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- Cartilage acidic protein 1, a new member of the beta-propeller protein family with amyloid propensityPublication . Anjos, Lliana; Morgado, Isabel; Guerreiro, Marta; Cardoso, João CR; Melo, Eduardo; Power, DeborahCartilage acidic protein1 (CRTAC1) is an extracellular matrix protein of chondrogenic tissue in humans and its presence in bacteria indicate it is of ancient origin. Structural modeling of piscine CRTAC1 reveals it belongs to the large family of beta-propeller proteins that in mammals have been associated with diseases, including amyloid diseases such as Alzheimer's. In order to characterize the structure/function evolution of this new member of the beta-propeller family we exploited the unique characteristics of piscine duplicate genes Crtac1a and Crtac1b and compared their structural and biochemical modifications with human recombinant CRTAC1. We demonstrate that CRTAC1 has a beta-propeller structure that has been conserved during evolution and easily forms high molecular weight thermo-stable aggregates. We reveal for the first time the propensity of CRTAC1 to form amyloid-like structures, and hypothesize that the aggregating property of CRTAC1 may be related to its disease-association. We further contribute to the general understating of CRTAC1's and beta-propeller family evolution and function. Proteins 2017; 85:242-255. (c) 2016 Wiley Periodicals, Inc.
- Thyroid hormone binding by recombinant sea bream transthyretin: the role of the N-terminal regionPublication . Morgado, Isabel; Sauer-Eriksson, A. Elisabeth; Power, DeborahTransthyretin (TTR) along with thyroxine-binding globulin (TBG) and albumin (ALB) constitute Thyroid Hormone-Binding Proteins (THBP) in vertebrates. These proteins bind and transport thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood. THBP are poorly characterized in fish and in the present study binding of THs by sea bream TTR was determined. Teleost TTR is a single polypeptide chain of 130 amino acids, but in common with mammals the functional form in the plasma is a tetramer. In contrast to mammalian TTR (127 amino acids), fish TTR has a longer N-terminal region and the latter has been proposed to influence TH binding. In the present study recombinant sea bream TTR (sbTTR, wild type), plus two recombinant N-terminal mutants were produced and purified. Binding of [I125]-T3 to the purified TTRs was confirmed by polyacrylamide gel electrophoresis under nondenaturing conditions followed by autoradiography and confirmed all bind THs as a tetramer. Ligand binding studies with labeled [I125]-T3 were performed and revealed that [I125]-T3 was displaced in a similar way from wild type and mutant TTRs by increasing concentrations of unlabeled T3. Similar Kd values were obtained for both T3 and T4 binding to wild type and mutant TTRs indicating that the N-terminal region does not seem to be important for the binding characteristics of sbTTR.
- The goitrogenic efficiency of thioamides in a marine teleost, sea bream (Sparus auratus)Publication . Campinho, Marco António; Morgado, Isabel; Pinto, Patricia IS; Silva, Nádia; Power, DeborahStudies on the role of thyroid hormones (THs) in teleost fish physiology have deployed the synthetic goitrogens, methimazol (MMI), propilthiouracil (PTU) and thiourea (TU) that are used to treat human hyperthyroidism. However, the action of the goitrogens, MMI, PTU and TU at different levels of the hypothalamic–pituitary–thyroid (HPT) axis in teleosts is largely unknown. The central importance of the hypothalamus and pituitary in a number of endocrine regulated systems and the cross-talk that occurs between different endocrine axes makes it pertinent to characterize the effects of MMI, PTU and TU, on several endpoints of the thyroid system. The marine teleost, sea bream (Sparus auratus) was exposed to MMI, PTU and TU (1 mg/kg wet weight per day), via the diet for 21 days. Radioimmunoassays (RIA) of plasma THs and ELISA of the TH carrier transthyretin (TTR) revealed that MMI was the only chemical that significantly reduced plasma TH levels (p < 0.05), although both MMI and PTU significantly (p < 0.05) reduced plasma levels of circulating TTR (p < 0.05). Histological analysis of the thyroid tissue revealed modifications in thyrocyte activity that explain the reduced circulating levels of THs. MMI also significantly (p < 0.05) up-regulated transcript abundance of liver deiodinase 1 and 2 while significantly (p < 0.05) decreasing TRb expression in the pituitary, all hallmarks of HPT axis action of goitrogens in vertebrates. The results indicate that in the sea bream MMI is the most effective goitrogen followed by PTU and that TU (1 mg/kg wet weight for 21 days) failed to have a goitrogenic effect. The study highlights the non-uniform effect of goitrogens on the thyroid axis of sea bream and provides the basis for future studies of thyroid disrupting pollutants.
