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- Characterization of the RofA regulon in the pandemic M1global and emergent M1UK lineages of Streptococcus pyogenesPublication . Zhi, Xiangyun; Vieira, Ana; Huse, Kristin K.; Martel, Paulo; Lobkowicz, Ludmila; Li, Ho Kwong; Croucher, Nick; Andrew, Ivan; Game, Laurence; Sriskandan, ShiraneeThe standalone regulator RofA is a positive regulator of the pilus locus in Streptococcus pyogenes. Found in only certain emm genotypes, RofA has been reported to regulate other virulence factors, although its role in the globally dominant emm1 S. pyogenes is unclear. Given the recent emergence of a new emm1 (M1 UK) toxigenic lineage that is distinguished by three non-synonymous SNPs in rofA, we characterized the rofA regulon in six emm1 strains that are representative of the two contemporary major emm1 lineages (M1 (global) and M1 (UK)) using RNAseq analysis, and then determined the specific role of the M1 (UK)-specific rofA SNPs. Deletion of rofA in three M1 (global) strains led to altered expression of 14 genes, including six non-pilus locus genes. In M1 (UK) strains, deletion of rofA led to altered expression of 16 genes, including nine genes that were unique to M1 (UK). Only the pilus locus genes were common to the RofA regulons of both lineages, while transcriptomic changes varied between strains even within the same lineage. Although introduction of the three SNPs into rofA did not impact gene expression in an M1 (global) strain, reversal of three SNPs in an M1 (UK) strain led to an unexpected number of transcriptomic changes that in part recapitulated transcriptomic changes seen when deleting RofA in the same strain. Computational analysis predicted that interactions with a key histidine residue in the PRD domain of RofA would differ between M1 (UK) and M1 (global). RofA is a positive regulator of the pilus locus in all emm1 strains but effects on other genes are strain- and lineage-specific, with no clear, common DNA binding motif. The SNPs in rofA that characterize M1 (UK) may impact regulation of RofA; whether they alter phosphorylation of the RofA PRD domain requires further investigation.
- FOXO family isoformsPublication . Santos, Bruno F; Grenho, Inês; Martel, Paulo; Ferreira, Bibiana; Link, WolfgangFOXO family of proteins are transcription factors involved in many physiological and pathological processes including cellular homeostasis, stem cell maintenance, cancer, metabolic, and cardiovascular diseases. Genetic evidence has been accumulating to suggest a prominent role of FOXOs in lifespan regulation in animal systems from hydra, C elegans, Drosophila, and mice. Together with the observation that FOXO3 is the second most replicated gene associated with extreme human longevity suggests that pharmacological targeting of FOXO proteins can be a promising approach to treat cancer and other age-related diseases and extend life and health span. However, due to the broad range of cellular functions of the FOXO family members FOXO1, 3, 4, and 6, isoform-specific targeting of FOXOs might lead to greater benefits and cause fewer side effects. Therefore, a deeper understanding of the common and specific features of these proteins as well as their redundant and specific functions in our cells represents the basis of specific targeting strategies. In this review, we provide an overview of the evolution, structure, function, and disease-relevance of each of the FOXO family members.
- Thermal adaptation and clinal mitochondrial DNA variation of European anchovyPublication . Silva, Goncalo; Lima, Fernando P.; Martel, Paulo; Castilho, RitaNatural populations of widely distributed organisms often exhibit genetic clinal variation over their geographical ranges. The European anchovy, Engraulis encrasicolus, illustrates this by displaying a two-clade mitochondrial structure clinally arranged along the eastern Atlantic. One clade has low frequencies at higher latitudes, whereas the other has an anti-tropical distribution, with frequencies decreasing towards the tropics. The distribution pattern of these clades has been explained as a consequence of secondary contact after an ancient geographical isolation. However, it is not unlikely that selection acts on mitochondria whose genes are involved in relevant oxidative phosphorylation processes. In this study, we performed selection tests on a fragment of 1044 bp of the mitochondrial cytochrome b gene using 455 individuals from 18 locations. We also tested correlations of six environmental features: temperature, salinity, apparent oxygen utilization and nutrient concentrations of phosphate, nitrate and silicate, on a compilation of mitochondrial clade frequencies from 66 sampling sites comprising 2776 specimens from previously published studies. Positive selection in a single codon was detected predominantly (99%) in the anti-tropical clade and temperature was the most relevant environmental predictor, contributing with 59% of the variance in the geographical distribution of clade frequencies. These findings strongly suggest that temperature is shaping the contemporary distribution of mitochondrial DNA clade frequencies in the European anchovy.
