Na última década, a análise da estrutura proteica baseada em teoria de redes/grafos tem emergido. A abstração da estrutura tridimensional proteica em forma de uma rede, leva em consideração os resíduos de aminoácidos e suas interações através do espaço, e apresenta um conjunto de conexões e propriedades mais complexas do que aquelas visualizadas apenas com a estrutura covalente. A análise da estrutura proteica identificou que as proteínas pertencem às redes de classes de \"mundo pequeno\" (small-world) e \"sem escala\" (scale-free), o que significa que seus resíduos de aminoácidos são altamente agregados e que existem poucas conexões entre 2 resíduos quaisquer da proteína. A identificação dos resíduos com alto grau de conexão, chamados centrais (\"resíduos hubs\"), é feita pela determinação do caminho mais curto que conecta um dado resíduo aos demais compreendidos nesta rede. A remoção destes resíduos centrais (hubs) afeta a integridade da rede de forma mais contundente diferentemente da remoção de resíduos que não são centrais. Até o momento estes \"resíduos hubs\" ainda não foram experimentalmente correlacionados com as propriedades enzimáticas de proteínas. Para tal finalidade, a estrutura terciária de uma β-glicosidase de Spodoptera frugiperda (Sfβgli) foi analisada como uma rede. Após calcular-se os caminhos médios entre todos os pares de aminoácidos da β-glicosidase, encontrou-se 11 resíduos centrais (\"resíduos hubs\"). Alinhamento de sequências e comparações estruturais indicaram alta conservação destes \"resíduos hubs\". Nosso objetivo foi produzir esta β-glicosidase mutando-se a maioria dos \"resíduos hubs\" e 3 aminoácidos não centrais (\"não hubs\"), expressar estes mutantes em E. coli, determinar suas propriedades enzimáticas como atividade catalítica e preferência pelo substrato e verificar a estabilidade destes mutantes em experimentos de inativação térmica. Os resultados obtidos sugerem que mutações nos \"resíduos hubs\" não afetam as propriedades catalíticas, contudo as enzimas com mutações nos \"resíduos hubs\" apresentaram uma menor estabilidade térmica. Estes resultados sugeriram que os \"resíduos hubs\" são relevantes na difusão da energia cinética (vibração) introduzida na estrutura desta β-glicosidase pelo seu aquecimento / In recent years, graph-theoretic approaches have established that protein structures can be modeled as complex networks of interacting residues. Proteins structures can be represented as small-world and scale-free networks that are usually highly clustered with few links connecting any pair of nodes. The identification of nodes with high connection degrees, called hubs, is made by determining the shortest path linking one amino acid to the further nodes comprising the network. Targeted removal of the hubs has greater affect on the integrity of the network structure in contrast to a random removal of amino acid residues comprising the network. Nevertheless these hubs had not previously been correlated with enzymatic properties. The tertiary structure of β-glycosidase from S. furgiperda (Sfβgly) was analyzed as a network. After calculating the averaged paths between all pairs of amino acid residues of Sfgly, we defined 11 hubs, which have the highest centrality on the network. Sequence alignment and structural comparison showed that these hubs residue are conserved among β-glycosidases. Our goal was to mutate most hubs and 3 ´non-hubs´ residues from Sfβgly, express these mutant enzymes in E. coli, test their enzymatic properties as catalytic efficiency and substrate preference, and verify the thermal stability of these mutants. The results implied that mutations in these hubs do not cause changes in catalytic properties although enzymes containing mutations in hubs showed lower thermal stability. Based on that, it was suggested that hub residues are important in the diffusion of kinetic energy (vibrations) introduced in the Sfβgly structure by heating

A catepsina L, uma cisteína proteinase da família da papaína, é a principal proteinase digestiva do besouro Tenebrio molitor. Estudos anteriores do nosso grupo mostraram que existem três catepsinas L no intestino médio do T. molitor, uma delas é lisossômica (CAL 1) e as outras duas são digestivas (CAL 2 e CAL 3). As estruturas 3D das enzimas digestivas foram recentemente elucidadas. Com o objetivo de estudar em detalhes as propriedades das enzimas digestivas, CAL 3 foi expressa como um zimógeno em E. coli, purificada por cromatografia de afinidade e autoativada em meio ácido. Foram realizados ensaios de atividade com 63 peptídeos FRET derivados da sequência Abz-KLRSSKQ-EDDnp em um espectrofluorímetro termostatizado a 30 ºC, monitorando-se continuamente a variação de fluorescência em 320 nm (λex) e 420 nm (λem). Os parâmetros kcat e KM obtidos foram utilizados na determinação da hidrofobicidade dos subsítios (H) e da função de cada subsítio através da razão das energias livres de ativação do complexo enzima-substrato (ΔG‡T) e de ligação da enzima com o substrato (ΔGs). Os resultados mostram que o subsítio S2 está envolvido prioritariamente em catálise e é bastante seletivo para substratos com resíduos hidrofóbicos em P2. Esse subsítio é o mais hidrofóbico dentre os analisados, encontrando-se num bolsão localizado no interior da enzima. O subsítio S\'2, por outro lado, é o que apresentou a menor especificidade dentre os analisados. Este subsítio está envolvido prioritariamente na ligação com o substrato e se localiza na superfície da enzima, o