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- The tumor suppressor p53 acquires oncogenic functions due to a translational switch during integrated stress responsePublication . Lacerda, Rafaela; Fonseca Costa, Inês; López-Iniesta, M.; Romão, Luísa; Candeias, Marco M.To cope with the stress stimuli to which they are often exposed, eukaryotic cells have developed adaptive pathways that restore cellular homeostasis. Under stress conditions there is an overall decrease of protein synthesis, and a concomitant induction of alternative mechanisms of mRNA translation initiation. The tumour suppressor protein p53 has been considered the guardian of the genome and a master regulator of many cellular functions. However, apart from the full-length p53 (FLp53), several p53 isoforms have been described so far. Some functions of shorter p53 isoforms have already been elucidated and they are different from and complement FLp53 activity, the most mutated gene in cancer. Here we show that the integrated stress response (ISR) leads to the specific induction of Δ160p53 isoform. Using bicistronic constructs we confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA that controls Δ160p53 isoform translation. Subjecting cells to endoplasmic reticulum stress showed that eIF2α phosphorylation is a key event leading to cap-independent expression of Δ160p53 during ISR. Additionally, cancer-specific mutations in p53 also enhanced cap-independent translation of Δ160p53 via Δ160p53IRES. An antisense morpholino oligo targeting Δ160IRES efficiently reduced Δ160p53 protein levels and significantly impaired oncogenic functions of Δ160p53. Our data support a model in which an IRES structure in the coding region of p53 is activated under stress conditions, leading to the expression of the oncogenic shorter Δ160p53 isoform, whose structure is affected by cancer-specific mutations in the p53 gene. A better understanding of Δ160p53IRES structure and function may be advantageous for a more efficient therapeutic targeting of p53.
- A first step to open the neuronal box of Gaucher CellsPublication . Ribeiro, Diogo; Duarte, Ana; Santos, Renato; Amaral, OlgaThis work focuses on the differentiation and gene expression characterization of neural progenitor cells obtained from human induced pluripotent cells (hiPSCs) reprogrammed from type 3 GD (GD3) fibroblasts. GD3 patient fibroblasts (from an international cell bank) were cultured and reprogramed as previously described (https://doi.org/10.1016/j.scr.2019.101595). The resulting hiPSCs were differentiated into pre-neuronal cells and, at this stage, they were examined. The gene expression behavior of all neurogenesis genes (NES, MAP2 and OTX2) was similar but higher expression was observed in GD3 hiPSCs than in GD3 neural progenitor cells. With this work, we can conclude that, when working with hiPSCs in the process of creating disease-specific cell models it is most important to carry out a general gene expression characterization of the different cell lines involved in all stages.
- Regulation of IRES-mediated translation in p53Publication . Fonseca Costa, Inês; Lacerda, Rafaela; López-Iniesta, M.; Romão, Luísa; Candeias, Marco M.The tumor microenvironment is characterized by several stresses impairing canonical translation. However, specific mRNAs harbouring internal ribosome entry sites (IRES), such as several tumour suppressors and oncogenes, can overcome this impairment. The tumor suppressor TP53 gene, an important transcription factor that ensures cellular homeostasis, is frequently mutated in human cancers. Over the years, several p53 isoforms have been identified, which in some cases result from alternative initiation of translation regulated by an IRES. Recently, we have associated mutant p53 “gain-of-function” cancer phenotype, such as enhanced cell survival, invasion, proliferation, and adhesion, with the expression of higher levels of shorter p53 isoforms, such as Δ160p53 isoform.1 Here, we used a bicistronic system containing two reporter luciferases (renilla luciferase and firefly luciferase) to assess IRES-mediated translation. Several p53 mRNA elements were tested in this system and, interestingly, we have found an inhibitory element of IRES-mediated translation. Overall, IRES-regulated translation in malignant cells is used to translate specific proteins that promote cancer progression. Thus, inhibiting translation of oncogenes via IRES could prevent the formation of tumor cells and their adaptation to unfavourable conditions in the tumor microenvironment. 1. Candeias, M. M., Hagiwara, M. & Matsuda, M. Cancer‐specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis. EMBO Rep. 17, 1542–1551 (2016).
