Browsing by Author "Costa, Paulo"
Now showing 1 - 10 of 17
Results Per Page
Sort Options
- An unexpected role for DIS3L2 over human NMD targetsPublication . Costa, Paulo; Saramago, Margarida; Viegas, Sandra; Arraiano, Cecília; Romão, LuísaThe nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological RNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5’ and 3’ ends. This is done by a process not yet completely understood that recruits decapping and 5’-to-3’ exonuclease activities, as well as deadenylating and 3’-to-5’ exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. However, DIS3L2 exoribonuclease activity is independent of the exosome. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, however nothing is known about their role in this process. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown in HeLa cells and measured the mRNA levels of various natural NMD targets. Our results show that some NMD targets are highly stabilized in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicated that these NMD targets are in fact direct DIS3L2 substrates. We also observed that DIS3L2-mediated decay depends on the activity of the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4). Together, our findings establish the role of DIS3L2 and uridylation in NMD.
- An unexpected role for DIS3L2 over human NMD targetsPublication . Costa, Paulo; Saramago, Margarida; Viegas, Sandra; Arraiano, Cecília; Santos, Hugo; Gama-Carvalho, Margarida; Romão, LuísaThe final step of cytoplasmic mRNA degradation proceeds in either a 5’-3’ direction, catalyzed by XRN1, or in a 3’-5’ direction catalyzed by the exosome and DIS3L2. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome. In humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. Important findings over the last years have shed a new light onto the mechanistic details of RNA degradation by these exoribonucleases. In addition, it has been shown that they are involved in growth, mitotic control and important human diseases, including cancer. For example, DIS3L2 inactivation was associated with mitotic abnormalities and altered expression of mitotic checkpoint proteins. In another study, DIS3 was found to be highly expressed in colorectal cancer (CRC), suggesting an oncogenic function. A major challenge in systems biology is to reveal the cellular networks that give rise to specific phenotypes. In this project, we aim to analyze how DIS3, DIS3L1 and DIS3L2 regulate the human transcriptome, and how their functional interactions modulate the transcriptional reprogramming of colorectal cancer cells. In order to unveil the role of these exoribonucleases in general mRNA decay, and/or in cytoplasmic mRNA surveillance mechanisms, such as nonstop- and nonsense-mediated decay (NSD and NMD), we performed their knockdown and measured the mRNA levels of various reporter transcripts (endogenous and exogenous), with emphasis in natural NMD targets. Our results show that DIS3 and DIS3L1 seem to be involved in the normal mRNA turnover, as well as in the NSD and NMD mechanisms. However, some natural NMD targets are resistant to these nucleases. On the other hand, DIS3L2 is not involved in the normal mRNA turnover or in NSD, being specifically involved in the degradation of some NMD targets. Presently, we are interested in identifying the transcript features implicated in the decision-making process of DIS3L2-mediated decay of natural NMD targets, as well as the corresponding mechanism. With this purpose, we performed a bioinformatics analysis of available transcriptomic data from DIS3, DIS3L1, DIS3L2+XRN1, XRN1, or UPF1 (a central player in NMD) knockdown experiments and identified transcripts differentially expressed in each condition. Results show some, but not total, redundancy between the upregulated transcripts, and this supports our experimental data.