que pode facilitar a acomodação de diferentes cadeias laterais em P\'2 do substrato, não oferecendo muitas restrições espaciais. O subsítio S1, hidrofílico, não é muito seletivo, o que pode ser consequência de sua localização na superfície da enzima. Esse subsítio está prioritariamente envolvido na ligação com o substrato. O subsítio S\'1, assim como S1, está localizado na superfície da enzima, é hidrofílico e não muito seletivo. No entanto, esse subsítio tem papel na catálise além de atuar na ligação do substrato. Numa análise inicial da estrutura 3D deste subsítio, sua função catalítica foi atribuída à presença de parte da cavidade oxiânica. Uma enzima com mutação no resíduo W187, pertencente à cavidade oxiânica e a S\'1, foi produzida e purificada, no entanto essa enzima não apresentou atividade. Uma análise mais aprofundada mostrou que a falta de atividade pode ser atribuída ao fato do resíduo de aminoácido mutado fazer parte de um cluster aromático essencial à estabilização da tríade catalítica. Os dados obtidos na caracterização de S\'1 e S\'2 permitem inferir que a acilação é o passo limitante da reação da CAL 3. Além disso, os resultados deste trabalho mostram que o conceito de hidrofobicidade de subsítios proposto anteriormente pelo grupo parece ser aplicável a subsítios que apresentem especificidades mais restritas. / Cathepsin L, a cysteine proteinase of the papain family, is the major digestive proteinase in the beetle Tenebrio molitor. Previous studies of our group showed that there are three cathepsins L in T. molitor midgut, one is lysosomal (CAL1) and two are digestive (CAL2 and CAL3). The 3D structures of the digestive enzymes were recently elucidated. With the aim to study in details the digestive enzymes specificities, CAL3 was expressed in E. coli as a zymogen, purified by affinity chromatography and autoactivated in acid conditions. Activity assays were performed in a thermostated spectrofluorometer at 30 ºC with 63 FRET peptides derived from the lead sequence Abz-KLRSSKQ-EDDnp, continuously monitoring the fluorescence changes at 320 nm (λex) and 420 nm (λem). The parameters kcat and KM were used in the determination of subsite hydrophobicity (H) and subsite role based on the ratio of complex enzyme-substrate activation energy (ΔG‡T) and free energy of substrate binding (ΔGs). The data obtained suggest that the S2 is mainly involved in catalysis and is very selective to substrates with hydrophobic residues in P2. This subsite is the most hydrophobic among the analyzed and is located in a pocket in the enzyme interior. S\'2, on the other hand, is the less selective subsite and is mainly involved in substrate binding and is located on the enzyme surface, what can ease the accommodation of different side chains located in P\'2 by not imposing many spatial restrictions. S1, is hydrophilic and not very selective, what may be a consequence of its location on the enzyme surface. This subsite is mainly involved in substrate binding. S\'1, just like S1, is located on the enzyme surface, is hydrophilic and not very selective. However this subsite has a role in catalysis besides the role in substrate binding. In an initial 3D structure analysis its catalytic function was attributed to the presence of a part of the oxyanion hole. An enzyme with mutation in the residue W187, which apparently belonged both to the oxyanion hole and S\'1, was produced and purified, but this enzyme was inactive. A better analysis showed that the lack of activity can be attributed to the fact that the mutated residue belongs to an aromatic cluster that is essential to the catalytic triad stabilization. The data obtained in S\'1 and S\'2 characterization suggest that acylation is the limiting step in CAL 3 reaction. The results presented in this work support the concept of subsite hydrophobicity previously proposed by our group, which seems to be true to subsites with more restrict specificities

INTRODUÇÃO: O tamanho ideal do fígado varia entre 0,8% a 4% do peso do receptor (graft to body weight ratio - GBWR). Em crianças com menos de 10 kg submetidas a transplante hepático com doador vivo encontramos frequentemente uma situação denominada large-for-size, que ocorre ao se implantar um enxerto com peso maior que 4% do peso corpóreo do receptor. A lesão de isquemia/reperfusão (LIR) é uma resposta inflamatória mediada pelas células de Kupffer, principais responsáveis pela liberação de espécies reativas de oxigênio no parênquima hepático. Além disso, a célula de Kupffer libera citocinas pró-inflamatórias como TNF-alfa, IL-1 e IL-6 que recrutam polimorfonucleares sistêmicos e linfócitos T-CD4+ perpetuando a lesão mesmo após o fluxo sanguíneo estar restabelecido. O hipofluxo portal pode piorar a LIR na situação large-for-size. Desta forma, um shunt mesentérico-cava tentando direcionar o fluxo para a veia porta poderia ser benéfico nesta situação. MÉTODOS: Dezesseis porcos LandraceLargewhite pesando entre 17 - 38 kg foram submetidos a transplante de fígado. Foram distribuídos em três grupos: controle (CTRL), large-for-size (LFS) e shunt (SHUNT). Foi aferido o fluxo venoso portal, arterial hepático e colhido material para estudo histológico e biomolecular uma hora após a reperfusão. A relação entre o fluxo venoso portal do receptor para o fluxo venoso portal doador (rFVP) foi utilizada para se correlacionar com a gravidade da isquemia/reperfusão. Foram realizadas biópsias uma hora após o transplante para microscopia óptica e TUNEL além de quantificar a