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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.
- A thyroid hormone regulated asymmetric responsive centre is correlated with eye migration during flatfish metamorphosisPublication . Campinho, Marco António; Silva, Nádia; Martins, Gabriel G.; Anjos, Liliana; Florindo, Claudia; Roman-Padilla, Javier; Garcia-Cegarra, Ana; Louro, Bruno; Manchado, Manuel; Power, DeborahFlatfish metamorphosis is a unique post-embryonic developmental event in which thyroid hormones (THs) drive the development of symmetric pelagic larva into asymmetric benthic juveniles. One of the eyes migrates to join the other eye on the opposite side of the head. Developmental mechanisms at the basis of the acquisition of flatfish anatomical asymmetry remain an open question. Here we demonstrate that an TH responsive asymmetric centre, determined by deiodinase 2 expression, ventrally juxtaposed to the migrating eye in sole (Solea senegalensis) correlates with asymmetric cranial ossification that in turn drives eye migration. Besides skin pigmentation that is asymmetric between dorsal and ventral sides, only the most anterior head region delimited by the eyes becomes asymmetric whereas the remainder of the head and organs therein stay symmetric. Sub-ocular ossification is common to all flatfish analysed to date, so we propose that this newly discovered mechanism is universal and is associated with eye migration in all flatfish.
- Vertebrate SLRP family evolution and the subfunctionalization of osteoglycin gene duplicates in teleost fishPublication . Costa, Rita; Brazona, Rute Sofia Tavares Martins; Capilla, E.; Anjos, Liliana; Power, DeborahBackground Osteoglycin (OGN, a.k.a. mimecan) belongs to cluster III of the small leucine-rich proteoglycans (SLRP) of the extracellular matrix (ECM). In vertebrates OGN is a characteristic ECM protein of bone. In the present study we explore the evolution of SLRP III and OGN in teleosts that have a skeleton adapted to an aquatic environment. Results The SLRP gene family has been conserved since the separation of chondrichthyes and osteichthyes. Few gene duplicates of the SLRP III family exist even in the teleosts that experienced a specific whole genome duplication. One exception is ogn for which duplicate copies were identified in fish genomes. The ogn promoter sequence and in vitro mesenchymal stem cell (MSC) cultures suggest the duplicate ogn genes acquired divergent functions. In gilthead sea bream (Sparus aurata) ogn1 was up-regulated during osteoblast and myocyte differentiation in vitro, while ogn2 was severely down-regulated during bone-derived MSCs differentiation into adipocytes in vitro. Conclusions Overall, the phylogenetic analysis indicates that the SLRP III family in vertebrates has been under conservative evolutionary pressure. The retention of the ogn gene duplicates in teleosts was linked with the acquisition of different functions. The acquisition by OGN of functions other than that of a bone ECM protein occurred early in the vertebrate lineage.
- Evolution and diversity of alpha-carbonic anhydrases in the mantle of the Mediterranean mussel (Mytilus galloprovincialis)Publication . Cardoso, João CR; Ferreira, Vinicius; Zhang, Xushuai; Anjos, Liliana; Félix, Rute; Batista, Frederico; Power, DeborahThe α-carbonic anhydrases (α-CAs) are a large and ancient group of metazoan-specific enzymes. They generate bicarbonate from metabolic carbon dioxide and through calcium carbonate crystal formation play a key role in the regulation of mineralized structures. To better understand how α-CAs contribute to shell mineralization in the marine Mediterranean mussel (Mytilus galloprovincialis) we characterized them in the mantle. Phylogenetic analysis revealed that mollusc α-CA evolution was affected by lineage and species-specific events. Ten α-CAs were found in the Mediterranean mussel mantle and the most abundant form was named, MgNACR, as it grouped with oyster nacreins (NACR). Exposure of the Mediterranean mussel to reduced water salinity (18 vs 37 ppt), caused a significant reduction (p < 0.05) in mantle esterase activity and MgNACR transcript abundance (p < 0.05). Protonograms revealed multiple proteins in the mantle with α-CA hydratase activity and mapped to a protein with a similar size to that deduced for monomeric MgNACR. Our data indicate that MgNACR is a major α-CA enzyme in mantle and that by homology with oyster nacreins likely regulates mussel shell production. We propose that species-dependent α-CA evolution may contribute to explain the diversity of bivalve shell structures and their vulnerability to environmental changes.
