O T. cruzi, é o agente causador da doença de Chagas. O estado redox NAD+/NADH intracelular é fundamental na manutenção do metabolismo celular. A GAPDH apresenta a função de proteção do telômero em mamíferos contra degradação, isto por causa de ligar se ao telômero. Aqui, mostramos que a GAPDH recombinante de T. cruzi (rTcGAPDH) interage com o DNA telomérico. A rTcGAPDH liga ao DNA de simples fita. Mostramos que a GAPDH liga ao DNA telomérico in vivo em células epimastigotas, onde a [NADH] é maior que [NAD+], mas a adição de NAD+ exógeno bloqueia esta interação. Corroborando a hipótese de que o equilíbrio NAD+/NADH determina a interação GAPDH-telômero, vimos que o tripomastigota tem maior [NAD+] intracelular que a [NADH] e a GAPDH não é capaz de ligar se ao DNA telomérico. Além disso, o NADH exógeno resgata a interação GAPDH-telómero nesta fase. É importante o equilíbrio NAD+/NADH desta interação em tripanosomas, sugerindo que a proteção do telômero do parasita pode ser regulada pelo estado metabólico das células. / The T. cruzi, is the causative agent of Chagas disease. The redox state of NAD+/NADH intracellular is critical in the maintenance of cellular metabolism. The GAPDH has the protection function of the telomere in mammals against degradation, because it is connecting to the telomere. Here we show the recombinant GAPDH of T. cruzi (rTcGAPDH) interacts with telomeric DNA. The rTcGAPDH binds to single-stranded DNA. We show GAPDH to bind to telomeric DNA in vivo epimastigotes cells, where [NADH] is greater than [NAD+], but the addition of exogenous NAD+ blocks this interaction. Corroborating the hypothesis that the NAD+/NADH balance determines the GAPDH-telomere interaction, we saw that the trypomastigote has higher [NAD+] that intracellular [NADH] and GAPDH is not able to connect to telomeric DNA. In addition, the exogenous NADH recovers the GAPDH-telomere interaction at this stage. It is important the NAD+/NADH balance this interaction in trypanosomes, suggesting that the protection of the telomere of the parasite can be regulated by the metabolic state of the cells.

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

Parkinson's disease (PD) is a slow-progressive neurodegenerative disorder affecting 5-6 million people around the globe. The disease is manifested by the rapid deterioration of dopaminergic cells in the substantia nigra portion of the brain; however, the pathological mechanism of selective dopaminergic neuronal death is unknown. A reduction in levels of 3,4-dihydroxyphenylacetaldehyde (DOPAL) is biologically critical as this aldehyde has been shown to be toxic to dopaminergic cells and is a highly reactive electrophile. Investigating neuronal protein targets is essential in determining the cause of toxicity. An essential protein-GAPDH (e.g., glyceraldehyde-3-phosphate dehydrogenase) is an abundantly expressed enzyme known for its glycolytic activity, and recent research has implicated its role in oxidative stress-mediated neuronal death. This work positively shows GAPDH as a target for DOPAL modification, and, for the first time, DOPAL is identified as a potent inhibitor for GAPDH enzymatic activity. LC-MS and other chemical probes (ie. thiol and amine modifiers) show that DOPAL modifies specific –Lys, -Arg, and –Cys residues in the cofactor binding-domain of GAPDH. The enzyme inhibition is also time and DOPAL dose-dependent. DOPAL has a unique structure, containing two reactive functional groups: an aldehyde and catechol ring. In-house syntheses of DOPAL analogues, containing the catechol group and lacking the aldehyde, and vice versa have been tested on GAPDH and do not inhibit or modify GAPDH. Therefore, both the catechol and aldehyde groups of DOPAL are specific to binding with GAPDH and are necessary to achieve modification and enzyme inhibition. In addition to finding a novel enzyme inhibited and modified by DOPAL, this work has also confirmed linking DOPAL levels to a fungicide associated with PD risk. This benzimidazole fungicide, benomyl was shown to inhibit ALDH2 in the SH-SY5Y neuroblastoma cell line via an increase in DOPAL and a decrease in DOPAC. The ratios of DOPAL and DOPAC, the product of ALDH, were measured by HPLC-ECD, and found that benomyl does inhibit ALDH2 in this dopaminergic cell model. The cytotoxicity of benomyl, DA, DOPAL and the combination of DA or DOPAL with benomyl was assessed by MTT assay. Surprisingly, the only toxic combination was the combination of DA or DOPAL with benomyl. In fact, this toxicity appears to be synergistic, as none of the single treatments are significantly toxic to the cells. This synergistic effect also affects GAPDH aggregation. The cell morphology is also drastically different in the presence of the combined treatments, compared to individual treatment of DA, DOPAL or benomyl; cells start to ebb and show apoptotic-like features at just 2h. A second class of pesticides, named chlorpyrifos and chlorpyrifos-oxon were tested for toxicity in PC6-3These compounds were toxic to these cells due to DOPAL accumulation reaching high levels in the 100 µM range. Exposure to environmental toxins such as pesticides and fungicides has long been linked to PD risk, but only recently to DOPAL levels. This work provides a novel mechanism by which fungicide exposure may stimulate PD pathogenesis.

