Browsing by Author "Santos, Vera"
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- Pesquisa e caracterização de mutações em genes relacionados com o metabolismo do ferro em individuos com hiperferritinemia gravePublication . Santos, Vera; Faustino, Paula; Silva, Bruno; Viegas-Crespo, AnaO Ferro desempenha um papel biológico muito importante nos organismos vivos. No entanto, tanto o seu excesso como a sua deficiência no organismo humano estão associados a consequências negativas para a saúde. A Hemocromatose Hereditária (HH) é dos distúrbios genéticos mais comuns em indivíduos de ascendência Norte-Europeia. Caracteriza-se por uma absorção excessiva de ferro a nível intestinal e sua consequente acumulação em vários órgãos. As complicações mais frequentes da doença são cirrose hepática, carcinoma hepatocelular, cardiomiopatias, diabetes, problemas endócrinos, artrite e hiperpigmentação da pele. O gene HFE é o principal associado à patologia (HH clássica, tipo I). Os genótipos mais comuns são a mutação C282Y em homozigotia e a heterozigotia composta desta com a mutação H63D. Têm surgido evidências de que mutações noutros genes possam estar envolvidas nesta patologia. Entre estes encontra-se o gene do Receptor 2 da Transferrina (TfR2) sendo a HH resultante (Hemocromatose do tipo III) caracterizada por sintomas clínicos idênticos aos da forma clássica. A Hemocromatose do tipo II, ou Hemocromatose Juvenil, é a forma mais grave da doença devido ao facto da sobrecarga de ferro ocorrer a um ritmo mais acelerado levando a problemas em idades jovens. Este tipo de HH ocorre devido a mutações no gene da Hemojuvelina, HJV (HH do tipo 2A) ou no da Hepcidina (HAMP) (HH do tipo 2B). O objectivo principal deste estudo consistiu na pesquisa e caracterização de mutações em genes relacionados com o metabolismo do ferro (HFE, TfR2, HJV, HAMP, BMP-6), em 11 indivíduos com hiperferritinemia muito grave (ferritina sérica > 2500 μg/L), cujos fenótipos não eram justificáveis pelos genótipos HFE apresentados. As sequências génicas foram amplificadas por metodologias de PCR e a análise mutacional dos fragmentos amplificados foi efectuada recorrendo à sequenciação automática. Nesta análise foram detectadas 9 alterações, sendo que 3 delas são identificadas pela primeira vez neste estudo (a P124L no gene HJV, a c.1281-197 T→C e a c.1542+4 G→A no gene BMP-6). Posteriormente foram realizados estudos in silico às sequências proteicas de modo a identificar possíveis alterações a nível estrutural recorrendo ao programa bioinformática PolyPhen-2 e aos modelos HumDiv e HumVar. Para além disso, também se procedeu à análise do impacto das alterações nucleotídicas a nível do splicing utilizando para esse fim o software Human Splicing Finder. Foi possível através desta pesquisa justificar a sobrecarga em ferro em 2 dos indivíduos em estudo. Num dos casos, colocamos a hipótese de que a conjugação da homozigotia para a mutação C282Y em HFE e da alteração R752H em heterozigotia no gene TfR2 seja o motivo da sobrecarga em ferro verificada. No outro caso, encontrou-se a alteração P124L em heterozigotia no gene HJV, que em co-herança com a homozigotia para a alteração C282Y, também poderá estar na origem da hiperferritinemia grave. A HH não clássica é caracterizada por uma grande heterogeneidade genética, que traduz mecanismos fisiológicos complexos inerentes à homeostase do ferro, ainda não completamente conhecidos. Com este estudo contribuímos para aumentar o conhecimento sobre a fisiopatologia deste tipo de Hemocromatose e esclarecemos as relações genótipo/fenótipo nalguns dos casos estudados.
- The expression of the soluble HFE corresponding transcript is up-regulated by intracellular iron and inhibits iron absorption in a duodenal cell modelPublication . Silva, Bruno; Ferreira, Joana; Santos, Vera; Baldaia, Cilénia; Serejo, Fátima; Faustino, PaulaBackground and aims: Dietary iron absorption regulation is a key-step for the maintenance of body iron homeostasis. Besides the HFE full-length protein, the HFE gene codes for alternative splicing variants responsible for the synthesis of a soluble form of HFE protein (sHFE). Here we aimed to determine whether sHFE transcript levels respond to different iron conditions in duodenal, macrophage and hepatic cell models, as well, in vivo, in the liver. Furthermore, we determined the functional effect of the sHFE protein on the expression of iron metabolism-related genes in a duodenal cell model. Methods: The levels of sHFE transcripts were measured in HuTu-80, activated THP1 and HepG2 cells, after holo-Tf stimulus, as well as in RNA from liver biopsies of chronic HCV patients. Moreover, the expression of iron metabolism-related genes was determined by RT-qPCR after the overexpression of sHFE protein. Results: Our results showed that the sHFE corresponding transcripts expression is up-regulated by intracellular iron, despite the total HFE levels not being affected. Peripheral blood iron biomarkers do not correlate with sHFE levels at the liver of HCV patients. Hephaestin and duodenal cytochrome b expressions are down-regulated by both endogenous full-length HFE and sHFE proteins. Exogenous sHFE stimulus also down-regulates hephaestin levels by an endocytosis dependent mechanism. Conclusions: sHFE is an important iron metabolism homeostasis regulator which levels vary accordingly to intracellular iron. It controls both hephaestin and duodenal cytochrome b expressions and consequently dietary iron absorption by the duodenum.
