Multidimensional Feature Engineering for Post-Translational Modification Prediction Problems

Protein sequence data has been produced at an astounding speed. This creates an opportunity to characterize these proteins for the treatment of illness. A crucial characterization of proteins is their post translational modifications (PTM). There are 20 amino acids coded by DNA after coding (translation) nearly every protein is modified at an amino acid level. We focus on three specific PTMs. First is the bonding formed between two cysteine amino acids, thus introducing a loop to the straight chain of a protein. Second, we predict which cysteines can generally be modified (oxidized). Finally, we predict which lysine amino acids are modified by the active form of Vitamin B6 (PLP/pyridoxal-5-phosphate.) Our work aims to predict the PTM\u27s from protein sequencing data. When available, we integrate other data sources to improve prediction. Data mining finds patterns in data and uses these patterns to give a confidence score to unknown PTMs. There are many steps to data mining; however, our focus is on the feature engineering step i.e. the transforming of raw data into an intelligible form for a prediction algorithm. Our primary innovation is as follows: First, we created the Local Similarity Matrix (LSM), a description of the evolutionarily relatedness of a cysteine and its neighboring amino acids. This feature is taken two at a time and template matched to other cysteine pairs. If they are similar, then we give a high probability of it sharing the same bonding state. LSM is a three step algorithm, 1) a matrix of amino acid probabilities is created for each cysteine and its neighbors from an alignment. 2) We multiply the iv square of the BLOSUM62 matrix diagonal to each of the corresponding amino acids. 3) We z-score normalize the matrix by row. Next, we innovated the Residue Adjacency Matrix (RAM) for sequential and 3-D space (integration of protein coordinate data). This matrix describes cysteine\u27s neighbors but at much greater distances than most algorithms. It is particularly effective at finding conserved residues that are further away while still remaining a compact description. More data than necessary incurs the curse of dimensionality. RAM runs in O(n) time, making it very useful for large datasets. Finally, we produced the Windowed Alignment Scoring algorithm (WAS). This is a vector of protein window alignment bit scores. The alignments are one to all. Then we apply dimensionality reduction for gains in speed and performance. WAS uses the BLAST algorithm to align sequences within a window surrounding potential PTMs, in this case PLP attached to Lysine. In the case of WAS, we tried many alignment algorithms and used the approximation that BLAST provides to reduce computational time from months to days. The performances of different alignment algorithms did not vary significantly. The applications of this work are many. It has been shown that cysteine bonding configurations play a critical role in the folding of proteins. Solving the protein folding problem will help us to find the solution to Alzheimer\u27s disease that is due to a misfolding of the amyloid-beta protein. Cysteine oxidation has been shown to play a role in oxidative stress, a situation when free radicals become too abundant in the body. Oxidative stress leads to chronic illness such as diabetes, cancer, heart disease and Parkinson\u27s. Lysine in concert with PLP catalyzes the aminotransferase reaction. Research suggests that anti-cancer drugs will potentially selectively inhibit this reaction. Others have targeted this reaction for the treatment of epilepsy and addictions

Monitoring Keap1-Nrf2 interactions in single live cells

AbstractThe transcription factor NF-E2 p45-related factor 2 (Nrf2) and its negative regulator Kelch-like ECH associated protein 1 (Keap1) control the expression of nearly 500 genes with diverse cytoprotective functions. Keap1, a substrate adaptor protein for Cullin3/Rbx1 ubiquitin ligase, normally continuously targets Nrf2 for degradation, but loses this ability in response to electrophiles and oxidants (termed inducers). Consequently, Nrf2 accumulates and activates transcription of its downstream target genes. Many inducers are phytochemicals, and cruciferous vegetables represent one of the richest sources of inducer activity among the most commonly used edible plants. Here we summarize the discovery of the isothiocyanate sulforaphane as a potent inducer which reacts with cysteine sensors of Keap1, leading to activation of Nrf2. We then describe the development of a quantitative Förster resonance energy transfer (FRET)-based methodology combined with multiphoton fluorescence lifetime imaging microscopy (FLIM) to investigate the interactions between Keap1 and Nrf2 in single live cells, and the effect of sulforaphane, and other cysteine-reactive inducers, on the dynamics of the Keap1–Nrf2 protein complex. We present the experimental evidence for the “cyclic sequential attachment and regeneration” or “conformation cycling” model of Keap1-mediated Nrf2 degradation. Finally, we discuss the implications of this mode of regulation of Nrf2 for achieving a fine balance under normal physiological conditions, and the consequences and mechanisms of disrupting this balance for tumor biology