- Apolipoprotein E isoforms and susceptibility to genetic generalized epilepsiesPublication . Chaves, João; Martins-Ferreira, Ricardo; Carvalho, Cláudia; Bettencourt, Andreia; Brás, Sandra; Chorão, Rui; Freitas, Joel; Samões, Raquel; Lopes, João; Ramalheira, João; Silva, Berta; Costa, Paulo; Martins Da Silva, António; Leal, BárbaraBackground: Apolipoprotein E (ApoE) is the main lipoprotein secreted in brain. It has a critical immunomodulatory function, influences neurotransmission and it is involved in repairing damaged neurons. ApoE e4 is an isoform of ApoE with altered function, and was previously associated with early onset epilepsy and refractoriness, both in animal models and in patients with focal epilepsies. There is a limited knowledge on ApoE’s role in Genetic Generalized Epilepsies (GGE). Aim: To determine if ApoE isoforms are risk factors for GGE development. Methods: A group of 337 GGE patients (193 F, 144 M, 33.6 ± 14.2 years) was compared with a group of 342 healthy individuals in a case-control genetic association study. ApoE genotyping was performed using PCR-RFLP. Results: The genotypic frequency of ApoE e3/e2 was lower in GGE patients relative to controls (6.5% in GGE vs. 11.7% in controls, p = 0.019, OR (95% CI) = 0.53 (0.305–0.905). No associations with other clinical data such as photosensitivity or age at disease onset were observed. Conclusion: Our results show that ApoE e3/e2 genotype may be a protective factor for GGE development. There is evidence that this genotype could be neuroprotective, preventing oxidative damage and promoting neuronal survival. Although replication studies are warranted, our data suggest that ApoE isoforms have a role in epileptogenic mechanisms regardless of the specific epileptic manifestations.
- Behind the curtain: unveiling DIS3L2 role in NMD and human cancerPublication . Costa, Paulo; Saramago, Margarida; Viegas, Sandra; Arraiano, Cecília; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance mechanism that targets and degrades mRNAs carrying premature translation-termination codons (PTCs), preventing the production of truncated proteins potentially harmful for the cells. In addition to this, several studies have shown that NMD regulates the levels of many physiological mRNAs that encode full-length proteins. Nevertheless, NMD is inhibited in tumor microenvironment and (de)regulates oncogenes and tumor suppressors in several types of cancer. In humans, the mRNA degradation pathways involve exonuclease proteins, such as the DIS3-like protein family (DIS3, DIS3L1 and DIS3L2); however, it is not known whether these proteins are involved in NMD. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown, by RNA interference, in HeLa cells and measured, by RT-qPCR, the mRNA levels and half-lives of various natural NMD targets. Our results show that some NMD targets are highly stabilized in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicate that these NMD targets are in fact direct DIS3L2 substrates. By performing DIS3L2, TUT4 and TUT7 triple knockdown, we also observed that DIS3L2-mediated decay depends on the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4) activity. Among the NMD targets regulated by DIS3L2, we highlight GADD45A. GADD45A is involved in cell cycle arrest, DNA damage response and apoptotic process. Furthermore, GADD45A deregulation is associated with several types of cancer, such as, esophageal, lung, bladder and pancreatic. Together, our findings establish the role of DIS3L2 and uridylation in NMD and in the regulation of oncogenes and tumor suppressor gene expression. These results might be highly relevant for the advance in diagnosis, prognosis and treatment of many human cancers.
- Genetic characterization of interleukins (IL-1α, IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12A, IL-12B, IL-15 and IL-18) with relevant biological roles in lagomorphsPublication . Neves, F.; Abrantes, J.; Almeida, T.; de Matos, A.L.; Costa, Paulo; Esteves, P.J.ILs, as essential innate immune modulators, are involved in an array of biological processes. In the European rabbit (Oryctolagus cuniculus) IL-1α, IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12A, IL-12B, IL-15 and IL-18 have been implicated in inflammatory processes and in the immune response against rabbit hemorrhagic disease virus and myxoma virus infections. In this study we characterized these ILs in six Lagomorpha species (European rabbit, pygmy rabbit, two cottontail rabbit species, European brown hare and American pika). Overall, these ILs are conserved between lagomorphs, including in their exon/intron structure. Most differences were observed between leporids and American pika. Indeed, when comparing both, some relevant differences were observed in American pika, such as the location of the stop codon in IL-1α and IL-2, the existence of a different transcript in IL8 and the number of cysteine residues in IL-1β. Changes at N-glycosylation motifs were also detected in IL-1, IL-10, IL-12B and IL-15. IL-1α is the protein that presents the highest evolutionary distances, which is in contrast to IL-12A where the distances between lagomorphs are the lowest. For all these ILs, sequences of human and European rabbit are more closely related than between human and mouse or European rabbit and mouse.
