117 research outputs found

    SOHSite: incorporating evolutionary information and physicochemical properties to identify protein S-sulfenylation sites

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    Distribution of KEGG pathway annotations for S-sulfenylated proteins. (DOCX 15 kb

    Pattern discovery in sequence databases : algorithms and applications to DNA/protein classification

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    Sequence databases comprise sequence data, which are linear structural descriptions of many natural entities. Approximate pattern discovery in a sequence database can lead to important conclusions or prediction of new phenomena. Traditional database technology is not suitable for accomplishing the task, and new techniques need to be developed. In this dissertation, we propose several new techniques for discovering patterns in sequence databases. Our techniques incorporate pattern matching algorithms and novel heuristics for discovery and optimization. Experimental results of applying the techniques to both generated data and DNA/proteins show the effectiveness of the proposed techniques. We then develop several classifiers using our pattern discovery algorithms and a previously published fingerprint technique. When we apply the classifiers to classify DNA and protein sequences, they give information that is complementary to the best classifiers available today

    A combined computational and experimental approach to human osteocalcin and GPCR Family C Group 6 Member A

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    INTRODUCTION. The use of mouse genetics recently highlighted coordination by endocrine regulation between bone, energy metabolism, and endogenous sex hormones. Osteocalcin, a bone matrix protein that regulates hydroxyapatite size and shape through its vitamin‑K-dependent γ‑carboxylated form, unraveled endocrinological functions in its circulating forms, that are not only simple markers of bone formation. Undercarboxylated osteocalcin administration regulates gene and protein expression in adipocytes, in pancreatic β cells, and in Leydig cells by a G protein-coupled receptor, GPRC6A receptor, with a potential gender selectivity. Total serum osteocalcin concentrations in humans are inversely associated with measures of glucose metabolism and cardiovascular risk factors; however, human data are inconclusive with regard to the role of uncarboxylated osteocalcin because most studies do not account for the influence of vitamin K or differentiate between the different γ‑carboxylated forms of osteocalcin. Intriguingly GPRC6A receptor mediates also the non-genomic effects of androgens and, although the information about SHBG-receptor structure is not conclusive, evidence seems to suggest that a G protein-coupled receptor is involved. AIM of the STUDY. Starting from clinical observations, we investigated the protein-protein interaction computational predictors of osteocalcin and SHBG with GPRC6A receptor, and we validated experimentally in vitro in a two-step approach. MATHERIAL and METHODS. Clinical and biochemical characteristics were studied in a cohort of 91 obese patients and healthy controls in a cross-sectional study. 3-D protein structure alignment analysis and protein docking analysis were resolved with a four (i.e. TM-align, FATCAT, TriangleMatch, TopMatch) and three (i.e. GRAMM-X, ZDOCK, PatchDock) different algorithms approach, respectively. ClickMD-min script was used as a molecular dynamic platform for computational analysis of the conformational structure of osteocalcin in presence or absence of Ca2+, developed with MOE software. Steered molecular dynamics simulations related to the carboxylation state were performed, and the data compared with the spectroscopic techniques results. Gene and protein expression analysis and cell-surface receptor binding assays (i.e. immunofluorescence and flow cytometry analysis) on HEK-293T cells experimentally validated the former results in vitro. RESULTS. Our study shows for the first time the existence of the competition for a specific binding site between osteocalcin and SHBG on human cells expressing GPRC6A receptor. The results are supported by a computational prediction analysis, describing amino acid residues of SHBG from Gly145 to Leu161 as a highly predicted interface. Our experiments describe the structure of human osteocalcin in its carboxylated and undercarboxylated forms for the first time. The influence of the binding of calcium on osteocalcin structure seems to be stronger than the presence of γ-carboxylated glutamic acid residues. Our clinical data show an imbalance between the γ‑carboxylated forms of osteocalcin in a cohort of hypogonadic obese an overweight patients, with decreased levels of SHBG and altered levels of the other sex hormones. CONCLUSIONS. The current two-step approach offers a target approach of investigation directly in humans, that has the potential to identify novel pathophysiological pathways as well as novel therapeutic possibilities. The computational basis of the possible binding of SHBG and osteocalcin has been experimentally validated and can directly lead to the synthesis of a peptide, whose physiological and therapeutic implications represent a feasible perspective for future research in an area of paramount importance, such as the cross-talk between bone, energy metabolism, and endogenous sex hormones

    DAROGAN: Enzyme function prediction from multiple sequence alignments

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    Evolution, gene regulation and functional analysis of BMP2 in fish

