Percorrer por autor "Pinto, Francisco"
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- Integrative network analysis for identification of new proteins involved in NMD or its regulationPublication . Nogueira, Gonçalo; Romão, Luísa; Pinto, FranciscoBackground: Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation termination codons (PTCs). NMD has been associated with many genetic diseases and some forms of cancer caused by nonsense or frameshift mutations that introduce PTCs. NMD is a complex process that relies on the involvement of numerous players which interact with each other in a highly organized manner. However, the interactions and connectivity among these components is only partly elucidated. Recent studies have shown that NMD is also involved in the regulation of apparently “normal” transcripts, suggesting a greater role in gene expression regulation.
- Integrative network approach to identify new players involved in NMD or its regulationPublication . Nogueira, Gonçalo; Pinto, Francisco; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation-termination codons (PTCs). The physiological importance of NMD is manifested by the fact that about one third of all genetic diseases and some forms of cancer are caused by nonsense or frameshift mutations that introduce PTCs, and NMD can modulate the clinical phenotype of these diseases. Noteworthy, in total, genetic diseases attributable to PTCs affect millions of patients worldwide. Recent studies have shown that NMD also targets mRNAs transcribed from a large subset of wild-type genes, shaping their levels. NMD is a complex process where several proteins interact with each other and cooperate to induce degradation of a given transcript. Although this pathway has been extensively studied, the interactions and connectivity among these components is only partly elucidated. Aiming to expand the knowledge about the NMD pathway, we are combining bioinformatics, network analysis and experimental work to identify new proteins involved in NMD or its regulation. Our work, begins with a network analysis approach that integrates publicly available data regarding different types of interactions: 1) protein-protein, 2) kinase-target, 3) phosphatase-target, 4) miRNA-target, 5) transcription factor-target, 6) gene co-expression and 7) ubiquitination-target. Additionally, our network include data regarding known NMD-targets and NMD-triggering features. The generated network will be used to find novel NMD-associated proteins, prioritizing candidates with simultaneous interactions with different mRNA processing pathways (mRNA splicing, mRNA transport, mRNA translation and mRNA decay). Following data integration, we will develop a scoring algorithm to select the most central proteins in the generated network, which can be essential to further understand NMD and its regulation. The predicted candidates will be experimentally validated and their role in NMD will be tested. Due to the diversity of regulatory links integrated in this workflow, we propose it can be applied to find molecular bridges between related biological processes and generate novel hypotheses about the molecular mechanisms co-regulating these phenomena.
- Integrative network approach to predict new NMD-associated proteinsPublication . Nogueira, Gonçalo; Romão, Luísa; Pinto, FranciscoIn our study, we apply such approach to predict new players involved in nonsense-mediated mRNA decay (NMD), a regulatory pathway of mRNA translation that recognizes and selectively degrades specific mRNAs. NMD is a complex process that relies on the involvement of numerous players, however the interactions and connectivity among these components is only partly elucidated.
- Multi-Network approach to predict new proteins involved in NMDPublication . Nogueira, Gonçalo; Pinto, Francisco; Romão, LuísaThe mechanism of nonsense-mediated decay (NMD) selectively degrades mRNAs carrying a premature translation-termination codon and regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. Although this complex process has been extensively studied along the years, the interactions and connectivity among NMD players is not completely understood. Additionally, some NMD mechanistical aspects suggest missing roles that can be played by proteins still not reported as involved in this pathway. To tackle this hypothesis, we developed a bioinformatic network-based approach to predict new proteins involved in NMD. Our approach consists in performing several queries to different types of publicly available data, in order to explore the ability of proteins to bridge related processes, while integrating data regarding protein-protein interactions, co-expression and co-regulation. We found that known NMD-factors have physical, regulatory and co-expression interaction signatures with related processes (mRNA translation, mRNA splicing, mRNA degradation and mRNA transport), which can be used to distinguish them from other proteins. We computed a scoring algorithm to rank NMD-neighbors according to the similarity to these signatures, generating a list of NMD candidates, that we aim to validate experimentally. Interestingly, some candidates were recently studied in NMD context and showed promising results. Furthermore, a cross-validation analysis indicated the robustness of the predictions provided by our method. On the road to developing a tool to apply this approach to other biological processes, we observed good cross-validations results for other RNA-related processes, suggesting this method’s usefulness in the RNA research area.
- Network biology approaches in the identification of novel pharmacological targets – the case of cystic fibrosisPublication . Loureiro, Cláudia; Matos, Ana; Santos, João; Farinha, Carlos; Jordan, Peter; Matos, Paulo; Pinto, FranciscoIn cystic fibrosis, the most common disease-causing mutation is F508del, which causes not only intracellular retention and degradation of CFTR, but also defective channel gating and decreased membrane stability of the small amount that reaches the plasma membrane (PM). Thus, pharmacological correction of mutant CFTR requires targeting of multiple cellular defects in order to achieve clinical benefit. Although small-molecule compounds have been identified and commercialized that can correct its folding or gating, an efficient retention of F508del CFTR at the PM has not yet been explored pharmacologically despite being recognized as a crucial factor for improving functional rescue of chloride transport. In ongoing efforts to determine the CFTR interactome at the PM, we used three complementary approaches: targeting proteins binding to tyrosine-phosphorylated CFTR, protein complexes involved in cAMP-mediated CFTR stabilization at the PM, and proteins selectively interacting at the PM with rescued F508del-CFTR but not wt-CFTR. Using co-immunoprecipitation or peptide–pull down strategies, we identified around 400 candidate proteins through sequencing of complex protein mixtures using the nano-LC Triple TOF MS technique. Key candidate proteins were validated for their robust interaction with CFTR-containing protein complexes and for their ability to modulate the amount of CFTR expressed at the cell surface of bronchial epithelial cells. Here, we describe how we explored the abovementioned experimental datasets to build a protein interaction network with the aim of identifying novel pharmacological targets to rescue CFTR function in cystic fibrosis (CF) patients. We identified and validated novel candidate proteins that were essential components of the network but not detected in previous proteomic analyses.