expressão dos genes da iNOS, eNOS, IL-6, BAX e BCL, c-fos/c-jun, ICAM e TNF-?. RESULTADOS: O GBWR foi maior nos grupos LFS e SHUNT (3,45 vs. 5,06 e 6,03; p=0,009). Quando comparamos isoladamente receptores e doadores por grupo houve diferença no fluxo portal somente nos grupos LFS (890 ± 203 mL / min vs. 458 ± 119 mL / min, p=0.0034) e SHUNT (1003 ± 5,3 mL/min vs. 495 ± 277 mL/min; p=0,0031). A rFVP foi próxima de 1 no grupo CTRL e significativamente maior que nos grupos LFS e SHUNT (p=0,03). O fluxo arterial hepático foi significativamente menor no grupo SHUNT (62,16 ± 31 mL / min) quando comparado com grupo CTRL (140.4 ± 53.88 mL / min) e LFS (142 ± 55.41 mL / min), p=0,02. A IL-6 e BAX foram menos expressas no grupo CTRL (p=0,05 e p=0,002). Houve uma relação linear entre a expressão dos genes BAX, TNF-alfa e eNOS com a rFVP (p=0,02; p=0,02 e p=0,009 respectivamente). Na avaliação histológica observou-se índice de isquemia e reperfusão semelhante entre os grupos CTRL e SHUNT mas menor em relação ao LFS, 3, 3,5 e 7 respectivamente (p=0,04). CONCLUSÕES: O transplante large-for-size acarreta hipofluxo portal relativo, e isto pode estar relacionado a um maior dano hepatocitário com acentuação da lesão de isquemia-reperfusão. A presença de um shunt entre a veia cava inferior e a veia mesentérica superior não conseguiu aumentar o influxo de sangue para o fígado de maneira persistente e pode estar implicado na diminuição do fluxo arterial hepático. No entanto, o aumento temporário do influxo portal pode estar relacionado a atenuação de algumas características da LIR no transplante large-for-size / INTRODUCTION: The optimum size of the liver graft for transplantation varies between 0.8% and 4% of the weight of the recipient (graft to body weight ratio - GBWR). In children with less than 10 kg who undergo liver transplantation with living donor a disorder called large-for-size (LFS) often occurs, when a graft weighing more than 4% of body weight of the recipient is implanted. Ischemia / reperfusion injury (IRI) is an inflammatory response mediated by Kupffer cells, mainly responsible for the release of reactive oxygen species in the liver parenchyma. In addition, Kupffer cells releases proinflammatory cytokines such as TNF-alfa, IL-1 and IL-6 that recruit systemic polymorphonuclear cells and T-CD4 + perpetuating this injury even after the blood flow is restored. Low portal flow may worsen IRI in LFS situation. So, a mesocaval shunt directing the flow to portal vein should be beneficial in this condition. METHODS: Sixteen Landrace/Largewhite pigs weighing approximately 20 kg (17-38 kg) underwent liver transplantation. They were divided into three groups: control (CTRL), large-for-size (LFS) and shunt (SHUNT). Portal venous flow was measured and liver and blood collected for histological and biomolecular study one hour after reperfusion. The relationship between the portal venous flow from the recipient to the donor portal venous flow (rFVP) was used to establish correlations with the parameters of ischemia and reperfusion. Biopsies were performed one hour after transplant to assess ischemia/reperfusion injury, TUNEL and quantify the following genes expression: iNOS, eNOS, IL-6, BAX and BCL c-fos / c-jun, ICAM, and TNF-alfa. RESULTS: GBWR was higher in groups LFS and SHUNT than CTRL (3.45 vs. 5.06 and 6.03; p=0.009). When compared separately by group recipients and donors difference in portal flow was observed only in LFS groups (890 ± 203 mL / min vs. 458 ± 119 mL / min, p=0.0034) and SHUNT (1003 ± 5.3 mL / min vs. 495 ± 277 mL / min, p=0.0031). rFVP was close to 1 in the CTRL group and significantly higher than the LFS and SHUNT groups (p= 0.03). Hepatic artery blood flow was significantly lower in SHUNT group (62.16 ± 31 mL / min) compared with CTRL group (140.4 ± 53.88 mL / min) and LFS (142 ± 55.41 mL / min), p= 0.02. IL-6 and BAX were expressed less in the CTRL group (p=0.05 and p=0.002). There was a linear relationship between the BAX, TNF-alfa and eNOS with rFVP (p=0.02, p=0.02 and p=0.009 respectively). Histological evaluation showed similar ischemia-reperfusion index between CTRL and SHUNT groups but lower values in compare to LFS group, 3, 3.5 and 7 respectively (p=0.04). CONCLUSIONS: The largefor-size liver transplantation leads to a relative low portal venous flow and this can be related to greater hepatocyte damage with accentuation of ischemia-reperfusion injury. The presence of a shunt between the inferior vena cava and the superior mesenteric vein could not increase the inflow of blood to the liver persistently and may be implicated in decreased hepatic artery blood flow. However, the temporary increase in portal inflow may be related to mitigation of some characteristics of IRI in large-for-size liver transplantation

