As Mucopolissacaridoses (MPS) são um grupo de doenças de armazenamento lisossômico causadas por deficiência de enzimas que catalisam a degradação gradual das glicosaminoglicanas (GAGs). GAGs (anteriormente chamadas de mucopolissacarídeos) são produtos de degradação das proteoglicanas que existem na matriz extracelular e tem efeito proteolítico. A classificação das MPS é baseada na deficiência enzimática específica. A MPS IVA é causada por mutações no gene que codifica a enzima GALNS (Nacetilgalactosamina-6-sulfatase), a qual desempenha um papel crucial na degradação do sulfato de queratano e condroitina-6-sulfatase. As mutações na enzima se resumem em três categorias: interrupção do sítio ativo, alterações no núcleo hidrofóbico e exposição da superfície, onde mutações missense na estrutura podem afetar gravemente a atividade da proteína GALNS, alterando seu núcleo hidrofóbico ou modificando seu enovelamento (folding). Com a falta de tratamentos efetivos, sendo em sua maioria paliativos, e tendo como base a estrutura já resolvida da GLANS selvagem, este trabalho teve como objetivo modelar 3 variantes da enzima GALNs, sendo uma mutação no sítio ativo, uma no núcleo hidrofóbico e uma na superfície. Foi usado o software MODELLER 9.12 para a modelagem comparativa, os softwares Prochek, PROSA II, ERRATv2, Verify3d, ProQ para a avaliação dos modelos, o software NAND 2.10, para simulação de dinâmica molecular e o software Chimera 1.10.1 para cálculo de superfícies eletrostáticas e hidrofobicidade da superfície. Os modelos apresentaram bons resultados segundo os softwares de avaliação e análise visual. Apresentaram poucas diferenças estruturais em relação à estrutura da GALNS selvagem, demonstraram estabilidade em simulação de dinâmica molecular. Entretanto, algumas diferenças foram observadas com relação à distribuição de cargas e hidrofobicidade no sítio ativo do modelo da variante com mutação no sítio ativo. Pôde ser concluído que as 3 mutações analisadas não causaram alterações estruturais significativas, não interferiram na estabilidade estrutural em simulação de dinâmica molecular, entretanto, foi demonstrado que mutações na região do sítio ativo podem interferir na função da enzima. / The Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by deficiencies in enzymes that catalyze the gradual glycosaminoglycans (GAGs) degradation. GAGs (formerly called mucopolysaccharides) are products of proteoglycan degradation that exist in the extracellular matrix and have proteolytic effect. The classification of MPS is based on the specific enzyme deficiency. MPS IVA is caused by mutations in the gene that encodes the GALNS enzyme (Nacetilgalactosamina-6-sulfatase), which plays a crucial role in the degradation of keratan sulfate and chondroitin-6-sulfatase. Mutations in the enzyme can be summarized in three categories: interruption of the active site, changes in the hydrophobic core and display surface, where missense mutations in the structure can seriously affect the activity of GALNS protein, changing its hydrophobic core or modifying its folding. With the lack of effective treatments, in its most palliative, and based on the wild GALNS structure already determined, this study aimed to model 3 variants of GALNS enzyme, a mutation in the active site, one in the hydrophobic core and a on the surface. 9.12 MODELLER was used for comparative modeling software, the software Prochek, Prose II, ERRATv2, Verify3d, ProQ models for the evaluation of the NAND 2.10 software, for molecular dynamics simulation and software Chimera 1.10.1 calculates electrostatic and hydrophobic surface. The models showed good results according to the evaluation software and visual analysis. Presented few structural differences from the wild GALNS structure and showed stability in molecular dynamics simulation. However, some differences were observed with respect to the charge distribution and hydrophobicity in the active site of the variants of the model with a mutation in the active site. It might be concluded that the three mutations analyzed did not cause significant structural changes and did not affect the structural stability in molecular dynamics simulation, however, it has been shown that mutations in the active site region may interfere with the function of this enzyme.