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    Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor β (TGFβ) superfamily with a central role in bone formation and mineralization. BMP2, a founding member of this family, has demonstrated remarkable osteogenic properties and is clinically used to promote bone repair and fracture healing. Lack of basic data on factors regulating BMP2 expression and activity have hampered a better understanding of its role in bone formation and bone-related diseases. The objective of this work was to collect new functional data and determine spatiotemporal expression patterns in a fish system aiming towards a better understanding of BMP2 function and regulation. Transcriptional and post-transcriptional regulation of gilthead seabream BMP2 gene was inferred from luciferase reporter systems. Several bone- and cartilage-related transcription factors (e.g. RUNX3, MEF2c, SOX9 and ETS1) were found to regulate BMP2 transcription, while microRNA 20a was shown to affect stability of the BMP2 transcript and thus the mineralogenic capacity of fish bone-derived host cells. The regulation of BMP2 activity through an interaction with the matrix Gla protein (MGP) was investigated in vitro using BMP responsive elements (BRE) coupled to luciferase reporter gene. Although we demonstrated the functionality of the experimental system in a fish cell line and the activation of BMP signaling pathway by seabream BMP2, no conclusive evidence could be collected on a possible interaction beween MGP and BMP2. The evolutionary relationship among the members of BMP2/4/16 subfamily was inferred from taxonomic and phylogenetic analyses. BMP16 diverged prior to BMP2 and BMP4 and should be the result of an ancient genome duplication that occurred early in vertebrate evolution. Structural and functional data suggested that all three proteins are effectors of the BMP signaling pathway, but expression data revealed different spatiotemporal patterns in teleost fish suggesting distinct mechanisms of regulation. In this work, through the collection of novel data, we provide additional insight into the regulation, the structure and the phylogenetic relationship of BMP2 and its closely related family members.O sistema esquelético confere suporte e proteção ao organismo, permite o armazenamento de minerais e desempenha funções hematopoiéticas. Um dos seus principais componentes é o osso, um tecido conectivo especializado e constituído por uma matriz extracelular extensamente mineralizada. O processo de mineralização envolve mecanismos extremamente complexos que estão sujeitos a um rigoroso controlo a nível molecular, no qual estão envolvidas várias proteínas responsáveis pela diferenciação celular e pela síntese de matriz extracelular. Dentro deste conjunto de proteínas, destacam-se alguns fatores de crescimento essenciais ao mecanismo de mineralização tecidular, como é o caso das proteínas morfogenéticas do osso (BMPs). As BMPs pertencem à superfamília de fatores de crescimento de transformação β (TGFβ) e estão envolvidas em vários processos durante a embriogénese, organogénese, proliferação e diferenciação celular e mecanismos de formação óssea. Atualmente estão descritos e caracterizados mais de vinte membros pertencentes a esta família, que foram divididos em várias subfamílias, de acordo com a semelhança da estrutura primária das suas proteínas. A subfamília BMP2/4/16 à qual pertence a BMP2 foi uma das primeiras a ser identificada e caracterizada. A BMP2 é uma das proteínas que possui uma maior capacidade osteogénica e é, desde há muito tempo, considerada um potencial agente terapêutico para o tratamento de doenças relacionadas com o osso, sendo mesmo utilizada em alguns casos clínicos de fraturas ósseas. O conhecimento dos processos de formação óssea, bem como os mecanismos de regulação da BMP2, são por isso de extrema importância para uma melhor compreensão dos processos subjacentes ao desenvolvimento e progressão de algumas doenças ósseas. Assim, a BMP2 tem sido alvo de vários estudos, quer a nível de conhecimento da sua função, quer a nível de mecanismos de ação e processamento. Sabe-se que a BMP2 é uma proteína secretada para a matriz extracelular onde, através de um mecanismo de sinalização molecular, é responsável pela regulação de vários processos. O mecanismo de sinalização inicia-se quando dímeros de BMP2 se ligam aos respetivos recetores, presentes na superfície da célula, ativando assim uma cascata de sinalização molecular. Através de diferentes intermediários intracelulares, envolvidos na cascata de sinalização, a BMP2 é responsável pela regulação transcricional de vários genes-alvo. No entanto, e apesar dos vários estudos que foram feitos nesta área, o conhecimento existente acerca deste assunto é ainda bastante escasso. Neste sentido, o objetivo principal deste trabalho foi a recolha de novos dados funcionais e estruturais, que permitam uma melhor compreensão da função da BMP2. Para tal, e de modo a complementar o conhecimento existente, utilizámos o peixe como modelo alternativo aos sistemas de mamíferos. O peixe é atualmente reconhecido como um modelo válido para estudos do esqueleto de vertebrados para o qual existem já várias ferramentas que permitem análises in silico, in vitro e in vivo. Este trabalho envolveu o estudo da regulação do gene da BMP2 de dourada, tanto a nível transcricional como a nível pós-transcricional. Numa primeira fase, foram identificados potenciais reguladores transcricionais da BMP2 de dourada, através da análise in silico da região reguladora do gene. Dentro dos potenciais reguladores transcricionais, foram identificados vários fatores de transcrição com funções descritas ao nível do osso e da cartilagem, nomeadamente o RUNX3, SOX9, MEF2C e ETS1, que foram posteriormente testados a nível funcional através de ensaios repórter de luciferase. Em paralelo, no decorrer da caracterização de reguladores pós-transcricionais da BMP2, através da análise da região 3’ não traduzida (3’UTR) do seu mRNA, foi possível identificar um local de ligação para o miR-20a, conservado ao longo da evolução. A fim de melhor compreender os mecanismos de ação da BMP2, neste trabalho investigámos também possíveis parceiros desta proteína. A caraterização da interação entre a BMP2 e a proteína Gla da matriz (MGP), um conhecido inibidor da calcificação, foi avaliada através do uso de um sistema de elementos de resposta às BMPs, acoplado a um gene repórter, a luciferase. Embora tenhamos demonstrado a funcionalidade do sistema através da ativação do mecanismo de sinalização celular pela BMP2 de dourada, não foram obtidos dados conclusivos no que diz respeito à interação entre a BMP2 e a MGP. Finalmente, abordamos o aspecto evolutivo dos membros da subfamília BMP2/4/16 através da análise da sua distribuição taxonómica entre vários organismos vertebrados, bem como as relações filogenéticas existentes entre os vários membros desta subfamília. Foi demonstrado que a BMP16 divergiu antes da BMP2 e BMP4 na linhagem dos vertebrados e foi, provavelmente, o resultado de uma duplicação genómica que terá ocorrido ancestralmente. Dados estruturais sugerem uma conservação funcional das três proteínas, facto que foi confirmado pela capacidade de ativação dos mecanismos de sinalização das BMPs. No entanto, e apesar da conservação ao nível da região codante dos genes das BMP2, BMP4 e BMP16, as regiões não traduzidas são substancialmente diferentes, apontando para uma regulação diferencial dos três genes, como é aliás sugerido pelos distintos padrões de expressão observados para a BMP2, BMP4 e BMP16, tanto em linguado como em peixe zebra. Ao longo deste trabalho foram recolhidos novos dados que permitem uma melhor compreensão da função e regulação da BMP2. Foram igualmente obtidas informações relevantes acerca da filogenia molecular dos membros da subfamília das BMP2/4/16 que contribuíram para uma melhor compreensão e interpretação da complexa história evolutiva desta subfamília. No seu conjunto, os resultados deste trabalho contribuem para uma validação do uso dos peixes como um modelo alternativo na investigação de mecanismos moleculares envolvidos no processo de mineralização tecidular.Universidade do Algarve, Departamento de Ciências Biomédica