- How the DIS3 proteins shape the human transcritpomePublication . Costa, Paulo; Santos, Hugo; Gama Carvalho, Margarida; Romão, LuísaThe final step of eukaryotic mRNA degradation proceeds in either a 5’-3’ direction, catalyzed by XRN1, or in a 3’-5’ direction catalyzed by DIS3, DIS3L1 (the catalytic subunits of the exosome) and/or DIS3L2 (exosome-independent). Important findings over the last years have shed a new light onto the mechanistic details of RNA degradation by these exoribonucleases. In addition, it has been shown that they are involved in growth, mitotic control and important human diseases, including cancer. With the aim of analyzing how DIS3, DIS3L1 and DIS3L2 regulate the human transcriptome, each one of these nucleases was depleted by RNA interference in HeLa cells and levels of several reporter mRNAs was monitored by RT-qPCR. Our results show that these exoribonucleases are target specific and not directly involved in a particular mRNA surveillance mechanism. In parallel, our bioinformatics analysis of available transcriptomic data from cells depleted of DIS3L1, DIS3L2, XRN1, or UPF1 (which has a central role in nonsense-mediated mRNA decay) has shown some, but not full, redundancy among the transcripts regulated by these nucleases, which supports our experimental data. Presently, we are exploring the molecular mechanisms underlying our observations.
- Immunogenetic predisposing factors for mesial temporal lobe epilepsy with hippocampal sclerosisPublication . Leal, Bárbara; Chaves, João; Carvalho, Cláudia; Bettencourt, Andreia; Brito, Cláudia; Boleixa, Daniela; Freitas, Joel; Brás, Sandra; Lopes, João; Ramalheira, João; Costa, Paulo; Silva, Berta; Martins Da Silva, AntónioPurpose: Neuroinflammation appears as an important epileptogenic mechanism. Experimental and clinical studies have demonstrated an upregulation of pro-inflammatory cytokines such as IL-1β and TNF-α, in mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS). Expression of these cytokines can be modulated by polymorphisms such as rs16944 and rs1800629, respectively, both of which have been associated with febrile seizures (FS) and MTLE-HS development. The human leukocyte antigen (HLA) system has also been implicated in diverse epileptic entities, suggesting a variable role of this system in epilepsy. Our aim was to analyse the association between immunogenetic factors and MTLE-HS development. For that rs16944 (-511 T>C, IL-1β), rs1800629 (-308 G>A, TNF-α) polymorphisms and HLA-DRB1 locus were genotyped in a Portuguese Population. Methods: We studied 196 MTLE-HS patients (108 females, 88 males, 44.7 ± 12.0 years, age of onset = 13.6 ± 10.3 years, 104 with FS antecedents) and 282 healthy controls in a case–control study. Results: The frequency of rs16944 TT genotype was higher in MTLE-HS patients compared to controls (14.9% in MTLE-HS vs. 7.7% in controls, p = 0.021, OR [95% CI] = 2.20 [1.13–4.30]). This association was independent of FS antecedents. No association was observed between rs1800629 genotypes or HLA-DRB1 alleles and MTLE-HS susceptibility. Also, no correlation was observed between the studied polymorphisms and disease age of onset. Conclusion: The rs16944 TT genotype is associated with MTLE-HS development what may be explained by the higher IL-1β levels produced by this genotype. High IL-1β levels may have neurotoxic effects or imbalance neurotransmission leading to seizures.