- A network integrative approach to unravel new links between NMD and mRNA processing pathwaysPublication . Nogueira, Gonçalo; Pinto, Francisco; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation termination codons (PTCs). This process has been associated with many genetic diseases and some forms of cancer caused by nonsense or frameshift mutations that introduce PTCs. Moreover, recent studies have shown that NMD is also involved in the regulation of a large number of transcripts, suggesting a major role in the control of gene expression. To further investigate the biological relevance of NMD and how this process can be modulated, we used a network analysis approach that integrates 1) protein-protein, 2) kinasetarget, 3) phosphatase-target, 4) miRNA-target, 5) transcription factors-target, 6) gene co-expression, 7) ubiquitination and 8) signaling interactions. The generated network was used to find novel NMD-associated proteins, prioritizing candidates with simultaneous interactions with different mRNA processing pathways (mRNA splicing, mRNA transport, mRNA translation and mRNA decay). Taking in account all information sources integrated in our network, we have created a scoring algorithm to identify new potentially important players in NMD, which can be essential to further understand the interplay between mRNA translation, PTC definition and NMD. Due to the diversity of regulatory links integrated in this workflow, we propose it can be applied to find molecular bridges between related biological processes and generate novel hypotheses about the molecular mechanisms co-regulating these phenomena.
- A network integrative approach to unravel new links between NMD and mRNA processing pathwaysPublication . Nogueira, Gonçalo; Pinto, Francisco; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation termination codons (PTCs). This process has been associated with many genetic diseases and some forms of cancer caused by nonsense or frameshift mutations that introduce PTCs. Moreover, recent studies have shown that NMD is also involved in the regulation of a large number of transcripts, suggesting a major role in the control of gene expression. To further investigate the biological relevance of NMD and how this process can be modulated, we used a network analysis approach that integrates 1) protein-protein, 2) kinasetarget, 3) phosphatase-target, 4) miRNA-target, 5) transcription factors-target, 6) gene co-expression, 7) ubiquitination and 8) signaling interactions. The generated network was used to find novel NMD-associated proteins, prioritizing candidates with simultaneous interactions with different mRNA processing pathways (mRNA splicing, mRNA transport, mRNA translation and mRNA decay). Taking in account all information sources integrated in our network, we have created a scoring algorithm to identify new potentially important players in NMD, which can be essential to further understand the interplay between mRNA translation, PTC definition and NMD. Due to the diversity of regulatory links integrated in this workflow, we propose it can be applied to find molecular bridges between related biological processes and generate novel hypotheses about the molecular mechanisms co-regulating these phenomena.
- NMD-associated proteins predicted by an integrative network approachPublication . Nogueira, Gonçalo; Pinto, Francisco; Romão, LuísaThe mechanism of nonsense-mediated decay (NMD) selectively degrades mRNAs carrying a premature translation-termination codon and regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. Although this complex process has been extensively studied along the years, the interactions and connectivity among NMD players is not completely understood. Additionally, some NMD mechanistical aspects suggest missing roles that can be played by proteins still not reported as involved in this pathway. To tackle this hypothesis, we developed a network-based approach to predict new proteins involved in NMD. Our approach explores the ability of proteins to bridge related processes, while integrating data regarding protein-protein interactions, co-expression and co-regulation by miRNAs and transcription factors. We found that known NMD-factors have physical, regulatory and co-expression interaction signatures with related processes (mRNA translation, mRNA splicing, mRNA degradation and mRNA transport), which can be used to distinguish them from other proteins. We computed a scoring algorithm to rank NMD-neighbors according to the similarity to these signatures, generating a list of NMD candidates, that we aim to validate experimentally. Interestingly, some candidates were recently studied in NMD context and showed promising results. Furthermore, a cross-validation analysis indicated the robustness of the predictions provided by our method. We propose that such approach can be applied to find molecular bridges between related biological processes and generate novel hypotheses about the molecular mechanisms co-regulating these phenomena.
- Nonsense-Mediated mRNA decay in development, stress and cancerPublication . Fernandes, Rafael; Nogueira, Gonçalo; da Costa, Paulo J.; Pinto, Francisco; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a well characterized eukaryotic mRNA degradation pathway, responsible for the identification and degradation of transcripts harboring translation termination codons in premature contexts. Transcriptome-wide studies revealed that NMD is not only an mRNA surveillance pathway as initially thought, but is also a post-transcriptional regulatory mechanism of gene expression, as it fine-tunes the transcript levels of many wild-type genes. Hence, NMD contributes to the regulation of many essential biological processes, including pathophysiological mechanisms. In this chapter we discuss the importance of NMD and of its regulation to organism development and its link to the cellular stress responses, like the unfolded protein response (UPR) and the integrated stress response (ISR). Additionally, we describe how tumor cells have explored both NMD functions to promote tumorigenesis. Using published data and databases, we have also performed a network-based approach that further supports the link between NMD and these (patho) physiological processes.