- Identification of mTOR and AGO1 IRES trans-acting factorsPublication . Marques, Rita; Lacerda, Rafaela; Romão, LuísaCancer is the second leading cause of death globally; therefore, its study is crucial to discover new therapies. Under stress, the regular process of protein synthesis (canonical translation) is impaired, while a back-up mechanism mediated by internal ribosome entry sites (IRES) continues to function, allowing the synthesis of proteins that maintain cellular viability. This also happens in cancer cells, contributing for their survival and consequent tumorigenesis. IRES-mediated translation and its regulation by IRES trans-acting factors (ITAFs) has been correlated to metastasis and chemotherapeutic drug resistance. Therefore, our main goal was to validate ITAFs and assess their significance in cancer onset, thus becoming candidates as novel therapeutic targets. A bicistronic reporter system, which contains a first cistron translated via canonical translation and a second one translated by IRES of mTOR1 and AGO12 was used to test IRES-driven translation initiation activity. Experiments were carried out in which several proteins (hnRNPs) were silenced by specific siRNAs to analyse their function as ITAFs of mTOR and AGO1 IRESs. Also, distinct drugs were applied to simulate endoplasmic reticulum (ER) or hypoxia stress, to evaluate their effect on IRES activity. The relative IRES activity was assessed by luminescence tests and the protein levels by Western blot. In general, knockdown of hnRNPK and hnRNPU seems to decrease the IRES activity by ~60% and ~30% respectively, while hnRNPC knockdown does not show a significant effect. Regarding the ER stress, hnRNPK knockdown seems to decrease even more the IRES activity, while hnRNPU depletion induces a significant increase. On the other hand, in hypoxia, the hnRNPs knockdowns do not significantly affect IRES activity. These results indicate that hnRNPK and hnRNPU may function as ITAFs of mTOR and AGO1 IRES activity in cells under ER stress. Our data can be decisive for a better understanding of carcinogenesis and suggest new therapeutic targets for cancer treatment. 1. Marques-Ramos, A., et.al. 2017. RNA. 23, 1712-1728 2. Lacerda, R. 2016. Faculdade de Ciências e Tecnologia da Universidade NOVA de Lisboa
- A machine learning approach for the prioritization of disease-associated genetic variants from ngs datasetsPublication . Martiniano, Hugo; Vilela, Joana; Marques, Ana R.; Santos, João; Rasga, Célia; Vicente, AstridOur objective is to develop variant prioritization methods using machine learning approaches to predict genotype-phenotype associations, that can detect the most likely causal variants associated to a given phenotype.
- Translational Regulation of the Human PERK by Upstream Open Reading FramesPublication . Fernandes, Rafael; Lopes, Pedro; Romão, LuísaUpstream open reading frames (uORFs) are cis-acting elements located within the 5’ leader sequence (5’UTR) of transcripts, which can regulate translation of the correspondent main open reading frame (mORF). During endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins activates the ER-resident PKR-like ER kinase (PERK), which results in phosphorylation of eIF2α to inhibit global mRNA translation, while allowing the selective uORF-mediated translation of downstream effectors responsible for stress resolution or, ultimately, cell death. The dual role of PERK in regulating cell fate was implicated in human diseases, like diabetes, neurodegenerative disorders and cancer. Moreover, mutations in the EIF2AK3 gene (encoding PERK) were associated to the rare genetic disease, Wolcott-Rallison Syndrome (WRS). In this work, we aimed to study the translational regulatory role of 5 AUG- and 3 non-AUG-uORFs identified in the PERK 5’UTR and assess its biological relevance. While uORF2 and the non-AUG-uORFs 5, 6 and 7 (numbered according to their distance to the 5’ end of the mRNA) do not seem to have a regulatory role, uORF1, uORF3, uORF4 and uORF8 together present a strong repressive effect over mORF translation in basal conditions. Curiously, we found that when PERK is overexpressed, it leads to the spontaneous activation of a portion of PERK in the absence of any stress stimulus, possibly highlighting the biological relevance of its uORF-mediated translational regulation. Conversely, during ER stress, increased bypass of uORF1 results in a modest degree of translational de-repression, which may help to counterbalance the increased rate of PERK protein turnover observed in these conditions. We also observed that alteration of the PERK uORFs by mutations found in WRS patients modify mORF expression, providing a possible link to the disease. Altogether, we highlight the importance of including 5’UTRs in the screening of disease-related mutations and the necessity of functional studies to assess their role in pathogenesis.