The current, grim world-TB scenario, with TB being the single largest infectious disease killer, warrants a more effective approach to tackle the deadly pathogen, Mycobacterium tuberculosis. The deadly synergy of this pathogen with HIV and the emergence of drugresistant strains of the organism present a challenge for disease treatment (Russell et al., 2010). Thus, there is a pressing need for newer drugs with faster killing-kinetics which can claim both the actively-multiplying and latent forms of this pathogen causing the oldest known disease to man. This thesis entitled “Mechanistic and Regulatory Aspects of the Mycobacterium tuberculosis Dephosphocoenzyme A Kinase” describes one such potential drug target, which holds promise in future drug development, in detail. The development of efficacious antimycobacterials now requires previously unexplored pathways of the pathogen and cofactor biosynthesis pathways present a good starting point. Therefore, the mycobacterial Coenzyme A (CoA) biosynthesis was chosen for investigation, with the last enzyme of this pathway, dephosphocoenzyme A kinase (CoaE) which was shown to be essential for M. tuberculosis survival, as the focus of the present study (Sassetti et al., 2003). This thesis presents a detailed biochemical and biophysical characterization of the enzymatic mechanism of mycobacterial CoaE, highlighting several hitherto-unknown, unique features of the enzyme. Mutagenic studies described herein have helped identify the critical residues of the kinase involved in substrate recognition, binding and catalysis. Further, a role has been assigned to the UPF0157 domain of unknown function found in the mycobacterial CoaE as well as in several organisms throughout the living kingdom. Detailed insights into the regulatory characteristics of this enzyme from this work further our current understanding of the regulation of the universal CoA biosynthetic pathway and call for the attribution of a greater role to the last enzyme in pathway regulation than has been previously accredited. The thesis begins with a survey of the current literature available on tuberculosis and where we stand today in our fight against this dreaded pathogen. Chapter 1 details the characteristic features of the causative organism M. tuberculosis, briefly describing its unique genome and the cellular envelope which the organism puts forward as a tough shield to its biology. This is followed by a brief description of the infection cycle in the host, the pathogen-host interplay in the lung macrophages, the deadly alliance of the disease with HIV and our current drug arsenal against tuberculosis. Further, emphasizing on the need for newer, faster-acting anti-mycobacterials, Chapter 1 presents the rationale for choosing the mycobacterial coenzyme A biosynthetic pathway as an effective target for newer drugs. A detailed description of our current understanding of the five steps constituting the pathway follows, including a comparison of all the five enzymatic steps between the human host and the pathogen. This chapter also sets the objectives of the thesis, describing the choice of the last enzyme of the mycobacterial CoA biosynthesis, dephosphocoenzyme A kinase, for detailed investigation. As described in Chapter 1, the mycobacterial CoaE is vastly different from its human counterpart in terms of its domain organization and regulatory features and is therefore a good target for future drug development. In this thesis, Rv1631, the probable mycobacterial dephosphocoenzyme A kinase annotated in the Tuberculist database (http://genolist.pasteur.fr/TubercuList), has been unequivocally established as the last enzyme of the tubercular CoA biosynthesis through several independent assays detailed in Chapter 2. The gene was cloned from the mycobacterial genomic DNA, expressed in E. coli and the corresponding recombinant protein purified via a single-step affinity purification method. The mechanistic details of the enzymatic reaction phosphorylating dephosphocoenzyme A (DCoA) to the ubiquitous cofactor, Coenzyme A, have been described in this chapter which presents a detailed biochemical and biophysical characterization of the mycobacterial enzyme, highlighting its novel features as well as unknown properties of this class of enzymes belonging to the Nucleoside Tri-Phosphate (NTP) hydrolase superfamily. The kinetics of the reaction have been biochemically elucidated via four separate assays and the energetics of the enzyme-substrate and enzymeproduct interactions have been detailed by isothermal titration Calorimetry (ITC). Further details on the phosphate donor specificity of the kinase and the order of substrate binding to the enzyme provide a complete picture of the enzymatic mechanism of the mycobacterial dephosphocoenzyme A kinase. Following on the leads generated in Chapter 2 on the unexpected strong binding of CTP to the enzyme but its inability to serve as a phosphate donor to CoaE, enzymatic assays described in Chapter 3 helped in the identification of a hitherto unknown, novel regulator of the last enzyme of CoA biosynthesis, the cellular metabolite CTP. This chapter outlines the remarkable interplay between the regulator, CTP and the leading substrate, dephosphocoenzyme A, possibly employed by the cell to modulate enzymatic activity. The interesting twist to the regulatory mechanisms of CoaE added by the involvement of various oligomeric forms of the enzyme and the influence of the regulator and the leading substrate on the dynamic equilibrium between the trimer and the monomer is further detailed. This reequilibration of the oligomeric states of the enzyme effected by the ligands and its role in activity regulation is further substantiated by the fact that CoaE oligomerization is not cysteine-mediated. Further, the effects of the cellular metabolites on the enzyme have been corroborated by limited proteolysis, CD and fluorescence studies which helped elucidate the conformational changes effected by CTP and DCoA on the enzyme. Thus, the third chapter discusses the novel regulatory features employed by the pathogen to regulate metabolite flow through a critical biosynthetic pathway. Results presented in this chapter highlight the