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

Metal toxicity is implicated in neurotoxicity, nephrotoxicity, aging and cancer. Protein oxidation resulting from oxidative stress is now known to be involved in metal toxicity. However, proteomic responses to metal induced oxidative stress have not been characterized. By using the yeast as a model, we characterized these changes occurring in response to sub-lethal doses of metals. Several proteins involved in protein synthesis, ribosome assembly decreased while antioxidant defenses, proteins involved in sulfur metabolism, and glutathione synthesis and ubiquitin increased following metal exposure. We also show that metals induced temporal and targeted protein oxidation independent of protein abundance. Among the targets were glycolytic enzymes and heat-shock proteins. As a consequence, glycolytic enzyme activities decreased whereas the levels and activities of the enzymes of the alternative pathway for glucose metabolism, pentose phosphate pathway (PPP) increased. True to prediction, we also found increased flow through the PPP as measured by elevated levels of NADPH and glutathione. NADPH and glutathione are crucial for maintaining the redox balance in the cell. Thus, rerouting of glucose metabolism into PPP is considered to be beneficial to the organism. Among the oxidation targets is a glycolytic protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that is required for apoptosis in neuronal cells. We show that not only is GAPDH required for metal induced apoptosis in yeast but also the levels of GAPDH transcript and protein increase in the cytosol and the nucleus in an isoform specific fashion. Such changes strongly implicate the role of GAPDH in yeast apoptosis. This work provides evidence for the involvement of targeted protein oxidation in metal toxicity, shows the overlaps and differences in the mechanism of copper and cadmium toxicity, allows comprehension of how metabolic processes respond to metal stress and explores the potential of GAPDH as a sensor of oxidative stress and mediator for apoptosis.

C-Phycocyanin (C-PC) from blue-green algae has been reported to have various pharmacological characteristics, including anti-inflammatory and anti-cancer activities. In this study, the beta-subunit of C-PC (ref to as C-PC/beta) was expressed and purified from bacteria E. coli BL-21. The recombinant C-PC/beta has been demonstrated to have anticancer properties. Under the treatment of 5 microM of the recombinant C-PC/beta, four different cancer cell lines accrued a high proliferation inhibition and apoptotic induction. The C-PC/beta interacts with membrane-associated beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been found. Under the treatment of the C-PC/beta, depolymerization of microtubulin and actin-filament was observed. The cells underwent apoptosis with increase of Caspase-3 and Caspase-8 activities. Cell cycle was arrested at G0/G1 phase under the treatment of C-PC/beta. In addition, the nuclear level of GAPDH decreased significantly. Inhibition of cancer cell proliferation and induction of apoptosis may potentate C-PC/beta as a promising cancer prevention or therapy agent.

C-Phycocyanin (C-PC) from blue-green algae has been reported to have various pharmacological characteristics, including anti-inflammatory and anti-cancer activities. In this study, the beta-subunit of C-PC (ref to as C-PC/beta) was expressed and purified from bacteria E. coli BL-21. The recombinant C-PC/beta has been demonstrated to have anticancer properties. Under the treatment of 5 microM of the recombinant C-PC/beta, four different cancer cell lines accrued a high proliferation inhibition and apoptotic induction. The C-PC/beta interacts with membrane-associated beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been found. Under the treatment of the C-PC/beta, depolymerization of microtubulin and actin-filament was observed. The cells underwent apoptosis with increase of Caspase-3 and Caspase-8 activities. Cell cycle was arrested at G0/G1 phase under the treatment of C-PC/beta. In addition, the nuclear level of GAPDH decreased significantly. Inhibition of cancer cell proliferation and induction of apoptosis may potentate C-PC/beta as a promising cancer prevention or therapy agent.