As Mucopolissacaridoses (MPS) são um grupo de doenças de armazenamento lisossômico causadas por deficiência de enzimas que catalisam a degradação gradual das glicosaminoglicanas (GAGs). GAGs (anteriormente chamadas de mucopolissacarídeos) são produtos de degradação das proteoglicanas que existem na matriz extracelular e tem efeito proteolítico. A classificação das MPS é baseada na deficiência enzimática específica. A MPS IVA é causada por mutações no gene que codifica a enzima GALNS (Nacetilgalactosamina-6-sulfatase), a qual desempenha um papel crucial na degradação do sulfato de queratano e condroitina-6-sulfatase. As mutações na enzima se resumem em três categorias: interrupção do sítio ativo, alterações no núcleo hidrofóbico e exposição da superfície, onde mutações missense na estrutura podem afetar gravemente a atividade da proteína GALNS, alterando seu núcleo hidrofóbico ou modificando seu enovelamento (folding). Com a falta de tratamentos efetivos, sendo em sua maioria paliativos, e tendo como base a estrutura já resolvida da GLANS selvagem, este trabalho teve como objetivo modelar 3 variantes da enzima GALNs, sendo uma mutação no sítio ativo, uma no núcleo hidrofóbico e uma na superfície. Foi usado o software MODELLER 9.12 para a modelagem comparativa, os softwares Prochek, PROSA II, ERRATv2, Verify3d, ProQ para a avaliação dos modelos, o software NAND 2.10, para simulação de dinâmica molecular e o software Chimera 1.10.1 para cálculo de superfícies eletrostáticas e hidrofobicidade da superfície. Os modelos apresentaram bons resultados segundo os softwares de avaliação e análise visual. Apresentaram poucas diferenças estruturais em relação à estrutura da GALNS selvagem, demonstraram estabilidade em simulação de dinâmica molecular. Entretanto, algumas diferenças foram observadas com relação à distribuição de cargas e hidrofobicidade no sítio ativo do modelo da variante com mutação no sítio ativo. Pôde ser concluído que as 3 mutações analisadas não causaram alterações estruturais significativas, não interferiram na estabilidade estrutural em simulação de dinâmica molecular, entretanto, foi demonstrado que mutações na região do sítio ativo podem interferir na função da enzima. / The Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by deficiencies in enzymes that catalyze the gradual glycosaminoglycans (GAGs) degradation. GAGs (formerly called mucopolysaccharides) are products of proteoglycan degradation that exist in the extracellular matrix and have proteolytic effect. The classification of MPS is based on the specific enzyme deficiency. MPS IVA is caused by mutations in the gene that encodes the GALNS enzyme (Nacetilgalactosamina-6-sulfatase), which plays a crucial role in the degradation of keratan sulfate and chondroitin-6-sulfatase. Mutations in the enzyme can be summarized in three categories: interruption of the active site, changes in the hydrophobic core and display surface, where missense mutations in the structure can seriously affect the activity of GALNS protein, changing its hydrophobic core or modifying its folding. With the lack of effective treatments, in its most palliative, and based on the wild GALNS structure already determined, this study aimed to model 3 variants of GALNS enzyme, a mutation in the active site, one in the hydrophobic core and a on the surface. 9.12 MODELLER was used for comparative modeling software, the software Prochek, Prose II, ERRATv2, Verify3d, ProQ models for the evaluation of the NAND 2.10 software, for molecular dynamics simulation and software Chimera 1.10.1 calculates electrostatic and hydrophobic surface. The models showed good results according to the evaluation software and visual analysis. Presented few structural differences from the wild GALNS structure and showed stability in molecular dynamics simulation. However, some differences were observed with respect to the charge distribution and hydrophobicity in the active site of the variants of the model with a mutation in the active site. It might be concluded that the three mutations analyzed did not cause significant structural changes and did not affect the structural stability in molecular dynamics simulation, however, it has been shown that mutations in the active site region may interfere with the function of this enzyme.