- Transthyretin and thyroid hormone transport in fish and the effect of endocrine disruptors on this processPublication . Morgado, Isabel; Power, Deborah; Melo, Eduardo P.
- Stanniocalcin 1 effects on the renal gluconeogenesis pathway in rat and fishPublication . Schein, Vanessa; Kucharski, Luiz C.; M Guerreiro, Pedro; Martins, Tiago Leal; Morgado, Isabel; Power, Deborah M.; Canario, Adelino V. M.; da Silva, Roselis S. M.The mammalian kidney contributes significantly to glucose homeostasis through gluconeogenesis. Considering that stanniocalcin 1 (STC1) regulates ATP production, is synthesized and acts in different cell types of the nephron, the present study hypothesized that STC1 may be implicated in the regulation of gluconeogenesis in the vertebrate kidney. Human STC1 strongly reduced gluconeogenesis from C-14-glutamine in rat renal medulla (MD) slices but not in renal cortex (CX), nor from C-14-lactic acid. Total PEPCK activity was markedly reduced by hSTC1 in MD but not in CX. Pck2 (mitochondrial PEPCK isoform) was down-regulated by hSTC1 in MD but not in CX. In fish (Dicentrarchus labrax) kidney slices, both STC1-A and -B isoforms decreased gluconeogenesis from C-14-acid lactic, while STC1-A increased gluconeogenesis from C-14-glutamine. Overall, our results demonstrate a role for STC1 in the control of glucose synthesis via renal gluconeogenesis in mammals and suggest that it may have a similar role in teleost fishes. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
- Regulation of transthyretin by thyroid hormones in WshPublication . Morgado, Isabel; Santos, C. R. A.; Jacinto, R.; Power, DeborahTransthyretin (TTR) is a thyroid hormone-binding protein (THBP) which in its tetrameric form transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. The principal site of production of TTR is the liver but in the sea bream TTR mRNA is also present in the heart, intestine and brain. The regulation of TTR is unstudied in Wsh and the normal circulating level of this THBP is unknown. The aim of the present study was to establish factors which regulate TTR production in Wsh. As a Wrst step a number of tools were generated; sea bream recombinant TTR (sbrTTR) and speciWc sbrTTR antisera which were used to establish an ELISA (enzyme-linked immunosorbent assay) for measuring TTR plasma levels. Subsequently, an experiment was conducted to determine the inXuence of THs on TTR production. Circulating physiological levels of TTR in sea bream determined by ELISA are approximately 3.8 gml¡1. Administration of T3 and T4 to sea bream signiWcantly increased (p< 0.001 and p<0.005, respectively) the concentration of circulating TTR (V11.5 gml¡1) in relation to control Wsh, but did not change gene transcription in the liver. Methimazol (MMI) an antithyroid agent, failed to signiWcantly reduce circulating THs below control levels but signiWcantly increased (p < 0.005) plasma TTR levels (approximately 10.8 gml¡1) and decreased (p< 0.05) transcription in the liver. Future studies will aim to elucidate in more detail these regulatory pathways.
- Hormone affinity and fibril formation of piscine transthyretin: the role of the N-terminalPublication . Morgado, Isabel; Melo, Eduardo P.; Lundberg, Erik; Estrela, Nídia L.; Sauer-Eriksson, A. Elisabeth; Power, DeborahTransthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild-type sea bream TTR (sbTTRWT) plus two mutants in which six (sbTTRM6) and twelve (sbTTRM12) N-terminal residues were removed. Ligandbinding studies revealed similar affinities for T3 (Kd=10.6±1.7nM) and T4 (Kd=9.8±0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3±15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine-T.In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.
- Disruption of thyroid hormone binding to sea bream recombinant transthyretin by ioxinyl and polybrominated diphenyl ethersPublication . Morgado, Isabel; Hamers, Timo; Van der Ven, Leo; Power, DeborahA number of chemicals released into the environment share structural similarity to the thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) and it is thought that they may interfere with the thyroid axis and behave as endocrine disruptors (EDs). One of the ways by which such environmental contaminants may disrupt the TH axis is by binding to TH transporter proteins. Transthyretin (TTR) is one of the thyroid hormone binding proteins responsible for TH transport in the blood. TTR forms a stable tetramer that binds both T4 and T3 and in fish it is principally synthesized in the liver but is also produced by the brain and intestine. In the present study, we investigate the ability of some chemicals arising from pharmaceutical, industrial or agricultural production and classified as EDs, to compete with [I125]-T3 for sea bream recombinant TTR (sbrTTR). Ioxinyl, a common herbicide and several polybrominated diphenyl ethers were strong inhibitors of [I125]-T3 binding to TTR and some showed even greater affinity than the natural ligand T3. The TTR competitive binding assay developed offers a quick and effective tool for preliminary risk assessment of chemicals which may disrupt the thyroid axis in teleost fish inhabiting vulnerable aquatic environments.