Prediction of Oxidation States of Cysteines and Disulphide Connectivity

Knowledge on cysteine oxidation state and disulfide bond connectivity is of great importance to protein chemistry and 3-D structures. This research is aimed at finding the most relevant features in prediction of cysteines oxidation states and the disulfide bonds connectivity of proteins. Models predicting the oxidation states of cysteines are developed with machine learning techniques such as Support Vector Machines (SVMs) and Associative Neural Networks (ASNNs). A record high prediction accuracy of oxidation state, 95%, is achieved by incorporating the oxidation states of N-terminus cysteines, flanking sequences of cysteines and global information on the protein chain (number of cysteines, length of the chain and amino acids composition of the chain etc.) into the SVM encoding. This is 5% higher than the current methods. This indicates to us that the oxidation states of amino terminal cysteines infer the oxidation states of other cysteines in the same protein chain. Satisfactory prediction results are also obtained with the newer and more inclusive SPX dataset, especially for chains with higher number of cysteines. Compared to literature methods, our approach is a one-step prediction system, which is easier to implement and use. A side by side comparison of SVM and ASNN is conducted. Results indicated that SVM outperform ASNN on this particular problem. For the prediction of correct pairings of cysteines to form disulfide bonds, we first study disulfide connectivity by calculating the local interaction potentials between the flanking sequences of the cysteine pairs. The obtained interaction potential is further adjusted by the coefficients related to the binding motif of enzymes during disulfide formation and also by the linear distance between the cysteine pairs. Finally, maximized weight matching algorithm is applied and performance of the interaction potentials evaluated. Overall prediction accuracy is unsatisfactory compared with the literature. SVM is used to predict the disulfide connectivity with the assumption that oxidation states of cysteines on the protein are known. Information on binding region during disulfide formation, distance between cysteine pairs, global information of the protein chain and the flanking sequences around the cysteine pairs are included in the SVM encoding. Prediction results illustrate the advantage of using possible anchor region information

Biophysical and Functional Characterization of the Persulfide Sensor CstR from Staphylococcus aureus

Thesis (Ph.D.) - Indiana University, Chemistry, 2015How cells regulate the intracellular bioavailable pool of sulfur while mitigating its toxic effects is poorly understood. Major components of this pool, collectively referred to as reactive sulfur species (RSS), include hydrogen sulfide (H2S) and low molecular weight per- and polysulfides and become deleterious at increasing concentrations. For example, H2S functions as a gaseous signaling molecule but at elevated levels can poison cytochrome c oxidase of the electron transport chain. The human pathogen Staphylococcus aureus contains an apparent complete sulfur oxidation system, termed the cst operon, which is under control of the transcriptional regulator CstR (Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor). In this work, sulfide stress, as exogenous H2S or RSS, is identified as a strong inducer of the cst operon. Failure to express this operon or deletions of enzymes within results in loss cellular viability under sulfide stress conditions. Biophysical investigations into the regualtor CstR revealed it does not react with hydrogen sulfide directly but rather reacts with the sulfane sulfur of an inorganic polysulfide or organic persulfide donor to form di-, tri, and tetrasulfide bonds between two conserved cysteine residues, Cys31 and Cys60' on opposite protomers as determined by high-resolution tandem mass spectrometry. These modifications result in negative regulation of cst operator DNA binding affinity. Interestingly, CstR displays reaction specificity compared to the structurally similar CsoR that is also found in S. aureus, which controls the expression of genes responsible for copper toxicity resistance. Comparative analysis of CstR and CsoR cysteine reactivity by pulsed-alkylation mass spectrometry reveals a striking difference in Cys31 reactivity (Cys41 in CsoR). These studies provide the framework for better understanding of sulfide homeostasis and stress resistance as well as identification of key differences in CstR and CsoR inducer selectivity

The Role of the S. cerevisiae Sco2p and Its Homologues in Antioxidant Defense Mechanisms

