Browsing by Author "Nolasco, Sofia"
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- Ageing affects the CD4+ T cell polarization and mucosal tropism induced by TLR2/TLR4-activated dendritic cellsPublication . Zúquete, Sara; Ferreira, Mariana; Delgado, Inês L.; Rosa, Maria teresa; Mendes, Ana Catarina; Santos, Dulce; Nolasco, Sofia; Graça, Luís; Leitão, Alexandre; Basto, Afonso P.Toll-like receptor (TLR)2 activation induces aldehyde dehydrogenase enzymes in non-mucosal dendritic cells (DCs), enabling them to metabolize vitamin A into all-trans retinoic acid, which induces the expression of mucosal-homing molecules (α4β7 and CCR9) in the activated T cells. Recently, we have shown that the simultaneous activation of non-mucosal DCs through TLR2 and TLR4 maintains such capacity while reinforcing the polarization of primed CD4+ T cells towards Th1. Here, we observed that TLR2/TLR4 stimulation of aged DCs leads to the production of less TNFα and more IL-10, and that CD4+ T cells primed by those DCs express lower levels of the mucosal homing receptor CCR9 and produce less type-1 (IFNγ) and more type-2 (IL-4 and IL-13) cytokines. These results emphasize the importance of considering the age-related alterations in DC function when developing novel immunomodulation strategies that rely on the DC-T cell crosstalk through stimulation of pattern recognition receptors.
- Balancing act: tubulin glutamylation and microtubule dynamics in Toxoplasma gondiiPublication . Delgado, Inês; Gonçalves, João; Fernandes, Rita; Zúquete, Sara; Basto, Afonso P.; Leitão, Alexandre; Soares, Helena; Nolasco, SofiaThe success of the intracellular parasite Toxoplasma gondii in invading host cells relies on the apical complex, a specialized microtubule cytoskeleton structure associated with secretory organelles. The T. gondii genome encodes three isoforms of both α- and β-tubulin, which undergo specific post-translational modifications (PTMs), altering the biochemical and biophysical proprieties of microtubules and modulating their interaction with associated proteins. Tubulin PTMs represent a powerful and evolutionarily conserved mechanism for generating tubulin diversity, forming a biochemical 'tubulin code' interpretable by microtubule-interacting factors. T. gondii exhibits various tubulin PTMs, including α-tubulin acetylation, α-tubulin detyrosination, Δ5α-tubulin, Δ2α-tubulin, α- and β-tubulin polyglutamylation, and α- and β-tubulin methylation. Tubulin glutamylation emerges as a key player in microtubule remodeling in Toxoplasma, regulating stability, dynamics, interaction with motor proteins, and severing enzymes. The balance of tubulin glutamylation is maintained through the coordinated action of polyglutamylases and deglutamylating enzymes. This work reviews and discusses current knowledge on T. gondii tubulin glutamylation. Through in silico identification of protein orthologs, we update the recognition of putative proteins related to glutamylation, contributing to a deeper understanding of its role in T. gondii biology.
- Balancing act: tubulin glutamylation and microtubule dynamics in Toxoplasma gondiiPublication . Delgado, Inês L.S.; Gonçalves, João; Fernandes, Rita; Zúquete, Sara; Basto, Afonso P.; Leitão, Alexandre; Soares, Helena; Nolasco, SofiaThe success of Toxoplasma gondii (intracellular parasite) host cell invasion relies on the apical complex, a specialized microtubule cytoskeleton structure associated with secretory organelles. The genome encodes three isoforms of both α- and β-tubulin which are altered by specific post-translational modifications (PTMs), changing the biochemical/biophysical proprieties of microtubules, and modulating their interaction with associated proteins. Tubulin PTMs are a powerful and evolutionarily conserved mechanism to generate tubulin diversity, forming a biochemical ‘tubulin code’ that can be ‘read’ by microtubule-interacting factors. The T. gondii tubulin PTMs are: α-tubulin acetylation, α-tubulin detyrosination, Δ5α-tubulin, Δ2α-tubulin, α- and β-tubulin polyglutamylation, and α- and α-tubulin methylation. Tubulin glutamylation is a key candidate to assist microtubule remodeling in Toxoplasma, being involved in the regulation of microtubule stability, dynamics, interaction with motor proteins, and severing enzymes. The correct balance of tubulin glutamylation is achieved by the coordinated action of polyglutamylases and deglutamylating enzymes. In this work, we will review and discuss the current knowledge on T. gondii tubulin glutamylation. By in silico identification of mammalian protein orthologs, we explored and updated the identification of putative proteins related to glutamylation, contributing to a better understanding of the role of tubulin glutamylation in T. gondii.
- Besnoitia besnoiti and Toxoplasma gondii: two apicomplexan strategies to manipulate the host cell centrosome and Golgi apparatusPublication . Cardoso, Rita; Nolasco, Sofia; Gonçalves, João; Cortes, Helder C.; Leitão, Alexandre; Soares, HelenaBesnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.
- Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modellingPublication . Marcelino, Eduardo; Martins, Tiago M.; Morais, Joana B.; Nolasco, Sofia; Cortes, Helder; Hemphill, Andrew; Leitão, Alexandre; Novo, CarlosBesnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.
- CCTαPublication . Soares, Helena; Nolasco, SofiaIn the cell, the correct folding of many proteins depends on the function of preexisting ones known as Molecular Chaperones (for a review see Hartl and Hayer-Hartl 2009). These, were defined as proteins that bind to and stabilize an otherwise unstable conformation of another protein, and by controlling binding and release, facilitate its correct fate in vivo, be it folding, oligomeric assembly, transport to a particular subcellular compartment, or disposal by degradation. Molecular chaperones do not convey steric information specifying correct folding: instead, they prevent incorrect interactions within and between nonnative peptides, thus typically increasing the yield but not the rate of folding reactions. Molecular chaperones are ubiquitous and comprise several protein families that are structurally unrelated (Hartl and Hayer-Hartl 2009). The Hsp70s and the Chaperonin families have been extensively studied.
- 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.
- Characterization of the Tubulin-binding cofactor B in Toxoplasma gondii, a zoonotic parasitePublication . Delgado, I.; Francisco, S.; Santos, D.; Zúquete, S.; Leitão, A.; Soares, Helena; Nolasco, SofiaTubulin cofactors participate in the folding, dimerization, and dissociation pathways of the tubulin dimer, being implicated in the control of tubulin proteostasis and consequently in the control of microtubule (MT) dynamics in vivo. We identified a gene coding for a Tubulin cofactor B in the T. gondii genome and characterized its protein (tgTBCB) in the tachyzoite stage.
- 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.
- 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.