- Induced Pluripotent Stem Cells derived cardiomyocytes from a patient with Fabry Disease: a work in process cell modelPublication . Duarte, Ana; Ribeiro, Diogo; Bragança, Jose; Amaral, OlgaFabry disease (FD) is one of the commonest Lysosomal Storage Disorders (LSDs) and is caused by mutations in the alpha-galactosidase A gene (GLA) from which results a deficient activity of the lysosomal hydrolase alphagalactosidase A (α-Gal A). This deficiency leads to progressive multisystemic accumulation of glycolipids, namely, globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3), in plasma and in a wide range of cells, particularly in the relevant cells affected by the disease like vascular endothelial cells, podocytes, cardiomyocytes, and arterial smooth muscle cells. Taking FD as a cardiac disorder, our aim was to differentiate induced pluripotent stem cells (iPSCs) reprogrammed from FD patients’ fibroblasts into cardiomyocytes, a cellular type usually targeted by this disease. Since only mature cardiomyocytes can fully recapitulate the same disease phenotypes present in vivo and serve as an accurate disease model, we have to surpass the immature state to achieve a complete disease model.
- Identification of neurotransmission and synaptic risk genes and disrupted biological processes in Autism Spectrum DisorderPublication . Vilela, Joana; Martiniano, Hugo; Marques, Ana Rita; Santos, João; Rasga, Célia; Oliveira, Guiomar; Vicente, Astrid M.The objective of this study is the identification of Single Nucleotide Variants (SNVs) in neurotransmission and synaptic genes (NS genes) that play a role in ASD etiology, and the biological processes and global networks affected.
- Interplay between glycemia and the genetics of ENOS and ACE genes for the susceptibility to the onset and development of hypertension on the Portuguese populationPublication . Aguiar, Laura; Ferreira, Joana; Matos, Andreia; Mascarenhas, Mário Rui; Menezes Falcão, Luiz; Faustino, Paula; Bicho, Manuel; Inácio, ÂngelaIntroduction: Hypertension is a multifactorial condition of anthropometric, physiologic, metabolic, genetic, and environmental nature. In Portugal, the mean prevalence of hypertension in the population is 45.5%. Objective: The aim of this study is to evaluate the contribution of anthropometric, physiological, and genetic factors (eNOS and ACE) to the development of hypertension in a Portuguese population. Methods: A case-control study was conducted in a sample of 377 individuals, 243 hypertensives, and 134 normotensives. The polymorphic analyses of intron 4 VNTR in the eNOS gene and the insertion/deletion (I/D) in ACE gene were performed by polymerase chain reaction (PCR). Results: High body mass index (BMI) values, high glycemia levels, and the 4a allele of the eNOS were associated with hypertension. Among the hypertensive group, the allele 4a (eNOS) was associated with high levels of HbA1c, and the D allele (ACE) with glycemia. Conclusion: Our results highlight the contribution of eNOS and ACE genes as important players for the onset and development of hypertension in the Portuguese population. We believe that a combinatory clinical approach including the traditional anthropomorphic and physiological parameters together with genetic studies can be more elucidative in establishing a susceptibility profile on multifactorial conditions as hypertension.
- Genetics as part of health science education: reflecting on a changing approachPublication . Amaral, Olga; Ferreira, FilipaGenetics goes beyond the human species and this should be the paramount power of genetics education. The current wave of interest in Science should be the force towards a change in the way information reaches the public. The prior underestimation of the understanding capabilities of the student and non-student populations resulted in a loss of engagement in science and might lead to misinformation. In the past decades, INSA has had an approach to communication in Health Science that depends on strong views and areas of expertise, various programmed activities and, as in research, it depends on expectations. Along the line of community involvement, GENEtic.COMunication (GENE.COM) is a project submitted to a competitive call launched by Ciencia Viva. With that project, the focus is on a niche of knowledge with great impact on Health, in society, as well as on personal and public budgets: the area of the rare genetic metabolic diseases. The project intended to contribute to the dissemination of information about Human Genetics, particularly in the area of rare genetic diseases. The aim was to communicate knowledge in an easily understandable way; inform health professionals, patients, students, teachers, as well as patient associations. This contact could provide the establishment of a dialogue between professionals working in the field and different publics. Furthermore, the switch to virtual tools relies on easier access to the information while implying diminished associated costs, such as transportation, time and space limitations. The problem may be how to make passive learning more engaging. As we know, assessment drives learning and there are simple and quick ways to assess the efficacy of the “take home message”, which may improve engagement and provide the needed feedback. Attempts to contribute to a more informed society should be disclosed and supported as they can lead to more positive attitudes and empowered choices. By providing digital contents in a way that they can be followed by various target audiences we also aim at reaching audiences that would not typically seek us.