- The physiological role of the soluble HFE isoform – a regulator of dietary iron absorption in the duodenumPublication . Silva, Bruno; Ferreira, Joana; Santos, Vera; Sousa, Patrícia; Baldaia, Cilénia; Serejo, Fátima; Faustino, PaulaObjective: Dietary iron absorption regulation is a key-step for body iron homeostasis. Once inside the enterocyte, iron is directed to the basolateral membrane being oxidized by hephaestin, which mediates iron efflux towards circulatory transferrin in cooperation with ferroportin. Besides the HFE full-length protein, the HFE gene codes for alternative splicing transcripts responsible for the synthesis of a soluble form of HFE protein (sHFE). The main objective of this work was to assess whether sHFE plays a role in iron absorption regulation in duodenum. In particular, we intended to determine if sHFE transcript levels respond to different iron conditions in duodenal cell models. Also, we aimed to investigate the functional effect of the sHFE protein on the expression of iron metabolism-related genes in duodenal cell models as well as, in ex-vivo, in duodenum biopsy samples. Methods: The levels of sHFE transcripts were measured in HuTu-80, Caco-2, and HT-29 cells, after holo-Tf stimulus. The expression of iron metabolism-related genes was determined after endogenous and exogenous overexpression of the sHFE protein. Moreover, expression levels of sHFE and HEPH were quantified by RT-qPCR in 6 RNA samples from duodenum biopsies of dyspepsia patients. Results: Our in vitro obtained results have shown that the sHFE transcripts expression is up-regulated by intracellular iron. Hephaestin and duodenal cytochrome b expressions are down-regulated by endogenous sHFE protein. Exogenous sHFE stimulus also down-regulates hephaestin levels by a clathrin-independent, dynamin-mediated and RhoA-regulated endocytosis mechanism. In agreement, our in ex-vivo studies revealed that HEPH expression correlates negatively with sHFE levels in the human duodenum. Conclusion: The sHFE is probably an important regulator of iron metabolism. Its levels vary according to the level of intracellular iron. It controls hephaestin expression and most likely the dietary iron absorption in the duodenum.
- The soluble form of HFE protein regulates hephaestin mRNA expression in the duodenum through an endocytosis-dependent mechanismPublication . Silva, Bruno; Ferreira, Joana; Santos, Vera; Baldaia, Cilénia; Serejo, Fátima; Faustino, PaulaDietary iron absorption regulation is one of the key steps for the maintenance of the body iron homeostasis. HFE gene expression undergoes a complex post-transcriptional alternative splicing mechanism through which two alternative transcripts are originated and translated to a soluble HFE protein isoform (sHFE). The first purpose of this study was to determine if sHFE transcript levels respond to different iron conditions in duodenal and macrophage cell models. In addition, we aimed to determine the functional effect of the sHFE protein on the expression of iron metabolism-related genes in a duodenal cell model as well as, in vivo, in duodenumbiopsy samples. Levels of sHFE transcripts were measured in HuTu-80, Caco-2, HT-29 and activated THP1 cells, after holo-Tf stimulus, and in total RNA from duodenum biopsies of functional dyspepsia patients. Also, the expression of several iron metabolism-related genes was determined after endogenous and exogenous overexpression of sHFE protein in a duodenal cell model. sHFE endocytosis mechanism was studied using endocytosis inhibitors. Our results showed that sHFE transcript expression was up-regulated after holo-Tf stimuli. Hephaestin and duodenal cytochrome b expressions were down-regulated by both endogenous HFE and sHFE proteins in a duodenal cell model. Exogenous sHFE was able to down-regulate hephaestin mRNA levels by a clathrin-independent, dynamin-mediated, and RhoA-regulated endocytosis mechanism. Moreover, HEPH levels negatively correlated with sHFE levels in the duodenum of functional dyspepsia patients. Thus, sHFE seems to be an important iron metabolism regulator playing a role in the control of dietary iron absorption in the duodenum.
- The soluble HFE isoform – a regulator of iron absorption in the duodenumPublication . Silva, Bruno; Ferreira, Joana; Santos, Vera; Sousa, Patícia; Baldaia, Cilénia; Serejo, Fátima; Faustino, PaulaObjective: Dietary iron absorption regulation is a key-step for body iron homeostasis. Once inside the enterocyte, iron is directed to the basolateral membrane being oxidized by hephaestin, which mediates iron efflux towards circulatory transferrin in cooperation with ferroportin. Besides the HFE full-length protein, the HFE gene codes for alternative splicing transcripts responsible for the synthesis of a soluble form of HFE protein (sHFE). The main objective of this work was to assess whether sHFE plays a role in iron absorption regulation in duodenum. In particular, we intended to determine if sHFE transcript levels respond to different iron conditions in duodenal cell models. Also, we aimed to investigate the functional effect of the sHFE protein on the expression of iron metabolism-related genes in duodenal cell models as well as, in vivo, in duodenum biopsy samples. Materials and Methods: The levels of sHFE transcripts were measured in HuTu-80, Caco-2, and HT-29 cells, after holo-Tf stimulus. The expression of iron metabolism-related genes was determined after endogenous and exogenous overexpression of the sHFE protein. Moreover, expression levels of sHFE and HEPH were quantified by RT-qPCR in 6 RNA samples from duodenum biopsies of dyspepsia patients. Results: Our in vitro results have shown that the sHFE transcripts expression is up-regulated by intracellular iron. Hephaestin and duodenal cytochrome b expressions are down-regulated by endogenous sHFE protein. Exogenous sHFE stimulus also down-regulates hephaestin levels by a clathrin-independent, dynamin-mediated and RhoA-regulated endocytosis mechanism. In agreement, we found that HEPH expression negatively correlates with sHFE levels in the human duodenum. Conclusion: sHFE may be an important iron metabolism homeostasis regulator which levels vary according to intracellular iron. It controls hephaestin expression and most likely the dietary iron absorption in the duodenum.