- Disruption of the thyroid system by diethylstilbestrol and ioxynil in the sea bream (Sparus aurata)Publication . Morgado, Isabel; Campinho, Marco António; Costa, Rita; Jacinto, R.; Power, DeborahSome environmental contaminants are thought to cause disruption of the thyroid system in vertebrates acting as endocrine disrupting chemicals (EDCs). Such chemicals may affect synthesis, transport and metabolism of thyroid hormones (THs). Ioxynil (IOX) and diethylstilbestrol (DES) are potential EDCs with strong affinity in vitro for sea bream transthyretin (TTR), a TH distributor protein (THDP). The aim of the present study was to establish how such chemicals influence the thyroid axis in sea bream (Sparus aurata). DES, IOX and propilthyouracil (PTU, a goitrogen) were administered in the diet to sea bream juveniles at 1 mg/kg fish (n = 14/treatment) for 21 days. After exposure plasma TH levels, quantified by RIA, were similar to those of control fish (p > 0.05) in all treatment groups. Analysis by quantitative PCR revealed that all treatments down-regulated TSH gene transcription (p < 0.05) in the brain and pituitary and deiodinase II and III transcription in the brain (p < 0.001). In contrast, PTU caused DII up-regulation in the liver (p < 0.05). Thyroid receptor beta (TR ) transcription was down-regulated in the pituitary by PTU (p < 0.001) and DES (p < 0.05). TTR plasma levels, quantified by ELISA, were elevated by all the chemicals including PTU (p < 0.001) which also increased TTR gene transcription in the liver (p < 0.05). Thyroid histology indicated follicular hyperstimulation in all treatments with marked hyperplasia, hypertrophy and colloid depletion in the PTU group. It appears therefore, that in vitro TTR-binders, IOX and DES, can strongly influence several components of the fish thyroid system in vivo but that the thyroid axis may have the ability to maintain or re-establish plasma TH homeostasis.
- Analysis of the transthyretin-like (TTL) gene family in Ostertagia ostertagi - comparison with other strongylid nematodes and Caenorhabditis elegansPublication . Saverwyns, H.; Visser, A.; Van Durme, J.; Power, Deborah; Morgado, Isabel; Kennedy, M. W.; Knox, D. P.; Schymkowitz, J.; Rousseau, F.; Gevaert, K.; Vercruysse, J.; Claerebout, E.; Geldhof, P.The transthyretin-like (ttl) gene family is one of the largest conserved nematode-specific gene families, coding for a group of proteins with significant sequence similarity to transthyretins (TTR) and transthyretin-related proteins (TRP). In the present study, we investigated the ttl family in Ostertagia ostertagi (a nematode of the abomasum of cattle). Mining of expressed sequence tag (EST) databases revealed the presence of at least 18 ttl genes in O. ostertagi (Oo-ttl), most of which are constitutively transcribed from the free-living, third larval stage onwards. The full-length cDNA of one of these genes (Oo-ttl-1) was amplified and cloned for recombinant expression. Western blot analysis using a specific antiserum showed that the native protein Oo-TTL-1 was highly present in the excretory–secretory (ES) products of adults of O. ostertagi. The protein was immunolocalized to the pseudocoelomic fluid of adult worms. A phylogenetic–bioinformatic analysis of all amino acid sequence data for TTL proteins from a range of strongylid nematodes showed that they could be divided into at least five different classes. This classification was based on conserved amino acids in the first TTL signature domain and the number and location of cysteine residues. The biological role(s) of the TTLs in nematode biology is still unclear. A theoretical threedimensional model of Oo-TTL-1 indicated that it had a similar structure to TTRs (i.e., containing b-sheets, arranged in a b-sandwich). In contrast to TTRs, competitive binding studies using recombinant Oo-TTL-1 indicated that the protein was devoid of any hydrophobic ligand- or thyroid hormone-binding properties. Finally, combinatorial analysis by double-stranded RNA interference of five ttl genes in the free-living nematode Caenorhabditis elegans did not reveal any visible phenotypes. More information on the transcription profile and tissue distribution of TTLs in nematodes is needed to provide new insights into the biological role of this gene family. 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.