- Chronic stress impairs the local immune response during cutaneous repair in gilthead sea bream (Sparus aurata, L.)Publication . Mateus, Ana; Anjos, Liliana; Cardoso, João CR; Power, DeborahScale removal in fish triggers a damage-repair program to re-establish the lost epidermis and scale and an associated local immune response. In mammals, chronic stress is known to delay wound healing and to modulate the cutaneous stress axis, but this is unstudied in teleost fish the most successful extant vertebrates. The present study was designed to test the hypothesis that chronic stress impairs cutaneous repair in teleost fish as a consequence of suppression of the immune response. The hypothesis was tested by removing the scales and damaging the skin on one side of the body of fish previously exposed for 4 weeks to a chronic crowding stress and then evaluating cutaneous repair for 1 week. Scale removal caused the loss of the epidermis although at 3 days it was re-established. At this stage the basement membrane was significantly thicker (p = 0.038) and the hypodermis was significantly thinner (p = 0.016) in the regenerating skin of stressed fish relative to the control fish. At 3 days, stressed fish also had a significantly lower plasma osmolality (p = 0.015) than control fish indicative of reduced barrier function. Chronic stress caused a significant down-regulation of the glucocorticoid receptor (gr) in skin before damage (time 0, p = 0.005) and of star at 3 and 7 days (p < 0.05) after regeneration relative to control fish. In regenerating skin key transcripts of cutaneous repair, pcna, colival and nunp9, and the inflammatory response, tgf beta 1, mpo and crtac2, were down-regulated (p < 0.05) by chronic stress. Irrespective of chronic stress and in contrast to intact skin many hyper pigmented masses, putative melanomacrophages, infiltrated the epidermis of regenerating skin. This study reveals that chronic stress suppresses the local immune response to scale removal and impairs the expression of key transcripts of wound healing. Elements of the stress axis were identified and modulated by chronic stress during cutaneous repair in gilthead seabream skin.
- CRTAC1 homolog proteins are conserved from cyanobacteria to man and secreted by the teleost fish pituitary glandPublication . Redruello, Begoña; Louro, Bruno; Anjos, Liliana; Silva, Nádia; Greenwell, Roger S.; Canario, Adelino V. M.; Power, DeborahCartilage acidic protein 1 (CRTAC1) gene expression is used as a marker for chondrocyte differentiation instem cell-based tissue engineering. It is also transcribed outside the skeleton where at least two different transcripts are expressed in lung and brain. In the pituitary gland of the teleost fish sea bream Sparus auratus, we have found a transcript with a high degree of sequence identity to CRTAC1 family members but lacking the EGF-like calcium-binding domain encoding sequence of CRTAC1 and designated it as CRTAC2. Database searches revealed many previously unidentified members of the CRTAC1 and CRTAC2 in phylogenetically distant organisms, such as cyanobacteria, bryophyta, lancelets, and diverse representatives of vertebrates. Phylogenetic analyses showed that the genes encoding CRTAC1 and CRTAC2 proteins coexist in teleost fish genomes. Structural prediction analysis identified the N-terminal region of the CRTAC1/CRTAC2 family members as a potential seven-bladed β -propeller structure, closely related to those of integrin α chains and glycosylphosphatidylinositol-specific phospholipase D1 protein families. This relationship is con fi rmed by phylogenetic analysis with the N-terminal domain of sea bream CRTAC2 as the most divergent sequence. Because teleost fi shes are the only phylogenetic group where both CRTAC1 and CRTAC2 genes are present, they occupy a pivotal position in studies of the mechanisms governing the speci fi c expression patterns of each gene/protein subfamily. This will be essential to elucidate their respective biological roles.
- Biochemical and biological characteristics of novel piscine forms of parathyroid hormone and parathyroid hormone related peptide: a proteomic approachPublication . Anjos, Liliana; Power, DeborahVertebrate bone remodeling and development is a fundamental physiological process which is regulated by multiple factors. Homologues of mammalian parathyroid hormone (PTH)/ parathyroid hormone related peptide (PTHrP) which have a role in skeletal homeostasis have been identified in teleosts but their function is poorly understood. This dissertation describes the biochemical characterization of PTH/ PTHrP family from an advanced teleost, sea bream. Recombinant His-tag PTHA\PTHB and PTH-L proteins were produced using a prokaryotic expression system. In silico analysis predicted a secondary structure for PTHA and PTHB composed of -helix and random coils which was confirmed by biophysical studies of the recombinant proteins. A reference proteome of acellular sea bream bone was generated using 2D electrophoresis coupled to MALDI-TOFF analysis and the way in which sea bream recombinant PTHrP modifies the bone proteome was established. Approximately 300 proteins were mapped in the bone proteome and 125 proteins were analysed of which 94.4% matched proteins in public databases (118). Comparison of control and PTHrP treated fish revealed 8 differentially expressed proteins and the results suggest that global proteome analysis identified novel PTHrP regulated pathways in fish bone. To establish the physiological function of piscine CRTAC2 (Cartilage acidic protein 2), which is a homologue of mammalian CRTAC1 (chondrocyte marker) a recombinant protein was produced. The secondary structure of the soluble recombinant protein contained 9% -helix, 32% - sheet and 58% unordered protein and it had a hyperthermostable tertiary structure. Western blot revealed CRTAC had a widespread tissue distribution and was abundantly expressed in kidney and liver and that the native protein exists in tissue extracts as high molecular weight aggregates. Preliminary physiological studies in fish revealed that sbCRTAC2 in common with collagen I favors outgrowth of epithelia and highlights a possible function which will be studied in the future.