The main aim of the study in this thesis was to use structure-based protocols to design new drugs for enzymes, DXS and DXR in the non mevalonate pathway. Another aim of this study was to identify the dimer interface in E.coli FabH as an allosteric binding site for designing new class of anti-infective drugs. We have attempted to identify potential inhibitors for DXS by docking the NCI Diversity set compounds, compound libraries available from GSK-MMV and St. Jude's Children's research center. FabH dimer interface has been identified as a potential target using SiteMap, Alanine mutagenesis and docking studies. The first chapter gives an overview of the computational methods. The next two chapters briefly introduce the biological targets in the author's study. Chapter two explains the importance of non-mevalonate pathway in microbes. Different enzymes in the non-mevalonate pathway are discussed and the importance of terpenoids in biological processes and also the use of terpenoids as drugs have been extensively discussed in this chapter. The crystal structures available for DXS and DXR are also discussed. Chapter three brings out the importance of FabH as an anti-infective target. Crystal structure of FabH E.coli is discussed and the importance of FabH as a dimer has been discussed in this chapter. Chapter 3 describes the methods, homology models generated, and analysis from docking studies. The homology models for PvDXS and PvDXR have been used in this study to identify potential inhibitors. Domain swapping and the structural organization of PvDXS before and after domain swaping are discussed. Identification of domain swaping in PvDXS using entropy changes has been extensively discussed. Chapter 4 focuses on FabH (Fatty Acid Biosynthesis, enzyme H also referred to as β-ketoacyl-ACP-synthase III) dimer interface as an allosteric target. SiteMap analysis and MD simulations on the FabH monomer and dimer structures revealed the dimer interface as a binding region. Further analyses were done by mutagenesis studies on the Phe87 residue, a key residue at the dimer interface region and validating the results using docking studies. NCI Diversity Set compounds were docked at the dimer interface of FabH, which revealed that compounds NSC91529 and NSC19803 docked best at the dimer interface region with the phenyl ring of both the compounds

Computational tools play an important role in the description of biological systems. Scientists describe and study structure, conformational changes and interactions between molecules in silico, often as a cheaper and faster alternative for biosynthesis. The simulated dynamic behavior in time of a molecular system is a straight forward source of information about substrate-enzyme interactions at the atomic level, and a powerful tool for the identification of molecular properties important in enzymatic reactions. Our study is focused on the computational investigation of structure and substrate specificity of hydrolases important in biotransformation. The computational work was performed in close collaboration with biochemists-experimentalists from Charles University and the Microbiological Institute of the Academy of Sciences of the Czech Republic. Hydrolases have great a potential in the chemoenzymatic synthesis of modified carbohydrates with regulated properties. Carbohydrates, as substrates of hydrolases, are important in normal functionality of many organisms. They have a dual role in immune response regulation: some carbohydrates (like GlcNAc and ManNAc) participate in activation and some (like GalNAc) in suppressing immunity; glycosidase deficiency is associated with a number of lysosomal disorders. We used homology modeling, computational docking and molecular dynamics simulation (MD) methods for the complex study of fungal hydrolases: alpha-galactosidase/alpha-N-acetylgalactosaminidase from Aspergillus niger; beta-N-acetylhexosaminidases (HEX) (from Aspergillus oryzae and Penicillium oxalicum); nitrilase from Aspergillus niger. Our structural study unambigously demonstrates that the enzyme encoded by genes variant A (aglA) from A. niger is able to accept alpha-N-acetylgalactosamine as its substrate and explains structural features responsible for its specificity. Homology models of HEXs from P. oxalicum and A. oryzae were built and compared. Homology models were used to study the role of protein glycosylation, disulfide bonds, dimer formation and interaction with natural and modified substrates. Model of nitrilase from Aspergillus niger helped to analyze multimer formation.

Magister Scientiae - MSc / Malaria remains the most important parasitic disease worldwide. It is estimated that over 500 million infections and more that 2.7 million deaths arising from malaria occur each year. Most (90%) of the infections occur in Africa with the most affected groups being children of less than five years of age and women. this dire situation is exacerbated by the emrggence of drug resistant strains of Plasmodium falciparum. The work reported in this thesis focuses on improving the purification of PfHPRT by investigating the characteristics of anion exchange DE-52 chromatography (the first stage of purification), developing an HPLC gel filtration method for examining the quaternary structure of the protein and possible end stage purification, and initialcrystalization trials. a homology model of the open, unligaded PfHPRT is constructed using the atoomic structures of human, T.ccruz and STryphimurium HPRT as templates. / South Africa