- Crystal structures of the free and sterol-bound forms of beta-cinnamominPublication . Rodrigues, Maria Luisa; Archer, Margarida; Martel, Paulo; Miranda, Sandra; Thomaz, Mónica; Enguita, Francisco J.; Baptista, Ricardo P.; Melo, Eduardo P.; Sousa, Nelson; Cravador, A.; Carrondo, Maria A.The crystal structure of the elicitin h-cinnamomin (h-CIN) was determined in complex with ergosterol at 1.1 A° resolution. h-CIN/ergosterol complex crystallized in the monoclinic space group P21, with unit cell parameters of a =31.0, b =62.8, c =50.0 A° and b =93.4- and two molecules in the asymmetric unit. Ligand extraction with chloroform followed by crystallographic analysis yielded a 1.35 A° structure of h-CIN (P43212 space group) where the characteristic elicitin fold was kept. After incubation with cholesterol, a new complex structure was obtained, showing that the protein retains, after the extraction procedure, its ability to complex sterols. The necrotic effect of h-CIN on tobacco was also shown to remain unchanged. Theoretical docking studies of the triterpene lupeol to h-CIN provided an explanation for the apparent inability of h-CIN to bind this ligand, as observed experimentally. D 2005 Elsevier B.V. All rights reserved.
- Binding modes of decavanadate to myosin and inhibition of the actomyosin ATPase activityPublication . Tiago, Teresa; Martel, Paulo; Gutiérrez-Merino, Carlos; Aureliano, M.Decavanadate, a vanadate oligomer, is known to interact with myosin and to inhibit the ATPase activity, but the putative binding sites and the mechanism of inhibition are still to be clarified. We have previously proposed that the decavanadate (V10O28 6−) inhibition of the actin-stimulated myosin ATPase activity is non-competitive towards both actin and ATP. A likely explanation for these results is that V10 binds to the so-called back-door at the end of the Pi-tube opposite to the nucleotide-binding site. In order to further investigate this possibility, we have carried out molecular docking simulations of the V10 oligomer on three different structures of the myosin motor domain of Dictyostelium discoideum, representing distinct states of the ATPase cycle. The results indicate a clear preference of V10 to bind at the back-door, but only on the “open” structures where there is access to the phosphate binding-loop. It is suggested that V10 acts as a “back-door stop” blocking the closure of the 50- kDa cleft necessary to carry out ATP-γ-phosphate hydrolysis. This provides a simple explanation to the non-competitive behavior of V10 and spurs the use of the oligomer as a tool to elucidate myosin back-door conformational changes in the process of muscle contraction.
- Evolution of matrix and bone gamma-carboxyglutamic acid proteins in vertebratesPublication . Laizé, Vincent; Martel, Paulo; Viegas, Carla; Price, P. A.; Cancela, LeonorThe evolution of calcified tissues is a defining feature in vertebrate evolution. Investigating the evolution of proteins involved in tissue calcification should help elucidate how calcified tissues have evolved. The purpose of this study was to collect and compare sequences of matrix and bone γ-carboxyglutamic acid proteins (MGP and BGP, respectively) to identify common features and determine the evolutionary relationship between MGP and BGP. Thirteen cDNAs and genes were cloned using standard methods or reconstructed through the use of comparative genomics and data mining. These sequences were compared with available annotated sequences (a total of 48 complete or nearly complete sequences, 28 BGPs and 20 MGPs) have been identified across 32 different species (representing most classes of vertebrates), and evolutionarily conserved features in both MGP and BGP were analyzed using bioinformatic tools and the Tree-Puzzle software. We propose that: 1) MGP and BGP genes originated from two genome duplications that occurred around 500 and 400 million years ago before jawless and jawed fish evolved, respectively; 2) MGP appeared first concomitantly with the emergence of cartilaginous structures, and BGP appeared thereafter along with bony structures; and 3) BGP derives from MGP. We also propose a highly specific pattern definition for the Gla domain of BGP and MGP. Previous Section Next Section BGP1 (bone Gla protein or osteocalcin) and MGP (matrix Gla protein) belong to the growing family of vitamin K-dependent (VKD) proteins, the members of which are involved in a broad range of biological functions such as skeletogenesis and bone maintenance (BGP and MGP), hemostasis (prothrombin, clotting factors VII, IX, and X, and proteins C, S, and Z), growth control (gas6), and potentially signal transduction (proline-rich Gla proteins 1 and 2). VKD proteins are characterized by the presence of several Gla residues resulting from the post-translational vitamin K-dependent γ-carboxylation of specific glutamates, through which they can bind to calcium-containing mineral such as hydroxyapatite. To date, VKD proteins have only been clearly identified in vertebrates (1) although the presence of a γ-glutamyl carboxylase has been reported in the fruit fly Drosophila melanogaster (2) and in marine snails belonging to the genus Conus (3). Gla residues have also been found in neuropeptides from Conus venoms (4), suggesting a wider prevalence of γ-carboxylation.
- Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectivesPublication . Marques, Cátia L.; Fernandez, Ignacio; Viegas, Michael; Cox, C. J.; Martel, Paulo; Rosa, Joana; Cancela, Leonor; Laizé, VincentBMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as bone inducers during osteogenesis. BMP16 is the most recent member of this subfamily and basic data regarding protein structure and function, and spatio-temporal gene expression is still scarce. In this work, insights on BMP16 were provided through the comparative analysis of structural and functional data for zebrafish BMP2a, BMP2b, BMP4 and BMP16 genes and proteins, determined from three-dimensional models, patterns of gene expression during development and in adult tissues, regulation by retinoic acid and capacity to activate BMP-signaling pathway. Structures of Bmp2a, Bmp2b, Bmp4 and Bmp16 were found to be remarkably similar; with residues involved in receptor binding being highly conserved. All proteins could activate the BMP-signaling pathway, suggesting that they share a common function. On the contrary, stage-and tissue-specific expression of bmp2, bmp4 and bmp16 suggested the genes might be differentially regulated (e.g. different transcription factors, enhancers and/or regulatory modules) but also that they are involved in distinct physiological processes, although with the same function. Retinoic acid, a morphogen known to interact with BMP-signaling during bone formation, was shown to downregulate the expression of bmp2, bmp4 and bmp16, although to different extents. Taxonomic and phylogenetic analyses indicated that bmp16 diverged before bmp2 and bmp4, is not restricted to teleost fish lineage as previously reported, and that it probably arose from a whole genomic duplication event that occurred early in vertebrate evolution and disappeared in various tetrapod lineages through independent events.
- Erratum: Mixed quantum-classical dynamics of an amide-I vibrational excitation in a protein a-helix [Phys. Rev. B 82, 174308 (2010)]Publication . Freedman, Holly; Martel, Paulo; Cruzeiro, LeonorIn the GROMACS codemodifications, instead of the nanometer unit for the distance that is standard in GROMACS, a unit of 1 °A was previously assumed. This led to dipole-dipole interactions between amide I vibrations at different sites and the interaction energies of the amide I vibration with the protein hydrogen bonds being overestimated, respectively, by three orders and by one order of magnitude.
- A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinonePublication . Martins, Ana M.; Latham, John A.; Martel, Paulo; Barr, Ian; Iavarone, Anthony T.; Klinman, Judith P.Pyrroloquinoline quinone is a prominent redox cofactor in many prokaryotes, produced from a ribosomally synthesized and post-translationally modified peptide PqqA via a pathway comprising four conserved proteins PqqB?E. These four proteins are now fairly well-characterized and span radical SAM activity (PqqE), aided by a peptide chaperone (PqqD), a dual hydroxylase (PqqB), and an eight-electron, eight-proton oxidase (PqqC). A full description of this pathway has been hampered by a lack of information regarding a protease/peptidase required for the excision of an early, cross-linked di-amino acid precursor to pyrroloquinoline quinone. Herein, we isolated and characterized a two-component heterodimer protein from the ?-proteobacterium Methylobacterium (Methylorubrum) extorquens that can rapidly catalyze cleavage of PqqA into smaller peptides. Using pulldown assays, surface plasmon resonance, and isothermal calorimetry, we demonstrated the formation of a complex PqqF/PqqG, with a K-D of 300 nm. We created a molecular model of the heterodimer by comparison with the Sphingomonas sp. A1 M16B Sph2681/Sph2682 protease. Analysis of time-dependent patterns for the appearance of proteolysis products indicates high specificity of PqqF/PqqG for serine side chains. We hypothesize that PqqF/PqqG initially cleaves between the PqqE/PqqD-generated cross-linked form of PqqA, with nonspecific cellular proteases completing the release of a suitable substrate for the downstream enzyme PqqB. The finding of a protease that specifically targets serine side chains is rare, and we propose that this activity may be useful in proteomic analyses of the large family of proteins that have undergone post-translational phosphorylation at serine.
- Mixed quantum-classical dynamics of an amide-I vibrational excitation in a protein a-helixPublication . Freedman, Holly; Martel, Paulo; Cruzeiro, LeonorAdenosine triphosphate sATPd is known to be the main energy currency of the living cell, and is used as a coenzyme to generate energy for many cellular processes through hydrolysis to adenosine diphosphate sADPd,although the mechanism of energy transfer is not well understood. It has been proposed that following hydrolysis of the ATP cofactor bound to a protein, up to two quanta of amide-I vibrational energy are excited and utilized to bring about important structural changes in the protein. To study whether, and how, amide-I vibrational excitations are capable of leading to protein structural changes, we have added components arising from quantum-mechanical amide-I vibrational excitations to the total energy and force terms within a moleculardynamics simulation. This model is applied to helical deca-alanine as a test case to investigate how its dynamics differs in the presence or absence of an amide-I excitation. We find that the presence of an amide-I excitation can bias the structure toward a more helical state.