The Sco proteins, present in all kind of organisms, are regarded as one of the key players in the cytochrome c oxidase (COX) assembly. However, experimental and structural data, such as the presence of a thioredoxin-like fold, suggest that Sco proteins may also play a role in redox homeostasis. Our current studies in S. cerevisiae have strongly suggested an antioxidant role to Sco2 protein (ySco2p). While the single deletion of SCO2 does not result in a distinctive phenotype, the concomitant deletion of superoxide dismutase 1 (SOD1) leads to an increased sensitivity to oxidative stress generating agents (paraquat, menadione, plumbagin) compared to the respective single mutants. Since S. cerevisiae is a good model to functionally characterize genes from more complex organisms, identification of such a phenotype has paved the way to test whether the Sco2 homologues from other organisms are able to substitute for the function of ySco2p. The Sco homologues from Homo sapiens, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster and Kluyveromyces lactis were integrated into the genome of the double deletion mutant. The functional complementation was tested by both growth and biochemical ROS assays. All homologues except for K. lactis K07152 and A. thaliana HCC1 were able to complement the phenotype, indicating their role in antioxidant defense. Interestingly, pathogenic human SCO2 point mutations failed to restore this function. The observation of non-functional homologues despite of the high sequence similarity to ySco2p strengthened our hypothesis on the importance of conserved aminoacid(s) for the defensive role. For this purpose, selected homologues were aligned and the conservation was judged not only based on identity but also similarity (e.g. charge, hydrophobicity). Interestingly, alignment results have pointed out an aminoacid site (located 15 aminoacids downstream of CxxxC motif) that a positively charged lysine is found only in the non-functional homologues. Subsequent mutagenesis analyses verified the functional importance of this aminoacid site (gain and loss of functions) and revealed the detrimental effect of positive charge on antioxidant function. In order to explain the observed functional change, further effort will be put into the calculations of the electrostatic potential and identifications of protein-protein interactions.:Contents List of figures x List of tables xii Abbreviations xiii 1 Introduction 1 1.1 ROS production 1 1.2 Oxidative stress 2 1.3 Antioxidant response 3 1.4 The thioredoxin fold: From structure to function 6 1.5 Sco proteins 7 1.5.1 Structural similarity of Sco proteins to antioxidant enzymes 8 1.5.2 Current knowledge about Sco proteins of S. cerevisiae 9 1.6 Background studies 10 1.7 Using yeast as a model 11 1.7.1 Cross-species complementation studies 11 1.7.2 Yeast model for human mitochondria studies 12 1.8 Aim of the study 12 2 Materials & Methods 14 2.1 Materials 14 2.1.1 Chemicals and Reagents 14 2.1.2 Equipments 16 2.1.3 Kits 17 2.1.4 Antibodies 18 2.1.5 Plasmid 18 