Filariasis and onchocerciasis are debilitating diseases affecting 120 million people globally. The massive socio-economic impact of these diseases energized the international community to declare a goal of eliminating filariasis 2020. This resulted in a dramatic increase in the efforts to eliminate filariasis and onchocerciasis, employing a strategy of mass drug administration (MDA). However, these programs rely upon the small arsenal of drugs. This leaves these programs vulnerable to failure in the face of developing resistance and local intolerance to the current drug regimens. Thus, new drugs against these infections are critically needed. A homologue of the ecdysone receptor (EcR), a master regulator of development in insects, has been identified in B. malayi. The potential of the EcR as a drug target has been underscored by work in the agricultural industry, where insecticides targeting the ecdysone developmental pathway are effective and non-toxic to non-target species. As the EcR is absent in humans, it represents an attractive potential chemotherapeutic target. The first study investigates the hypothesis that the ecdysone receptor controls the embryogenesis and molting in the filarial parasite. In-vitro embryogram and in-vivo phenotypic studies were conducted to delineate the effect of 20-hydroxyecdysone on the Brugia malayi parasites. The results suggest that the hormone accelerates embryogenesis and causes precocious molts, resulting in the death of the parasite. Further, transcriptomic and proteomic analysis of the ecdysone treated worms provided evidence that the up-regulated genes participate in embryogenesis. Based upon the validation of the ecdysone receptor as a potential drug target, subsequent studies focused on the development of a drug discovery model to screen for agonists and antagonists of the B. malayi ecdysone receptor. A stable cell line was created to aid the high throughput screening to rapidly identity agonist and antagonist compounds. A total of 7 agonists and 2 antagonists were identified. A homology model of the BmEcR ligand-binding domain was created as an alternate method for virtual screening of small molecules as well as to study the ligand-receptor interactions. The hits identified with the assay were docked in the active site of the BmEcR homology model providing an excellent correspondence of data between the molecular assay and the virtual screening method.

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-04-20T16:40:36Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao_SheylaSilvaLima_2015.pdf: 4958893 bytes, checksum: 21511e1c9e1a86ea210befeb33c91543 (MD5) / Made available in DSpace on 2016-04-20T16:40:36Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao_SheylaSilvaLima_2015.pdf: 4958893 bytes, checksum: 21511e1c9e1a86ea210befeb33c91543 (MD5) Previous issue date: 2015-02-24 / FACEPE / Ciclotídeos são uma classe de peptídeos antimicrobianos (AMPs - do inglês Antimicrobial peptide) cíclicos de plantas, compostos de, aproximadamente, 30 resíduos de aminoácidos, sendo seis cisteínas conservadas e conectadas por três pontes de dissulfeto. Sua expressão é constitutiva, tendo sua principal função na defesa vegetal contra patógenos, que podem causar perdas significativas em culturas importantes para a agricultura, como no caso da família Poaceae que apresenta destacada importância econômica no Brasil e no mundo. Nesse estudo foi conduzida uma busca por genes relacionados a ciclotídeos vegetais, disponíveis em bancos de dados de acesso restrito e público, com vistas ao isolamento e caracterização in silico desses peptídeos. Através da busca nos genomas de Hevea brasiliensis, Manihot esculenta, Ricinus communis, Sorghum bicolor e Zea mays; bem como no transcriptoma de Vigna unguiculata foi verificado que apenas o genoma de Zea mays apresentou dois possíveis genes codificadores de ciclotídeos. Assim, primers foram desenhados para o isolamento destes genes em milho. Além da espécie Z. mays, as espécies Triticum aestivum (trigo) e Secale cereale (centeio), foram utilizadas para a tentativa de isolamento a partir dos pares de primers desenhados. Foram obtidos 19 fragmentos (amplicons), sendo quatro deles (zm315, zm316, zm317, sc359) com o domínio ciclotídeo, os três primeiros de milho e o último de centeio. Essas quatro sequências foram, então, submetidas a uma caracterização in silico, para predição da estrutura secundaria, terciaria e função predita. Verificou-se que esses peptídeos apresentam as seis cisteínas conservadas, três pontes dissulfeto e o padrão de aminoácidos entre as cisteínas, similar aos encontrados em ciclotídeos. Ainda foi possível a predição de algumas propriedades físico-químicas e modelagem por homologia para as quatro proteínas, o que mostrou a qualidade e confiabilidade dos modelos. Sugere-se que dois dos ciclotídeos isolados (zm315, zm316) pertençam a uma nova classe de peptídeos lineares, mas com características de ciclotídeos. / Cyclotides are a class of cyclic antimicrobial peptides (AMPs) present on plants, composed by approximately 30 amino acid residues, including six conserved cysteines connected by three disulphide bridges. Its expression is constitutive, with main function on plant defense against pathogens, that may cause significant losses in important cultivars, as in the case of Poaceae, a family that presents economic importance for the agriculture in Brazil and worldwide. This study performed a search for genes related to plant cyclotides, available in restricted and public access databases, aimed at their in silico isolation and characterization. Searching for these peptides in Hevea brasiliensis, Manihot esculenta, Ricinus communis, Sorghum bicolor, Vigna unguiculata and Zea mays genomes, we obtained two possible genes encoding Cyclotides in Z. mays. Thus, primers were designed for the isolation of these genes in maize as well in wheat (Triticum aestivum) and rye (Secale cereale) species. We obtained 19 amplicons and four of them (zm315, zm316, zm317, sc359) presented cyclotide domain. These four sequences were then subjected to in silico characterization, for predicting their secondary and tertiary structures, as well their function. It was found that these peptides present six conserved cysteines, three disulphide bridges and the amino acid pattern between the cysteines similar to those found in cyclotides. It was also possible to predict some physical chemical properties and also building a 3D protein by homology modeling for the four peptides, presenting high quality and reliability. Our analysis indicates that two isolated cyclotides (zm315, zm316) appear to belong to a new class of linear peptides, but with cyclotide features.

Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered NO responses. Specifically, the mis-expressing atdgk4 pollen tubes have growth rates that were less affected by NO and showed reduced bending angles when challenged by an NO source. Further works on atdgk4 knockout/knockdown mutants will reveal the biological functions of ATDGK4 in NO and/or cGMP signaling in the pollen, and in the broader fertilization process.

Protein-protein and protein-peptide interactions play a central role in various aspects of the structural and functional organization of the cell. While the most complete structural characterization is provided by X-ray crystallography, many biological interactions occur in complexes that will not be amenable to direct experimental analysis. Therefore, it is important to develop computational docking methods that start from the structures of component proteins and predict the structure of their complexes, preferably with accuracy close to that provided by X-ray crystallography. This thesis details three applications of computational protein modeling, including the study of antibody maturation mechanisms, and the development of protocols for peptide-protein interaction prediction and template-based modeling of protein complexes. The first project, a comparative analysis of docking an antigen structure to antibodies across a lineage, reveals insights into antibody maturation mechanisms. A linear relationship between near-native docking results and changes in binding free energy is established, and used to investigate changes in binding affinity following mutation across two antibody-antigen systems: influenza and anthrax. The second project demonstrates that a motif-based search of available protein crystal structures is sufficient to adequately represent the conformational space sampled by a flexible peptide, compared to that of a rigid globular protein. This observation forms the basis for a global peptide-protein docking protocol that has since been implemented into the Structural Bioinformatics Laboratory’s docking web server, ClusPro. Finally, as structure availability remains a roadblock to many studies, researchers turn to homology modeling, in which the desired protein sequence is modeled onto a related structure. This is particularly challenging when the target is a protein complex, further restricting template availability. To address this problem, the third project details the development of a new template-based modeling protocol to be integrated into the ClusPro server. The implementation of a novel template-based search enables users to model both homomeric and heteromeric complexes, greatly expanding ClusPro server functionality. / 2020-08-30T00:00:00Z

Similarity searches are an essential component to most bioinformatic applications. They form the bases of structural motif identification, gene identification, and insights into functional associations. With the rapid increase in the available genetic data through a wide variety of databases, similarity searches are an essential tool for accessing these data in an informative and productive way. In our chapter, we provide an overview of similarity searching approaches, related databases, and parameter options to achieve the best results for a variety of applications. We then provide a worked example and some notes for consideration. Homology detection is one of the most basic and fundamental problems at the heart of bioinformatics. It is central to problems currently under intense investigation in protein structure prediction, phylogenetic analyses, and computational drug development. Currently discriminative methods for homology detection, which are not readily interpretable, are substantially more powerful than their more interpretable counterparts, particularly when sequence identity is very low. Here I present a computational graph-based framework for homology inference using physiochemical amino acid properties which aims to both reduce the gap in accuracy between discriminative and generative methods and provide a framework for easily identifying the physiochemical basis for the structural similarity between proteins. The accuracy of my method slightly improves on the accuracy of PSI-BLAST, the most popular generative approach, and underscores the potential of this methodology given a more robust statistical foundation.