Browsing by Author "Carmona, Bruno"
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- 5' and 3' UTR thymidylate synthase polymorphisms modulate the risk of colorectal cancer independently of the intake of methyl group donorsPublication . Carmona, Bruno; Guerreiro, Catarina Sousa; Cravo, Marília; Leitão, Carlos Nobre; Brito, MiguelThymidylate synthase, as a rate-limiting step in DNA synthesis, catalyses the conversion of dUMP into dTMP using 5,10-methylenotetrahydrofolate as the methyl donor. Two polymorphisms have been described in this gene: a repeat polymorphism in the 5' promoter enhancer region (3R versus 2R) and a 6 bp deletion in the 3' unstranslated region. Both of these may affect protein levels. The present case control study was aimed at investigating the influence of these two polymorphisms on the development of colorectal cancer (CRC), as well as their potential interaction with folate, vitamin B6 and vitamin B12 intake. A total of 196 cases and 200 controls, matched for age and sex distribution, were included in the study. No association was found between CRC and the 28 bp repeat polymorphism, but it was observed that individuals with the 6 bp/del and del/del genotypes had a significantly lower risk of developing the disease (OR=0.47; 95% CI 0.30-0.72). A combined genotype (2R/2R; 6 bp/del+del/del) was also found, which was associated with an even lower risk of developing of the disease (OR=0.42; 95% CI 0.26-0.69). No significant interaction between these polymorphisms and vitamin intake was observed. These results indicate for the first time that the 6 bp/del allele might be a protective factor in the development of CRC, independent of the intake of methyl group donors.
- Centrosome positioning and development of ciliopathies: role of the human centrosomal protein TBCCD1Publication . Carmona, Bruno; Camelo, Carolina; Mehraz, Manon; Lemullois, Michel; Ferreira, David C.; Nolasco, Sofia; Lince-Faria, Mariana; Marinho, H. Susana; Bettencourt-Dias, Mónica; Tassin, Anne-Marie; Soares, HelenaAims/Context: Primary cilia are specialized microtubule-based signaling organelles that convey extracellular signaling and cellular polarity into a cellular response. Defects in primary cilia assembly/function cause severe diseases known as ciliopathies, typified by clinical manifestations, like infertility, obesity, brain problems, blindness, and kidney cysts. Primary cilia assembly entails centrosome migration to the plasma membrane where a centriole docks, maturates into a basal body (BB), and assembles the cilia axoneme. The human centrosomal TBCCD1 is a critical factor in centrosome positioning previously identified by us. Our aim is to discover the mechanisms/signals required for the correct positioning of the centrosome during cilia assembly, and how these mechanisms, when compromised, are related to ciliopathies. Methods: The proximity-dependent identification (BioID) assay was used to screen for TBCCD1 interactors. Immunofluorescent and super-resolution microscopy, as well as Western blot, were used to study the levels and cellular localization of the identified TBCCD1 interactors in human RPE1 cells overexpressing or depleted of TBCCD1. To study the impact of TBCCD1 knockdown in motile cilia the ciliate Paramecium, containing ∼3,000 motile cilia, was used. Results: Our BioID screen for TBCCD1 interactors identified several well-known proteins encoded by ciliopathy genes, e.g. the centrosomal protein OFD1 involved in the Orofacial-Digital Syndrome. We show that TBCCD1 knockdown and overexpression in RPE1 cells affects OFD1 distribution. Super-resolution microscopy shows TBCCD1 is localized at the distal region of the centrosome and that its depletion dramatically affects the centrosome subdistal protein CEP170, a component of cilia basal feet. In Paramecium, the TBCCD1 knockdown causes abnormal BB-associated structures organization and anomalous BB positioning/anchoring defects. Conclusions: Our data support a role for TBCCD1 in the maintenance of centrosome structure and in BB anchoring at the cell membrane during ciliogenesis. TBCCD1 is emerging as a novel protein with a role in human ciliopathies.