    The Distinctive Regulatory Mechanisms of Bacterial Acetyl-CoA Carboxylase

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    Metabolic Regulation is a complex system used to control cellular metabolism in response to conditions in the cell’s environment. For most enzymes, the cell can rely upon a minimal amount of regulation; however, critical enzymes, such as acetyl-CoA carboxylase, must be regulated at multiple levels. Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In bacteria, acetyl-CoA carboxylase forms a complex of three subunits–biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase–which catalyze the carboxylation of acetyl-CoA to form malonyl-CoA via two half-reactions. In the first half-reaction, biotin covalently linked to biotin carboxyl carrier protein is carboxylated by biotin carboxylase. Carboxyltransferase catalyzes the second half-reaction where the carboxyl group is transferred from carboxybiotin to acetyl-CoA, forming malonyl-CoA. As acetyl-CoA carboxylase is a critical enzyme in cell growth, it is subject to multiple forms of regulation. This dissertation describes two distinct mechanisms of regulation of acetyl-CoA carboxylase. Previous reports showed that the downstream product of fatty acid synthesis, palmitoyl-acyl carrier protein, inhibits catalysis by acetyl-CoA carboxylase via negative feedback regulation. This dissertation reports the acetyl-CoA carboxylase complex was found to exhibit a pronounced hysteresis when inhibited by palmitoyl-acyl carrier protein. Alternatively, palmitoyl-acyl carrier protein does not inhibit either half–reaction. Structure-function studies of palmitoyl-acyl carrier protein demonstrate the inhibitory moiety is the cofactor pantothenic acid. This dissertation also reports that the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase regulates catalysis. The biotin carboxyl carrier protein in E. coli is composed of two domains: the N-terminal domain, and the C-terminal domain. The C-terminal domain contains the biotin cofactor, and acts as a substrate, while the function of the N-terminal domain was unknown. In vivopull-down assays demonstrated the N-terminal domain is not required for formation of the catalytic complex, whereasin vitroanalyses demonstrated that the N-terminal domain binds biotin. Thus, it is likely the role of the N-terminal domain of biotin carboxyl carrier protein in bacterial acetyl-CoA carboxylase is to bind and stabilize the carboxybiotin intermediate during catalysis