2.1.6 Primers 19 2.1.7 S. cerevisiae strains 22 2.1.8 Media 22 2.2 Methods 24 2.2.1 Cultivation of S. cerevisiae cells 24 2.2.1.1 Culture conditions 24 2.2.1.2 Preparation of glycerol stocks 24 2.2.2 Molecular Biology Methods 24 2.2.2.1 S. cerevisiae genomic DNA isolation 24 2.2.2.2 RNA isolation 25 2.2.2.2a Cultured mammalian cells (HEK293) 25 2.2.2.2b Drosophila melanogaster 25 2.2.2.3 RNA purity and concentration determination 25 2.2.2.4 Reverse transcription 25 2.2.2.5 Polymerase chain reaction 25 2.2.2.5a Standard PCR 25 2.2.2.5b Overhang PCR 26 2.2.2.5c Overlap extension PCR 27 2.2.2.5d Site-directed mutagenesis by overlap extension PCR 27 2.2.2.6 DNA agarose gel electrophoresis 28 2.2.2.7 DNA gel extraction and clean-up 29 2.2.2.8 DNA sequencing 29 2.2.2.9 Southern blotting 29 2.2.2.9a DNA preparation 29 2.2.2.9b Blotting 30 2.2.2.9c Preparation of a DIG-labelled probe 30 2.2.2.9d Hybridization of the DIG-labelled probe to DNA 30 2.2.2.9e Detection of hybridized DIG-labelled URA3 probe 31 2.2.2.10 Yeast transformation 32 2.2.2.11 Growth assay 32 2.2.3 Protein methods 33 2.2.3.1 Isolation of crude mitochondria from yeast 33 2.2.3.2 SDS-PAGE 33 2.2.3.3 Protein transfer 34 2.2.3.4 Colloidal Coomassie gel staining 34 2.2.3.5 Protein detection 35 2.2.3.6 Stripping the membrane and reprobing 35 2.2.4 Biochemical methods 35 2.2.4.1 Methylene Blue staining 36 2.2.4.2 Quantification of ROS 36 2.2.4.2a Amplex Red staining 36 2.2.4.2b Lipid peroxidation assay 36 2.2.5 Bioinformatics 37 2.2.6 Statistical Analysis 37 3 Results 40 3.1 Selection of homologues by bioinformatic analysis 40 3.2 Generation of recombinant strains 42 3.3 Confirmation of site-specific integration by check PCR 44 3.4 Verification of single site integration by Southern Blotting 44 3.5 Analysis of the functional homology between selected homologues and ySCO2 45 3.5.1 Complementation assay in solid media 45 3.5.2 Complementation assay in liquid media 47 3.6 Determination of cell viability 48 3.7 Quantification of ROS 51 3.7.1 Quantification of extracellular H2O2 51 3.7.2 Quantification of lipid peroxidation 53 3.8 Investigation of the expression and subcellular localization of homologues 55 3.9 Investigation of the impact of pathogenic hSCO2 mutations on its antioxidant role 58 3.10 Mutational analysis of ySCO2 60 3.11 Identification of functionally important residues 61 3.12 Prediction of salt bridges 65 3.13 Alanine mutagenesis 66 4 Discussion 68 4.1 Functional homology between the selected homologues and ySCO2 68 4.1.1 A. thaliana homologues, HCC1 & HCC2 68 4.1.2 H. sapiens homologues, hSCO1 & hSCO2 69 4.1.3 D. melanogaster homologue, SCOX 70 4.1.4 Yeast homologues, K07152 & SpSCO1 70 4.2 The localization and expression pattern of homologues 71 4.3 The impact of pathogenic hSCO2 mutations on its antioxidant role 72 4.4 Mutational analysis of ySCO2 73 4.5 Attempts to understand the underlying reason(s) behind charge-related functional change 74 4.6 Potential mechanisms associated with the antioxidant action of ySco2p 78 5 Summary 81 6 References 8

Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response

Tese de Doutoramento em Ciências Veterinárias na especialidade de Sanidade [email protected] besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor a vaccine to be used in Europe is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neospora caninum and Toxoplasma gondii, which are closely related to B. besnoiti, PDI plays an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we presented the nucleotide sequence of the B. besnoiti PDI gene and a 3D theoretical model was built by comparative homology using Swiss-Model server. B. besnoiti expresses a PDI with 471 amino acids, structurally similar to human and yeast PDIs, with four thioredoxin-like domains a, b, b’, a’ and a C-terminal extension c. The a and a’ domains present the characteristic active site pattern CxxC, in this case CGHC and CGYC, respectively. Analysis of the phylogenetic tree for PDI within the phylum Apicomplexa reinforced the close relationship among B. besnoiti, N. caninum and T. gondii. Recombinant B. besnoiti PDI (recBbPDI) and truncated versions corresponding to domains a, b, b’ and a’c were expressed in a heterologous system. Mice were immunized with recBbPDI for the production of monoclonal antibodies (mAbs) by hybridoma technology and four mAbs were produced and characterized. RecBbPDI and domain a’c (recBb-a’c) were functionally active and exhibited a dose dependent cross-linking activity of insulin. In the presence of bacitracin, tocinoic acid, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 4-chloromercuribenzoic acid (pCMBA) activity of both enzymes was inhibited, in a dose dependent manner. The same happened with recBbPDI in the presence of mAbs (with the exception of T8a), but not with recBb-a’c, whose activity was not sensitive to the presence of mAbs. In vitro proliferation of B. besnoiti tachyzoites was diminished in the presence of PDI inhibitors and anti-PDI mAbs, indicating that this enzyme seems to intervene in the process of host cell adhesion/invasion. In this way, considering the inhibitions obtained, both in the host cell invasion ability and in the enzyme catalytic activity, PDI can represent a potential target for addressing the treatment and/or prevention of besnoitiosis. The panel of monoclonal antibodies here developed represents an important tool for future studies.RESUMO - A enzima isomerase de dissulfureto como alvo terapêutico contra a besnoitiose bovina. Caracterização molecular e estudo do seu papel na infeção e na resposta imunitária - Besnoitia besnoiti, o agente etiológico da besnoitiose bovina, pertence ao filo Apicomplexa, família Sarcocystidae, subfamília Toxoplasmatinae, estando filogeneticamente próximo dos géneros Neospora e Toxoplasma. A besnoitiose bovina é uma doença severa mas geralmente não fatal, endémica em vastas áreas tropicais e subtropicais de África e responsável por elevadas perdas económicas. Na Europa, após as primeiras descrições há mais de um século, em França e Portugal, a doença recebeu pouca atenção até finais do século XX, altura em que a sua incidência começou a aumentar com relatos em Portugal, Espanha, Itália e França. Mais recentemente o avanço geográfico da doença é relatado com casos na Grécia, Suíça, Alemanha, Hungria, Croácia e Irlanda. A fase aguda da doença é marcada por alterações respiratórias, febre, anasarca, diarreia e, por vezes, aborto. Na fase crónica, o animal apresenta grande espessamento da pele (elefantíase) com soluções de continuidade, muitas vezes complicadas por infecções oportunistas, má condição corporal e, nos machos, orquite necrosante e aspermia. Apesar de ser conhecida há mais de um século, assim como a sua etiologia parasitária, a besnoitiose bovina mantém muitos aspectos da sua epidemiologia desconhecidos, incluindo o ciclo de vida do seu agente etiológico. Outras espécies do género Besnoitia têm um ciclo heteroxeno mantido por uma relação presa-predador entre pequenos mamíferos e o gato. Acredita-se que o ciclo de vida de B. besnoiti seja semelhante, mas até à data ainda não foi identificado um hospedeiro definitivo. A transmissão homoxena de bradizoítos e taquizoítos de B. besnoiti foi comprovada experimentalmente, em bovinos e em diversos animais de laboratório, e uma maior incidência da doença nos meses mais quentes do ano, quando existe maior actividade de insetos hematófagos, sugere um papel destes na transmissão de B besnoiti. No entanto, o verdadeiro modo de transmissão deste parasita na natureza mantém-se desconhecido. A doença pode atingir uma elevada taxa de morbilidade, o que, associado à gravidade das manifestações clínicas e à inexistência de vacina segura ou tratamento eficaz, justifica a necessidade de aprofundar o conhecimento sobre a biologia deste parasita, tentando desenvolver novas abordagens à terapêutica e à formulação de vacinas. A enzima isomerase de dissulfureto (PDI, do inglês protein disulfide isomerase), uma das mais abundantes do retículo endoplasmático, catalisa a formação de pontes dissulfureto e o rearranjo de emparelhamentos dissulfureto incorrectos, permitindo que as proteínas adquiram a sua correta conformação tridimensional. As suas funções (ou disfunções) têm sido implicadas em doenças neurodegenerativas como Alzheimer ou Parkinson ou na capacidade proliferativa de células