fact that greater importance should be attributed to the last step of CoA biosynthesis in the overall pathway regulation mechanisms than has been previously accorded. The availability of only three crystal structures for a critical enzyme like dephosphocoenzyme A kinase (those from Escherichia. coli, Haemophilus influenzae and Thermus thermophilus) is indeed surprising (Obmolova et al., 2001; O’Toole et al., 2003; Seto et al., 2005). In search of a structural basis for the dynamic regulatory interplay between the leading substrate, DCoA and the regulator, CTP, a computational approach was adopted. Interestingly, the mycobacterial enzyme, unlike its other counterparts from the prokaryotic kingdom, is a bi-domain protein of which the C-terminal domain has no assigned function. Thus both the N- and C-terminal domains were independently modeled, stitched together and energy minimized to generate a three-dimensional picture of the mycobacterial dephosphocoenzyme A kinase, as described in Chapter 4. Ligand-docking analyses and a comprehensive analysis of the interactions of each ligand with the enzyme, in terms of the residues interacted with and the strength of the interaction, presented in this chapter provide interesting insights into the CTP-mediated regulation of CoaE providing a final confirmation of the enzymatic inhibition effected by CTP. These homology modeling and ligand-docking studies reveal that CTP binds the enzyme at the site overlapping with that occupied by the leading substrate, thereby potentially obscuring the active site and preventing catalysis. Further, very close structural homology of the modeled full-length enzyme to uridylmonophosphate/cytidylmonophosphate kinases, deoxycytidine kinases and cytidylate kinases from several different sources, with RMSD values in the range of 2.8-3 Å further lend credence to the strong binding of CTP detailed in Chapter 2 and the regulation of enzymatic activity described in Chapter 3. Computational analyses on the mycobacterial CoaE detailed in this chapter further threw up some interesting features of dephosphocoenzyme A kinases, such as the universal DXD motif in these enzymes, which appears to play a crucial role in catalysis as has been assessed in the next chapter. It is interesting to note that the P-loop-containing nucleoside monophosphate kinases (NMPK), with which the dephosphocoenzyme A kinases share significant homology, have three catalytic domains, the nucleotide-binding domain, the acceptor substrate-binding domain and the lid domain. Computational analyses detailed in Chapter 4 including the structural and sequential homology studies, helped in the delineation of the three domains in the mycobacterial enzyme as well as highly conserved residues potentially involved in crucial roles for substrate binding and catalysis. Therefore important residues from all three domains of the mycobacterial CoaE were chosen for mutagenesis to study their contributions to catalysis. Conservative and non-conservative replacements of these residues detailed in Chapter 5 helped in the identification of crucial residues involved in phosphate donor, ATP binding (Lys14 and Arg140); leading substrate, DCoA binding (Leu113); stabilization of the phosphoryl transfer reaction (Asp32 and Arg140) and catalysis (Asp32). Thus, the results reported here present a first attempt to identify the previously unknown functional roles of highly conserved residues in dephosphocoenzyme A kinases. Chapter 5 also delineates the dependence of this kinase on the divalent cation, magnesium, for catalysis, describing a comparison of the kinetic activity by the wild type and the mutants, in the presence and absence of Mg2+. Therefore, this chapter presents a thorough molecular dissection of the roles played by crucial amino acids of the protein and the results herein can serve as a good starting point for targeted drug development approaches. As described above, another unusual characteristic of the mycobacterial CoaE is the fact that it carries a domain of unknown function, UPF0157, C-terminal to the N-terminal dephosphocoenzyme A kinase domain. The function of this unique C-terminal domain carried by the mycobacterial CoaE has been explored in Chapter 6. The failure of the Nterminal domain (NTD) to be expressed and purified in the soluble fraction in the absence of a domain at its C-terminus (either the mycobacterial CoaE CTD or GST from the pETGEXCT vector) pointed out a possible chaperonic activity for the CTD. A universal chaperonic activity by this domain in the cell was ruled out by carrying out established chaperone assays with insulin, abrin and -crystallin. In order to delineate the CTD sequence involved in the NTD-specific chaperoning activity, deletion mutagenesis helped establish the residues 35-50 (KIACGHKALRVDHIG) of the CTD in the N-terminal domain-specific assistance in folding. Chapter 6 further details the several other potential roles of the mycobacterial CTD probed, including the 4’-phosphopantethienyl transfer, SAM-dependent methyltransferase activity, activation of the NTD via phospholipids among others. Thus the results presented in this chapter are a first attempt at investigating the role of this domain found in several unique architectures in several species across the living kingdom. Chapter 7 is an attempt to stitch together and summarize the results presented in all the preceding chapters, giving an overview of our present understanding of the mycobacterial CoaE and its novel features.