    Identification and Characterization of Novel Transporters Involved in the CO2 Concentrating Mechanism of Chlamydomonas reinhardtii

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    The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) which helps in successful acclimation to low CO2 conditions. One of the main aspects of the CCM is bringing in inorganic carbon (Ci) into the cell as bicarbonate using Ci transporters. Current models of the CCM postulate that a series of ion transporters bring HCO3- from outside the cell to the thylakoid lumen where the carbonic anhydrase, CAH3, dehydrates accumulated HCO3- to CO2, raising the CO2 concentration for Rubisco. Previously, HCO3- transporters have been identified at both the plasma membrane (HLA3 and LCI1) and the chloroplast envelope (LCIA/NAR1.2), but the transporter thought to be on the thylakoid membrane has not been identified. The goal of this thesis has been to find the putative thylakoid transporter using a bioinformatics approach to identify candidate proteins followed by characterization of the interesting transporters using RNAi. Nine candidate proteins were identified from the Chlamydomonas proteome which had transporter like domains and were upregulated in low CO2. Three of the candidates are the paralogous genes (BST1, BST2, BST3) belonging to the bestrophin family, which are not only upregulated in low CO2 conditions but their expression is controlled by CIA5, a transcription factor known to control Ci transporters of the CCM. YFP fusions demonstrate that all three proteins are located on the thylakoid membrane and interactome studies indicate that they might associate with other chloroplast CCM components. A single mutant defective in BST3, still grows normally on low CO2, indicating that the three bestrophin-like proteins may have redundant functions. Therefore, an RNAi approach was adopted to reduce the expression of all three genes at once. RNAi mutants with reduced expression of BST1-3, were unable to grow at low CO2 concentrations, exhibited a reduced affinity for inorganic carbon compared to the wild type cells, and exhibited reduced inorganic carbon uptake. We propose that these bestrophin-like proteins are essential components of the CCM and deliver HCO3- accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen Another mutant , A144 was discovered to be a part of a large-scale mutagenesis project to select for mutants that have been transformed with a paromomycin cassette and grow slowly under low CO2 growth conditions. Using a modified form of the adapter PCR method, the location of the insert in A144 was found to be in a gene that codes for a protein with homology of fungal translational elongation factor (eEF3). This mutant cannot adapt to light after prolonged exposure to darkness and has a significantly lower rate of respiration than wild type. Thus we propose that EF3 putatively aids in the synthesis of stress proteins for dark adaptation

    A Bioinformatics Study of Protein Conformational Flexibility and Misfolding: a Sequence, Structure and Dynamics Approach

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    This PhD Thesis titled "A Bioinformatics Study of Protein Conformational Flexibility and Misfolding: a Sequence, Structure and Dynamics Approach" comprises the results and conclusions obtained by us from the study of three different but somehow related research projects, covering aspects of the phenomenon of protein local conformational instability, its relationship with protein function, evolvability and aggregation, and the effect of genetic variations on protein conformational instability related to Conformational Diseases. These projects include the prediction of putative prion proteins in complete proteomes and the study of prion biology from a genomic perspective, the prediction of conformationally unstable protein regions and the existence of a structural framework for linking conformational instability to folding and function, and the establishment of a rationale for assessing the connection among mutations and disease phenotypes in Conformational Diseases.Esta tesis doctoral comprende los resultados y conclusiones obtenidos por nosotros a partir del estudio de tres proyectos de investigación diferentes pero de alguna manera relacionados, cubriendo los aspectos del fenómeno de la inestabilidad conformacional local de la proteína, su relación con la función de la proteína, la capacidad de evolución y agregación, y el efecto de las variaciones genéticas en la inestabilidad conformacional de la proteína relacionados con las enfermedades conformacionales. Estos proyectos incluyen la predicción de presuntas proteínas priónicas en proteomas complejos y el estudio de la biología de priones desde una perspectiva genómica, la predicción de las regiones de proteínas conformacionalmente inestables y la existencia de un marco estructural para la vinculación de la inestabilidad conformacional del plegado y la función, y el establecimiento de una razón fundamental para la evaluación de la relación entre las mutaciones y fenotipos de la enfermedad en enfermedades conformacionales
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