therefore, a single gene can originate multiple mRNA isoforms which causes a dramatic increase in the variability of the expected proteome. Noteworthy, phenotypic variability and disease susceptibility in human populations are related to alternative splicing. Published work from our group identified a new human centrosomal protein, TBCC domain-containing 1 (TBCCD1). Our studies revealed that this gene undergoes alternative splicing producing at least two transcripts encoding proteins. Here we analyze the differential functions of the two splicing variants (TBCCD1v1 and TBCCD1v2). Both variants present distinct cellular localization being TBCCD1v1 essentially centrosomal, whereas TBCCD1v2 is cytoplasmatic. The screening for TBCCD1v2 proximity interactome using BioID identified 19 proteins that functionally group in kinetochore, MT/cilia, and DNA-binding proteins. Striking, the overexpression of TBCCD1v2 decreases the levels of the kinetochore protein CENP-M, a protein upregulated in tumors. On the other hand, the TBCCD1v1 is involved in MT organization and is required to maintain the distal structure of the mother centriole. Our BioID screening for TBCCD1v1 interactors revealed 82 distinct proteins including several well-known proteins encoded by ciliopathy genes. A wider analysis of how TBCCD1v1 levels impact cellular physiological proteome showed that the group of proteins presenting fold changes in their levels vs control cells is enriched in proteins involved in focal adhesions, namely HSPA5/GRP-78/BiP, PDIA3, RPS10, MSN, TGM2, and PPP1R12A. Together our results show that we are still far from having a complete picture of the functional importance of TBCCD1 and how its deregulation may be associated not only with the development of ciliopathies but also with more common diseases like cancer.
- The nuclear levels of thioredoxin reductase 1, gamma-H2AX, and yap are modulated by primary cilia in response to high glucose levelsPublication . Marques, Rita; Paiva, Mariana; Ginete, Catarina; Nolasco, Sofia; Marinho, Susana H.; Veiga, Luisa; Brito, Miguel; Soares, Helena; Carmona, BrunoDiabetes is a condition characterized by impaired regulation of blood glucose levels, leading to various complications such as hypertension, cardiovascular disease, and retinopathy. Diabetic retinopathy (DR), caused by a disrupted retinal blood barrier, is associated with oxidative stress resulting from dysregulated glucose levels in the retina. The primary cilium, an organelle involved in energy balance and glucose homeostasis, has been implicated in the development of various diseases known as ciliopathies, which include overlapping phenotypes such as obesity, diabetes, and retinopathy. This study aims to investigate the impact of high glucose levels on primary cilia assembly in retinal pigment epithelium (RPE-1) cell cultures and explore the role of cilia in the cellular response to high glucose levels. RPE-1 cells were grown in media supplemented with different glucose concentrations (5 mM, 25 mM, and 5 mM glucose + 20 mM mannitol), and cilia assembly was induced before or after glucose supplementation. The results revealed that glucose supplementation did not affect the number of ciliated cells, but cells supplemented with 25 mM glucose exhibited shorter cilia. To understand the role of cilia in response to high glucose levels, the nuclear levels of thioredoxin reductase 1 (TRXR1), a key enzyme involved in combating oxidative stress triggered by hyperglycemia, were evaluated. Additionally, γH2AX, a marker of DNA breaks and cellular senescence, and YAP, a Hippo pathway effector, were examined. It was observed that glucose supplementation, particularly at high levels (25 mM), influenced the nuclear levels of TRXR1, γH2AX, and YAP. Notably, the presence of cilia modulated the cellular response to high glucose levels, modulating the levels of these proteins. These preliminary findings indicate that primary cilia significantly influence the cellular response to high glucose concentrations, which are known to induce oxidative stress and potentially contribute to the development of DR.