P. americana e T. molitor são capazes de secretar β-1,3-glucanases no tubo digestivo, pelas glândulas salivares e pelo epitélio do ventrículo, respectivamente. As laminarinases majoritárias de P. americana (LIQ1, 42kDa; LAM_P, 45kDa), A. flavolineata (LAM_A, 45kDa) e T. molitor (LAM_T, 50kDa) foram purificadas até a homogeneidade. Essas enzimas têm diferentes especificidades, padrões de ação e resíduos envolvidos em catálise, fazendo parte dos E.C. 3.2.1.6 - endo-β-1,3(4)-glucanase (LIQ1), E.C. 3.2.1.39 - endo-β-1,3-glucanase (LAM_P) ou E.C. 3.2.1.58 - exo-β-1,3-glucanase (LAM_A e LAMT). O papel dessas enzimas é digerir β-glucanas de fungos e de cereais. LAM_P e LAMA são inibidas por laminarina, pela formação de complexos enzima-substrato não-produtivos. LIQ1, LAM_P e LAM_A são enzimas processivas, com diferentes graus de ataque múltiplo e produzem série distintas de oligossacarídeos. LAM_A possui um sítio acessório de ligação para laminarina, o qual pode estar envolvido no mecanismo de processividade. Quitinases digestivas de insetos podem ser diferentes das descritas até o momento. A. flavolineata e T. molitor possuem sistemas celulásicos completos. Os três insetos apresentam proteínas de baixo peso molecular capazes de ligar-se a celulose ou a pachyman. O ancestral dos hexapoda provavelmente possuía β-1,3 e β-1,3(4) glucanases digestivas associadas a um hábito detritívoro. / P. americana salivary glands and T. molitor midgut epithelium actively secrete laminarinases into the midgut. The major laminarinases from P. americana (LIQ1, 42kDa and LAM_P, 45kDa), A. flavolineata (LAM_A, 45kDa) and T molitor (LAM_T, 50kDa) were purified until homogeneity. These enzymes have different specificities, action patterns and activesite catalytic groups, and correspond to E.C.s 3.2.1.6 - endo-β-1,3(4)-glucanase (LIQ1), 3.2.1.39 - endo-β-1,3-glucanase (LAM_P) or 3.2.1.58 -exo-β-1,3-glucanase (LAM_A and LAM_T). Their physiological role is fungai and cereal β-glucan digestion. LAM_P and LAM_A are inhibited by excess substrate (non-productive enzyme-substrate complexes). LIQ1, LAM_P and LAMA have different multiple attack degrees and produce different oligosaccharides. LAM_A has a second substrate binding site, probably involved with processivity. T. molitor digestive chitinase is different from other insect chitinases. A. flavolineata and T. molitor can hydrolyse cristalline cellulose efficiently. The three studied insects have cellulose or pachyman-binding proteins with low molecular weights. Hexapoda ancestors probably had digestive β-1,3 and β-1,3(4)-glucanases and a detritivore habit.

Echinometra lucunter, o ouriço-do-mar responsável por 50% dos acidentes por animais marinhos, causa inflamação e dor quando os espinhos entram na pele, efeitos atribuídos ao trauma mecânico, além de acidentes por ingestão de ovas. O líquido celômico e o extrato aquoso de espinhos foram fracionados e purificados até a obtenção de moléculas puras, que foram testadas em modelos de inflamação. Foram feitas análises histológicas do espinho e de atividade enzimática do extrato de espinho. Foi isolada uma molécula do espinho e um peptídeo do líquido celômico, que causaram inflamação e dor. Foi verificada atividade enzimática de catepsina B/X. Foi observada uma estrutura histológica organizada no espinho, com células entre a porção calcificada, algumas contendo grânulos eletrodensos com conteúdo protéico, típicas secretoras. Conclui-se que o espinho e o líquido celômico de E. lucunter possuem toxinas inflamatórias, que participam do envenenamento e o espinho tem células secretoras de toxinas. A catepsina pode auxiliar no mecanismo de reparação do espinho, quando quebrado. / Echinometra lucunter, the sea urchin responsible for 50% of marine animals accidents, cause inflammation and pain by the spine penetration, effects attributed to the mechanical trauma. Accidents were reported after the ingestion of raw. The celomic fluid and spines were fractionated and purified, procedure repeated until pure molecules were obtained, tested for inflammation models. Histological analyses and enzymatic assays were performed. A molecule from spines and a peptide from the celomic fluid caused inflammatory effects. Moreover, a cathepsin B/X activity could be identified in the spines. An organized histological structure in the spine was observed, with cells embedded in a calcified matrix, as well as granulous cells displaying proteic contents, typical of secretory cells. It was possible to conclude that the spine and the celomic fluid of E. lucunter do contain inflammatory toxins that prolong the spine puncturing event itself, and the spine possesses a toxin secretory structure. The cathepsin would be present in a mechanism of tissue remodeling.