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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.
- Molecular mechanisms of cilia related diseases [editorial]Publication . Carvalhal, Sara; Carmona, Bruno; Tassin, Anne-Marie; Gonçalves, JoãoEukaryotic cilia are fascinating evolutionarily conserved microtubule-based organelles that protrude from the cell surface. In vertebrates, multiple types of motile and primary (immotile) cilia fulfill motility and signaling functions, critical for embryonic development and homeostasis of adult tissues. Importantly, perturbed cilia assembly and functions are associated with a growing number of diseases. This Research Topic gathers an update on recent progress made in understanding the molecular mechanisms of cilia-related diseases. Critically, understanding disease development has been facilitated by advances in technology. For example, the importance of omics techniques for monitoring the progression of cilia-associated rare diseases is showcased in the work of (Jeziorny et al.). This study applied an untargeted metabolomic approach using LC-QTOF-MS to study patients with Alström (ALMS) and Bardet-Biedl (BBS), which shared defective primary ciliary structures and found common molecular fingerprints between ALMS and BBS, and alterations in various lipid metabolites when comparing obese and healthy participants.
- 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.
- 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 maintenance of centriole appendages and motile cilia basal body anchoring relies on TBCCD1Publication . Carmona, Bruno; Camelo, Carolina; Mehraz, Manon; Lemullois, Michel; Faria, Mariana Lince; Coyaud, Étienne; Marinho, H. Susana; Gonçalves, João; Nolasco, Sofia; Pinto, Francisco; Raught, Brian; Tassin, Anne-Marie; Koll, France; Soares, HelenaCentrosomes are organelles consisting of two structurally and functionally distinct centrioles, with the mother centriole having complex distal (DA) and subdistal appendages (SDA). Despite their importance, how appendages are assembled and maintained remains unclear. This study investigated human TBCCD1, a centrosomal protein essential for centrosome positioning, to uncover its localization and role at centrioles. We found that TBCCD1 localizes at both proximal and distal regions of the two centrioles, forming a complex structure spanning from SDA to DA and extending inside and outside the centriole lumen. TBCCD1 depletion caused centrosome mispositioning, which was partially rescued by taxol, and the loss of microtubules (MTs) anchored to centrosomes. TBCCD1 depletion also reduced levels of SDA proteins involved in MT anchoring such as Centriolin/CEP110, Ninein, and CEP170. Additionally, TBCCD1 was essential for the correct positioning of motile cilia basal bodies and associated structures in Paramecium. This study reveals that TBCCD1 is an evolutionarily conserved protein essential for centriole and basal body localization and appendage assembly and maintenance. A BioID screening also linked TBCCD1 to ciliopathy-associated protein networks.
- Cílios e sinalização celular na obesidadePublication . Carmona, BrunoAims of the study: Determine the functional relevance of cilia in the cellular response to high glucose levels. Using retinal pigment epithelium cells: assess the impact of high glucose levels on cilia assembly and morphology; study the role of cilia in the cellular response to glucose-induced stress; understand how the cilia-associated signaling pathways can be involved.
- Tubulin post-translational modifications: the elusive roles of acetylationPublication . Carmona, Bruno; Marinho, H. Susana; Matos, Catarina Lopes; Nolasco, S.; Soares, HelenaMicrotubules (MTs), dynamic polymers of α/β-tubulin heterodimers found in all eukaryotes, are involved in cytoplasm spatial organization, intracellular transport, cell polarity, migration, division, and cilia biology. MTs functional diversity depends on the differential expression of distinct tubulin isotypes and is amplified by a vast number of different post-translational modifications (PTMs). The addition/removal of PTMs to α- or β-tubulins is catalyzed by specific enzymes and allows combinatory patterns largely enriching the distinct biochemical and biophysical properties of MTs, creating a code read by distinct proteins, including microtubule-associated proteins (MAPs), which allow cellular responses. This review is focused on tubulin-acetylation, whose cellular roles continue to generate debate. We travel through the experimental data pointing to α-tubulin Lys40 acetylation role as being an MT stabilizer and a typical PTM of long-lived MTs, to the most recent data, suggesting that Lys40 acetylation enhances MT flexibility and alters the mechanical properties of MTs, preventing MTs from mechanical aging characterized by structural damage. Additionally, we discuss the regulation of tubulin acetyltransferases/desacetylases and their impacts on cell physiology. Finally, we analyze how changes in MT acetylation levels have been found to be a general response to stress and how they are associated with several human pathologies.