neoplásicas. Sabe-se hoje que, para além da sua localização maioritariamente reticular, a PDI está presente à superfície das células, onde participa em diversas funções biológicas, como a ativação e agregação plaquetária, a adesão leucocitária ou a internalização de agentes patogénicos como alguns vírus e clamídias. Em diversos parasitas do filo Apicomplexa, incluindo Neospora caninum e Toxoplasma gondii, foram já identificadas PDIs na superfície dos taquizoítos e foi demonstrado que a inibição desta proteína, através de drogas ou anticorpos específicos, tem um efeito negativo sobre a capacidade de proliferação dos parasitas, evidenciando o papel que esta parece desempenhar no processo de adesão/invasão da célula hospedeira. Por outro lado, a PDI parece ter um papel relevante na resposta imunitária do hospedeiro, uma vez que foi identificada como uma proteína imunodominante e que foi demonstrada a presença de anticorpos anti-T. gondii-PDI e anti-N. caninum-PDI no repertório inato do homem e da vaca, respetivamente. Em conjunto, estas evidências sublinham a importância da PDI no processo de invasão celular e na resposta imunitária do hospedeiro. Neste trabalho determinámos a sequência completa do gene da PDI em B. besnoiti, clonámos o respectivo cDNA e expressámos a proteína recombinante, assim como versões truncadas da mesma, num sistema heterólogo. A PDI de B. besnoiti (BbPDI) pertence à superfamília das tiorredoxinas (cluster 00388), estando incluída na família PDI_a (cluster defined cd02961) e subfamília PDI_a_PDI_a’_c (cd02995). Construímos um modelo tridimensional da BbPDI por homologia de comparação usando o software disponível no servidor Swiss-Model e tendo como modelo a estrutura cristalográfica da Tapasina-ERp57 (código do PDB: 3F8U chain C). Verificámos que B. besnoiti expressa uma PDI composta por 471 aminoácidos, com a organização típica descrita para a PDI humana e de levedura, ou seja os 4 domínios estruturalmente semelhantes à tiorredoxina, domínio a, b, b’, a’, com uma extensão C-terminal c. Os domínios activos a e a’ apresentam os característicos centros ativos CxxC, no caso de B. besnoiti com a sequência aminoacídica CGHC e CGYC, respectivamente. A análise filogenética para a PDI colocou a B. besnoiti no mesmo ramo que a N. caninum e T. gondii. Com a PDI recombinante de B. besnoiti (recBbPDI), imunizámos murganhos com o objetivo final de produzir anticorpos monoclonais (mAbs) anti-PDI, pela tecnologia de hibridomas. Obtivemos 4 mAbs, que foram caracterizados no reconhecimento das versões truncadas de PDI, correspondentes aos domínios a, b, b’ e a’c e na capacidade de reconhecerem cruzadamente a PDI de N. caninum e T. gondii. Todos os anticorpos produzidos, T8a, S4a, R60b e S16p, reconhecem a proteína recombinante e a natural de B. besnoiti, tanto em condições desnaturantes como em condições nativas. Dois mAbs (T8a e S4a) reconhecem o domínio a’c, um anticorpo reconhece o domínio b’ (R60b) e o outro (S16p) não reconhece nenhuma versão truncada da proteína. O anticorpo T8a é específico para a PDI de B. besnoiti, enquanto os restantes reagem cruzadamente com a PDI de N. caninum e T. gondii, quando testados por western blot e ELISA. Foi avaliada a actividade cinética da proteína recombinante pelo método da agregação da insulina. Este consiste na avaliação turbidimétrica da precipitação da insulina como resultado da formação de pontes dissulfureto, ação que é catalisada pela PDI. Foram avaliadas as versões truncadas dos domínios ativos (a e a’c) e a proteína integral, tendo-se verificado ausência de atividade para a versão truncada correspondente ao domínio a. Contudo, a versão truncada a’c (recBb-a’c) e recBbPDI mostraram-se funcionalmente ativas. Na presença de bacitracina, ácido tocinóico, ácido ditionitrobenzoico (DTNB) e ácido p-cloromercurobenzóico (pCMBA) a atividade destas enzimas foi, de uma forma geral, inibida de um modo dose-dependente. O mesmo aconteceu com a atividade catalítica da recBbPDI quando testada na presença dos mAbs produzidos (excepto mAb T8a), mas não na actividade de recBb-a’c, que não foi inibida por nenhum dos mAbs. Verificou-se a presença de PDI no produto de secreção/excreção de taquizoítos e, para avaliar o envolvimento desta no processo de invasão da célula hospedeira, foram colocados taquizoítos de B. besnoiti sobre um tapete confluente de células Vero na presença das drogas e dos mAbs acima mencionados. Após uma hora, removeram-se os inibidores e os taquizoítos em suspensão e as culturas foram incubadas por mais onze horas para permitir a fácil visualização dos vacúolos parasitóforos, por imunofluorescência indireta. A presença de inibidores da PDI e de mAbs anti-PDI permitiram diminuir a taxa de invasão de B. besnoiti, demonstrando a acção desta enzima no processo de adesão/invasão da célula hospedeira. Tendo em conta os resultados de inibição obtidos, quer na capacidade de invasão de célula hospedeira, quer na atividade cinética da enzima, a PDI pode de facto representar um alvo para o desenvolvimento de estratégias terapêuticas e/ou profilácticas contra a besnoitiose bovina. O painel de anticorpos monoclonais aqui desenvolvidos representa uma ferramenta importante para estudos futuros

Multicofactor proteins: structure, prediction, function

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