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The negative heath consequences of smoking are widely recognized, but there are still about 20% of the people in United States using tobacco products. Cigarette smoke condensate (CSC), the particulate matter of cigarette smoke, is comprised of thousands of chemicals (e.g., nicotine). Secondary only to bacterial plaque, cigarette smoking is a major risk factor for periodontal disease. Human gingival fibroblasts (HGFs) are the main cellular component of periodontal connective tissues. During the development of periodontal disease, collagen degradation occurs. Collagen is the major extracellular matrix component of the gingiva. The major extracellular matrix degrading enzymes produced by the HGFs are the matrix metalloproteinases (MMPs). The MMPs are mainly modulated by the tissue inhibitors of metalloproteinases (TIMPs). In this dissertation, three studies aimed at understanding the effects of tobacco on human gingival fibroblasts and their mechanisms have been conducted: the effects of CSC on HGF-mediated collagen degradation; comparison of the effects of CSC on HGFs with that of nicotine; and the combined effects of CSC and bacteria on HGFs. The cell proliferation of HGFs decreased and cytotoxicity increased in HGFs treated with increasing concentrations of CSC. CSC increased the collagen degrading ability of the HGFs by altering the production and localization of MMPs and TIMPs. Nicotine is one of the major components and the most pharmacologically active agent in tobacco. The percentage of nicotine in the CSC was 2.4%. CSC (100 µg/ml) increased the collagen degrading ability of the HGFs by affecting membrane associated MMP-2, MMP-14, and TIMP-2, but the level of nicotine in the CSC may only play a limited role in this process. Porphyromonas gingivalis (P. gingivalis) is an opportunistic pathogen involved in periodontal disease. The combined effects of CSC and P. gingivalis supernatant increased HGF-mediated collagen degradation by destroying the balance between the MMPs and TIMPs at the protein and mRNA levels. This project demonstrated that tobacco (with or without P. gingivalis) increased HGF mediated collagen degradation, as seen in the periodontal disease, through altering the MMPs and TIMPs.

Lisozimas são enzimas que fazem parte do mecanismo de defesa contra bactérias, no entanto lisozimas com função digestiva também são encontradas no trato digestivo de vertebrados e no intestino médio de insetos. As lisozimas digestivas de insetos são do tipo c e assim compartilham semelhanças estruturais e mecanísticas com a lisozima da clara de ovo de galinha (HEWL). Entretanto, para desempenhar sua função digestiva, as lisozimas de insetos apresentam algumas propriedades particulares entre as quais se destaca um pH ótimo mais ácido em relação às lisozimas não-digestivas. Para elucidar as bases moleculares dessa diferença no pH ótimo, duas lisozimas digestivas (lisozima 1 AAQ20048 e lisozima 2 AAQ20047) da larva de Musca domestica (mosca Diptera Cyclorrhapha), clonadas em Pichia pastoris e purificadas, foram caracterizadas estruturalmente e cineticamente com o substrato sintético (MUQ3) e natural (cápsulas de Micrococcus lysodeikticus). Foi observado que o efeito do pH na atividade das lisozimas 1 e 2 sobre o MUQ3 é uma curva com formato de sino e pH ótimo mais ácido que o da HEWL. Essas curvas foram reflexos da diminuição simultânea dos valores de pKas do nucleófilo e do doador de prótons. Estruturas cristalográficas das lisozimas digestivas de Musca domestica foram obtidas a 1,9 Å e análise comparativa com a estrutura terciária da HEWL revelou resíduos de aminoácidos no ambiente do nucleófilo (N46) e do doador de prótons (S106 e T107) que podem estar envolvidos na modulação das constantes de ionização dos resíduos essenciais à catálise. Esses resíduos foram substituídos via mutagênese sítio-dirigida por D, V e A respectivamente e três mutantes simples (N46D, S106V e T107A) e um triplo (N46DS106V- T107A) foram produzidos e purificados. Caracterização revelou que as contribuições individuais da N46, S106 e T107 foram pequenas e próximas do limite de detecção da técnica utilizada. Por outro lado, o conjunto dos 3 aminoácidos foi responsável pelo pH ótimo ácido frente ao substrato sintético, elevando os valores de pKas do nucleófilo e doador de prótons para valores muito semelhantes ao da HEWL. Diferentemente, essa tripla mutação não foi suficiente para elevar o pH ótimo da lisozima 2 sobre cápsulas de Micrococcus lysodeikticus para valores próximos àqueles de HEWL, sugerindo que as bases moleculares do pH ótimo frente ao substrato natural e sintético são diferentes. Uma comparação estrutural entre lisozima 1 e HEWL sugere que os resíduos de aminoácidos carregados na superfície dessas lisozimas sejam importantes para determinação do pH ótimo. A investigação dessa hipótese foi feita substituindo 5 aminoácidos neutros e 1 ácido, via mutagênese sítio-dirigida, por resíduos básicos. A caracterização do mutante sêxtuplo revelou um aumento significativo nos valores de pH ótimo da lisozima 1, indicando que a redução da basicidade da superfície das lisozimas digestivas é determinante para seus pHs ótimos ácidos. / Lysozymes are enzymes that are part of the defence mechanism against bacteria, however lysozymes with digestive function are also found in the digestive tract of vertebrates and in the insect midgut. The digestive lysozymes from insects are c type, so they share similar structural and mechanistic characteristics with hen egg-white lysozyme (HEWL). However, to perform their digestive function, insect lysozymes present some particular properties among them a more acidic pH optimum than that of non-digestive lysozymes. To elucidate the molecular basis of this pH optimum difference, two digestive lysozymes (lysozyme 1 AAQ20048 and lysozyme 2 AAQ20047) from Musca domestica larvae (housefly Diptera Cyclorrhapha), cloned in Pichia pastoris and purified, were