- Four stanniocalcin genes in teleost fish: Structure, phylogenetic analysis, tissue distribution and expression during hypercalcemic challengePublication . Schein, V; Schein, Vanessa; Cardoso, João CR; Pinto, Patricia IS; Anjos, Liliana; Silva, Nadia; Power, Deborah; Canario, Adelino V. M.Stanniocalcin (STC), first isolated from the corpuscles of Stannius (CS) of teleost fishes and a systemic regulator of mineral metabolism, is present in all vertebrates as two isoforms, STC1 and STC2, encoded by separate genes. Here we show that the genome of Tetraodon nigroviridis, and other teleosts, possess duplicate genes for each STC isoform, designated stc1-a and -b, and stc2-a and -b. Stc1-a was cloned from CS, stc2-a from muscle and the two novel cDNAs, stc1-b and stc2-b, from brain. However, stc2-b was isolated as a conjoined (read-through) transcript with bod1 (bi-orientation defective 1, or FAM44B), and two additional alternative conjoined transcripts were also isolated. The predicted STC products shared the typical vertebrate 10 conserved cysteine residues and N-linked glycosylation motifs, in addition to specific features. Gene structure was generally conserved with four exons and three introns with the exception of stc1-a which gained an extra intron in exon three, originating one extra exon. Gene order and synteny is also maintained across vertebrates and the cpeb4 gene identified in the homologue region of the chordate Ciona was linked to vertebrate stc2 but not stc1. Immunohistochemistry in different species revealed that STC1-A was found only in CS and in a few cells in kidney. STC1-B had a restricted expression and was more prominent in the gills. STC2-A was detected in a variety of tissues, including pituitary, with most abundant immunoreaction in kidney cells and gill rakers and the CS was negative. Expression of stc1-a in CS of Tetraodon was 15-fold (p < 0.05) up-regulated 2 h after transfer from 2.9 mM Ca2+ to 10 mM Ca2+ water and down-regulated after 12 hours to 11-fold lower than 2.9 mM Ca2+ fish (p < 0.05). With the exception of stc1-a in CS, low expression levels and high individual variation were generally found for the expression of stc transcripts in kidney and gills, with no statistically significant changes in response to the hypercalcemic shock. In conclusion, both stc1 and stc2 genes are represented by paralogues in teleosts genomes and the analysis performed suggests that only stc1-a in the CS is involved in extracellular calcium regulation. The widespread distribution of stcs in fish tissues supports pleiotropic roles.
- High pressure processing of European sea bass (Dicentrarchus labrax) fillets and tools for flesh quality and shelf life monitoringPublication . Tsironi, Theofania; Anjos, Liliana; Pinto, Patricia IS; Dimopoulos, George; Santos, Soraia; Santa, Cátia; Manadas, Bruno; Canario, Adelino; Taoukis, Petros; Power, DeborahThe effects of high pressure (HP:600 MPa, 5 min, 25 °C) on European sea bass fillets were investigated using microbiological, physicochemical and sensory indices, and “omics” technologies. HPP led to more than a 5 log(cfu/g) reduction in initial bacterial total viable counts and altered the bacterial microbiome, reducing the proportion of food spoilage genera. Lightness and hardness of the fish flesh significantly increased after HPP and were associated with modified muscle tissue histology, with fibers appearing fused and more compact in comparison to the unprocessed control. Sensory evaluation (based on a lower limit of 5 for overall acceptability scoring) indicated a shelf life of 11 days for untreated control samples and 2 months for the HP-treated fillets. Quantitative SWATH proteomics revealed 281 proteins that had modified levels between control and HP-processed fish flesh. The metagenomics and proteomics provided detailed insight into how the change in HP-processed sea bass fillets is linked to the modifications in the microbiome and proteome.
- Molecular cloning and functional characterization of a monoterpene synthase isolated from the aromatic wild shrub Thymus albicansPublication . Filipe, Alexandra; Cardoso, João; Miguel, Maria Graca; Anjos, Liliana; Trindade, Helena; Figueiredo, Ana Cristina; Barroso, Jose; Power, Deborah; Marques, N T.The essential oil of Thymus albicans Hoffmanns. & Link, a native shrub from the Iberian Peninsula, is mainly composed of monoterpenes. In this study, a 1,8-cineole synthase was isolated from the 1,8-cineole chemotype. A partial sequence that lacked the complete plastid transit peptide but contained an extended C-terminal when compared to other related terpene synthases was generated by PCR and Rapid Amplification of cDNA Ends (RACE). The predicted mature polypeptide was 593 amino acids in length and shared 78% and 77% sequence similarity with the homologue 1,8-cineole synthase from Rosmarinus officinalis and Salvia officinalis, respectively. The putative protein possessed the characteristic conserved motifs of plant monoterpene synthases including the RRx(8)W and DDxxD motifs and phylogenetic analysis indicated that the amplified 1,8-cineole synthase bears greater sequence similarity with other 1,8-cineole synthases from Lamiaceae family relative to the terpene synthases from the genus Thymus. Functional expression of the recombinant protein in Escherichia coli revealed that in the presence of geranyl diphosphate (GPP) 1,8-cineole was the major product but that its production was too low for robust quantification. Other minor conversion products included a-pinene, beta-pinene, sabinene and beta-myrcene suggesting the isolated 1,8-cineole synthase may be a multi-product enzyme. To our knowledge, this is the first report of a functionally characterized monoterpene synthase from Thymus albicans.