- Cilia distal domain: diversity in evolutionarily conserved structuresPublication . Soares, Helena; Carmona, Bruno; Nolasco, Sofia; Melo, Luís Viseu; Gonçalves, JoãoEukaryotic cilia are microtubule-based organelles that protrude from the cell surface to fulfill sensory and motility functions. Their basic structure consists of an axoneme templated by a centriole/basal body. Striking differences in ciliary ultra-structures can be found at the ciliary base, the axoneme and the tip, not only throughout the eukaryotic tree of life, but within a single organism. Defects in cilia biogenesis and function are at the origin of human ciliopathies. This structural/functional diversity and its relationship with the etiology of these diseases is poorly understood. Some of the important events in cilia function occur at their distal domain, including cilia assembly/disassembly, IFT (intraflagellar transport) complexes' remodeling, and signal detection/transduction. How axonemal microtubules end at this domain varies with distinct cilia types, originating different tip architectures. Additionally, they show a high degree of dynamic behavior and are able to respond to different stimuli. The existence of microtubule-capping structures (caps) in certain types of cilia contributes to this diversity. It has been proposed that caps play a role in axoneme length control and stabilization, but their roles are still poorly understood. Here, we review the current knowledge on cilia structure diversity with a focus on the cilia distal domain and caps and discuss how they affect cilia structure and function.
- 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.
- 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.
- Colchicine blocks tubulin heterodimer recycling by tubulin cofactors TBCA, TBCB, and TBCEPublication . Nolasco, Sofia; Bellido, Javier; Serna, Marina; Carmona, Bruno; Soares, Helena; Zabala, Juan CarlosColchicine has been used to treat gout and, more recently, to effectively prevent autoinflammatory diseases and both primary and recurrent episodes of pericarditis. The anti-inflammatory action of colchicine seems to result from irreversible inhibition of tubulin polymerization and microtubule (MT) assembly by binding to the tubulin heterodimer, avoiding the signal transduction required to the activation of the entire NLRP3 inflammasome. Emerging results show that the MT network is a potential regulator of cardiac mechanics. Here, we investigated how colchicine impacts tubulin folding cofactors TBCA, TBCB, and TBCE activities. We show that TBCA is abundant in mouse heart insoluble protein extracts. Also, a decrease of the TBCA/β-tubulin complex followed by an increase of free TBCA is observed in human cells treated with colchicine. The presence of free TBCA is not observed in cells treated with other anti-mitotic agents such as nocodazole or cold shock, neither after translation inhibition by cycloheximide. In vitro assays show that colchicine inhibits tubulin heterodimer dissociation by TBCE/TBCB, probably by interfering with interactions of TBCE with tubulin dimers, leading to free TBCA. Manipulation of TBCA levels, either by RNAi or overexpression results in decreased levels of tubulin heterodimers. Together, these data strongly suggest that TBCA is mainly receiving β-tubulin from the dissociation of pre-existing heterodimers instead of newly synthesized tubulins. The TBCE/TBCB+TBCA system is crucial for controlling the critical concentration of free tubulin heterodimers and MT dynamics in the cells by recycling the tubulin heterodimers. It is conceivable that colchicine affects tubulin heterodimer recycling through the TBCE/TBCB+TBCA system producing the known benefits in the treatment of pericardium inflammation.