- Immunogenetic protective factors in Genetic Generalized EpilepsyPublication . Chaves, João; Martins-Ferreira, Ricardo; Ferreira, Ana Marta; Brás, Sandra; Carvalho, Cláudia; Bettencourt, Andreia; Samões, Raquel; Monteiro, Fábio; Freitas, Joel; Chorão, Rui; Lopes, João; Ramalheira, João; Silva, Berta; Costa, Paulo; Martins Da Silva, António; Leal, BárbaraBackground: Genetic Generalized Epilepsies (GGEs) are a heterogeneous group of syndromes characterized by generalized seizure activity that affects both hemispheres, with mainly genetic causes. Neuroinflammation has been established as an important mechanism in epileptogenesis. The ability to develop an appropriated immune response is strongly determined by immunogenetic factors. In this setting, our aim was to evaluate potential associations between GGEs and immunogenetic factors. Methods: The rs16944 (IL-1β -511 T > C) polymorphism and the HLA-DRB1 locus were genotyped in a Portuguese GGE population. Association with two clinicopathological features, photosensitivity and refractoriness, was investigated. This case-control study included 323 GGE patients (187 F, 136 M, 34.0 ± 13.9 years of age), 145 of which with JME diagnosis (88 F, 57 M, 34.1 ± 14.0 years), and 282 healthy controls (174 F, 108 M, 37.7 ± 11.6 years). Results: Decreased frequencies of the HLA-DRB1*09 and DRB1*13 alleles were observed in the GGE population. HLA-DRB1*07 frequency was increased in JME. Rs16944 allelic frequencies were similar between patients and controls. Conclusions: These results, not entirely consistent with previous reports, suggest that HLA molecules may have a complex role in epileptogenesis.
- Microglial innate memory and epigenetic reprogramming in neurological disordersPublication . Martins-Ferreira, Ricardo; Leal, Bárbara; Costa, Paulo; Ballestar, EstebanMicroglia are myeloid-derived cells recognized as brain-resident macrophages. They act as the first and main line of immune defense in the central nervous system (CNS). Microglia have high phenotypic plasticity and are essential for regulating healthy brain homeostasis, and their dysregulation underlies the onset and progression of several CNS pathologies through impaired inflammatory responses. Aberrant microglial activation, following an inflammatory insult, is associated with epigenetic dysregulation in various CNS pathologies. Emerging data suggest that certain stimuli to myeloid cells determine enhanced or attenuated responses to subsequent stimuli. These phenomena, generally termed innate immune memory (IIM), are highly dependent on epigenetic reprogramming. Microglial priming has been reported in several neurological diseases and corresponds to a state of increased permissiveness or exacerbated response, promoted by continuous exposure to a chronic pro-inflammatory environment. In this article, we provide extensive evidence of these epigenetic-mediated phenomena under neurological conditions and discuss their contribution to pathogenesis and their clinical implications, including those concerning potential novel therapeutic approaches.
- Obstructive sleep apnoea syndrome and HLA in the North of PortugalPublication . Silva, Luís; Lopes, João; Ramalheira, João; Cunha, Daniela; Carvalho, Cláudia; Bettencourt, Andreia; Bras, Sandra; Costa, Paulo; Silva, M. Berta; Martins-da-Silva, António[ENG] Introduction. The obstructive sleep apnoea syndrome (OSAS) is a common, complex and polygenic disease with diverse aetiologies interacting to produce a single phenotype. OSAS occurs throughout the entire lifespan and familial aggregation has been suggested. Several predisposing factors, as age, gender and obesity have been described. Associations between HLA polymorphisms and sleep disorders are confirmed, in European and Non-European descendent populations. However the associations found between HLA alleles and OSAS have not been consistent and have no informative value for sleep disorder classification. Aims. To explore the genetic association of HLA with OSAS in a northern Portuguese population and to evaluate the role of obesity in the context of HLA in OSAS. Patients and methods. A cohort of 131 patients with OSAS was studied. Patients followed up in an Outpatient Sleep Clinic were assessed by clinical history, night sleep polygraphic recording, multiple sleep latency test (when necessary for differential diagnosis), laboratorial and demographic studies. A control population (CP) of 223 healthy individuals was used for comparison. HLA-DRB1 genotyping was performed using a polymerase chain reaction with sequence specific primers methodology. Results. In this cohort, the HLA-DRB1*03 allele was identified as a susceptibility factor for OSAS (24% OSAS vs. 15% CP; p = 0.025; odds ratio = 1.861; 95% CI = 1.081-3.205). No significant differences were found for other HLA-DBR1* alleles. Conclusion. HLA-DRB1*03 is a susceptibility factor for OSAS in Portuguese population. Key words. Gender. HLA-DRB1*03. Obesity. Obstructive sleep apnoea syndrome (OSAS). Risk factors. Sleep disorders.