structurally and kinecticly characterized with synthetic (MUQ3) and natural (lyophilized cells of Micrococcus lysodeikticus) substrates. It was observed that the pH effect on the activity of lysozymes 1 and 2 upon MUQ3 is a bell shaped curve exhibiting a more acidic pH optimum than that of HEWL. These curves result from simultaneous decrease of pKas values of the nucleophile and proton donor. Crystallographic structures of these digestive lysozymes from Musca domestica were obtained at 1.9 Å and comparative analysis with the terciary structure of HEWL revealed amino acid residues in the catalytic nucleophile (N46) and proton donor environment (S106 and T107) that may be involved in the modulation of ionization constants of those catalytic residues. N46, S106, and T107 were replaced via site-directed mutagenesis by D, V and A respectively and three simple (N46D, S106V and T107A) and one triple (N46D-S106V-T107A) mutants were produced and purified. Their characterization revealed that the individual contributions of N46, S106 and T107 were small and close to the detection borderline of the technique utilized. On the other hand, a set of these 3 amino acids was responsible by acidic pH optimum upon synthetic substrate, increasing the pKas values of nucleophile and proton donor to similar values to that of the HEWL. Differently, this triple mutation was not enough to increase the pH optimum of lysozyme 2 upon lyophilized cells of Micrococcus lysodeikticus to values close to those of HEWL, suggesting that the molecular bases of pH optimum upon natural and synthetic substrates are different. A structural comparison between lysozyme 1 and HEWL suggests that the charged amino acid residues on the surface of these lysozymes are important for pH optimum determination. The investigation of this hypothesis was done replacing 5 neutral and 1 acidic amino acids, via site-directed mutagenesis, by basic residues. The characterization of this mutant revealed a significant increase in the pH optimum values of lysozyme 1, suggesting that the reduction of basicity on the surface of the digestive lysozymes is a important factor in the determination of their acidic pH optimum.

La drépanocytose est une hémoglobinopathie génétique ayant pour conséquences une anémie hémolytique chronique et sévère et des crises vaso-occlusives itératives. Cette pathologie s’accompagne également d’une intolérance à l’effort et d’altérations de la fonction et du tissu musculaire. Récemment, nous avons pu montrer, par une étude contrôlée et randomisée, l’innocuité et les bénéfices fonctionnels d’un programme d’entrainement en endurance, d’intensité modérée, chez des patients drépanocytaires. L’objectif de ce travail doctoral a été d’évaluer les effets de ce programme d’entrainement sur les caractéristiques musculaires de quarante patients drépanocytaires homozygotes. L’analyse des biopsies musculaires rapporte des adaptations tissulaires chez les patients entrainés, illustrées par une augmentation de la surface des myocytes, une amélioration de leur capacité oxydative, une augmentation du nombre de microvaisseaux sans modification de leur tortuosité, laissant supposer une meilleure oxygénation musculaire. L’excellente tolérance de ce mode d’entrainement semble reposer sur une plus faible mobilisation des voies anaérobies comme en témoigne la stabilité des activités enzymatiques associées à la glycolyse lactique et l’absence de modification du contenu musculaire des protéines impliquées dans la régulation du pH. Par ailleurs, cet entrainement n’a pas engendré de dégradation tissulaire notable. Ainsi, cet entrainement a non seulement apporté des bénéfices fonctionnels, mais également réduit les dysfonctionnements tissulaires musculaires. Cette thérapie par l’exercice peut donc être considéré comme une stratégie adjuvante prometteuse pour les patients drépanocytaires. / Sickle cell disease is a genetic hemoglobinopathy resulting in chronic and severe hemolytic anemia and iterative vaso-occlusive crisis. This pathology is also accompanied by exercise intolerance and alterations in muscle function and tissue. Recently, we demonstrated, through a randomized controlled study, the safety and functional benefits of a moderate-intensity endurance exercise training program in sickle cell disease patients. The objective of this doctoral work was to evaluate the effects of this training program on the muscle characteristics of forty homozygous sickle cell disease patients. The analysis of muscle biopsies reported tissue adaptations in trained patients, illustrated by an increase in the surface area of myocytes, an improvement in their oxidative capacity, an increase in the number of microvessels without modification of their tortuosity, suggesting a better muscle oxygenation. The excellent tolerance of this training mode seems to be based on a lower mobilization of the anaerobic pathways, as shown by the stability of the enzymatic activities associated with lactic glycolysis and the lack of any modification of the muscle protein content involved in pH regulation. Moreover, this training did not result in any significant tissue degradation. Thus, this training provided functional benefits, but also reduced muscle tissue dysfunctions. This exercise therapy can therefore be considered a promising adjuvant strategy for sickle cell disease patients.