- From centrosomal microtubule anchoring and organization to basal body positioning: TBCCD1 an elusive proteinPublication . Carmona, Bruno; Camelo, Carolina; Mehraz, Manon; Lemullois, Michel; Ferreira, David C.; Nolasco, Sofia; Lince-Faria, Mariana; Marino, H. Susana; Bettencourt-Dias, Mónica; Tassin, Anne-Marie; Koll, France; Soares, HelenaCilia are microtubule-based organelles that protrude from the cell surface and fulfill critical motility and sensory functions being required for normal embryonic development and for homeostasis of human adult tissues. Cilia loss or dysfunction is associated with human ciliopathies. At their base cilia have a centriole/basal body (BB), which can be derived from the centrosome and assembles the ciliary axoneme. This process requires the correct positioning/anchoring of the centrosome’s mother centriole/BB to the cell membrane. A clear picture of the different signals and players involved in centrosome positioning/anchoring is still not available. Published work from our group identified a new centrosomal TBCC domain-containing human protein (TBCCD1) that is involved in centrosome correct positioning and primary cilia assembly. In mammalian cells, TBCCD1 is observed at pericentriolar satellites, in basal bodies of primary and motile cilia and at primary cilia ciliopathy hot domain, the transition zone. Super-resolution microscopy shows that TBCCD1 is localized at the distal region of the centrosome and its depletion dramatically affects the centrosome subdistal protein CEP170, a component of primary and motile cilia basal feet. By doing a proximity-dependent biotin identification (BioID-MS) screen for TBCCD1 interactors several well-known proteins encoded by ciliopathy genes were identified, e.g. the centrosomal proteins OFD1 and Moonraker/KIAA0753 associated with Digital Syndrome 1 and Joubert syndrome, respectively. OFD1 and Moonraker are required for the maintenance of centrosome structure and both proteins localization is dramatically disturbed by TBCCD1 depletion. To clarify the role of human TBCCD1 in cilia biogenesis we used the ciliate Paramecium. Noteworthy, in Paramecium TBCCD1 knockdown causes abnormal basal body associated rootlets organization, anomalous BB positioning/anchoring defects. Our data using human cells and the ciliate Paramecium support a role of TBCCD1 in centrosome structure maintenance and BB anchoring at the cell membrane. The Paramecium phenotypes confirm that TBCCD1 is a new candidate to a ciliopathic gene probably by founding the TBCCD1/Moonraker/OFD1 functional conserved module required for cilia assembly.
- 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.
- Functional interactions of the ciliopathy-involved protein KIAA0753 with the regulator of centrosome positioning TBCCD1Publication . Ferreira, David C.; Carmona, Bruno; Nolasco, Sofia; Marinho, H. Susana; Soares, Helena
- How many cell polarity related genes are conserved from Tetrahymena to metazoa?Publication . Carmona, Bruno; Soares, HelenaCell polarity can be seen as an asymmetric distribution and spatial arrangement of biomolecules, cellular components (e.g., membrane domains and organelles such as the Golgi apparatus, mitochondria, cilia, and others) and cytoskeleton such that, their specific positioning in the cell, in close relationship with their functions, generates a structural/functional asymmetry that can be conserved and transmitted to new cells during cell division. In fact, cell polarity controls the morphology from single cells to whole tissues. Cellular organizational/functional asymmetry is required for a variety of cell functions in both unicellular and multicellular organisms such as correct symmetric and asymmetric cell division, differentiation, motility and cell migration. Moreover, in mammalian cells, polarity can be challenged by environmental cues, and cells are able to remodel their intrinsic polarity. The ciliate Tetrahymena thermophila is a highly differentiated cell organism that possesses a permanent anterior-posterior axis and left-right asymmetry. Tetrahymena cells are also characterized by a complex cortex where basal bodies are longitudinally arranged in close association with cytoskeleton appendages and networks originating a complex pattern. The molecular mechanisms that control the formation and regeneration of this complex cortical patterning in each daughter cell after cytokinesis are still not well understood. We have shown that the Tetrahymena Mob1 protein is essential for maintenance and regeneration of cell polarity, proper cell proportions, correct division plane placement, and finally to cytokinesis completion. At the time, Mob1 was already described as a member of the mitotic exit network, a signaling cascade that controls mitosis to interphase transition. In metazoans, Mob1 is a member of the Hippo signaling pathway, a major conserved mechanism governing cell contact inhibition and organ size control. Due to its cell features Tetrahymena emerges as a good model to address the regulatory mechanisms underlying cell polarity/morphogenesis/and cell division. To test this idea we went throughout the Tetrahymena genome looking for genes already described to be involved in cell polarity in other unicellular and multicellular model organisms. Interestingly, some of these core genes involved in cell polarity appear to be conserved in this ciliate. In this presentation we will discuss our findings.
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