Made available in DSpace on 2014-06-03T19:33:07Z (GMT). No. of bitstreams: 4 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 44.pdf: 10707792 bytes, checksum: c5125ab7f60ab003fc772f74c8c4517f (MD5) 44.pdf.txt: 668966 bytes, checksum: 99d869753275b20698a78896e6240b59 (MD5) 44.pdf.jpg: 1554 bytes, checksum: 1e3f2b67d245dd5c72e348d82e4541ba (MD5) Previous issue date: 2004 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil. / BUSCA DE PRODUTOS NATURAIS BIOATIVOS EM PLANTAS DAS FAMÍLIAS MYRTACEAE (Siphoneugena densiflora Berg) E VERBENACEAE (Vitex polygama Cham.). Neste trabalho estão sendo descritas trinta e sete substâncias identificadas de V. polygama Cham. e trinta e duas de S. densiflora Berg, provenientes do estudo fitoquímico de seus respectivos extratos. Destas, quatro substâncias isoladas de S. densiflora são destacadas por serem inéditas na literatura, a saber: 6βhidroximaslinato de βD-glucopiranosila; 4-O-α-L-2 -O-acetilramnopiranosídeo do ácido elágico e seu regioisômero com o grupo acetila na posição 3 e a siphoneugenina. São relatadas as atividades inibitórias enzimáticas de várias das substâncias identificadas, e de seus extratos de origem, sobre as enzimas gliceraldeído -fosfatodesidrogenase glicossomal (gGAPDH), de Trypanosoma cruzi; adenina fosforribosiltransferase (APRT), de Leishmania tarentolae; e pectinase, do fungo Leucoagaricus gongylophorus, simbionte da formiga cortadeira Atta sexdens rubropilosa. Dentre todas as substâncias testadas, os taninos castalagina e casuarinina se revelaram os mais promissores inibidores com valores de CI50 de 7,5 e 1,8 µM sobre a gGAPDH e 3,3 e 1,8 µM sobre a APRT, respectivamente. Como os taninos podem precipitar proteínas e causar uma ação inibitória não específica, foram realizados estudos sobre a interação deles com os reagentes dos ensaios enzimáticos. São relatadas as atuações biológicas in vitro de algumas substâncias sobre as formas tripomastigotas de T. cruzi; sobre as lagartas de 1º e 2º instar da mariposa, praga do milho, Spodoptera frugiperda; sobre as bactérias Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Micrococcus roseus e sobre os fungos Leucoagaricus gongylophorus, Candida albicans, Cryptococcus laurentii, Sacharomyces cerevisae e Trichosporon cutaneum. A mistura dos flavonóis glicosilados 3-O + 4 -O-β-D-glucopiranosil quercetina apresentou a maior atividade tripanocida (1,1 mM; 97 % lise contra 0,6 mM; 100 % lise da violeta genciana), ressaltando-se que sua CI50 sobre a gGAPDH foi de 20 µM. Os extratos metanólicos e hidrometanólicos dos galhos, caule, folhas e cascas da raiz de S. densiflora causaram 100 % de morte das lagartas de S. frugiperda, a uma concentração de 1000 ppm. Do extrato metanólico de folhas de S. densiflora foram isolados dois flavonóides: quercetina e quercitrina que, a uma concentração de 100 ppm, provocaram 78 e 85 % de morte das lagartas. Os taninos casuarinina e 4-O-α-raminopiranosídeo do ácido elágico, na mesma concentração, atuaram principalmente como fagoinibidores. O extrato hidrometanólico de folhas de V. polygama teve uma atividade inseticida de 60 % sobre S. frugiperda a uma concentração de 1000 ppm. Dele foram isoladas as misturas das flavonas di-C-glicosiladas carlinosídeo, schaftosídeo e seus respectivos isômeros, os quais apresentaram atividade inseticida relatada na literatura (SIMMONDS, 2001). O extrato hidrometanólico de folhas de S. densiflora apresentou uma atividade bactericida (halo de inibição de 11 mm) tão intensa quanto a do antibiótico tetraciclina (10 e 12 mm) para as bactérias P. aeruginosa e M. roseus. Das substâncias ensaiadas, apenas a mistura do cafeoil-6-O-a + β-D-glicopiranosídeo foi relativamente ativa sobre o fungo L. gongylophorus causando 60 % de inibição de seu crescimento, a uma concentração de 50 µg/mL. Também está sendo narrado o desenvolvimento de um método, usando-se CLAE, para a análise e quantificação da 20-hidroxiecdisona presente no extrato metanólico de galhos de V. polygama, em virtude de suas inúmeras atividades biológicas e grande uso e importância na indústria farmacêutica.

Made available in DSpace on 2016-06-02T20:34:17Z (GMT). No. of bitstreams: 1 2694.pdf: 3100677 bytes, checksum: e539b70bd59d8ae168c5b5d00b544881 (MD5) Previous issue date: 2009-06-05 / Universidade Federal de Sao Carlos / Theoretical methods in computational chemistry were used to study the gGAPDH enzyme from Trypanosoma cruzi. This protozoan is responsible by the Chagas diseases. Molecular dynamics simulations were performed to obtain the time evolution of the gGAPDH enzyme in the holo (with the cofactor NAD+) and apo (without the cofactor) forms in aqueous solution. The calculations were performed in the NpT ensemble with T = 300K e p = 1bar. In these simulations little conformational changes were observed in both holo and apo forms of the enzyme along 20.0 ns simulation time. Docking calculations were carried out to fit some drugs in enzyme active site using the ensemble docking methodology. Therefore, multiple enzyme conformations of both holo and apo forms were obtained at time intervals of 2.0 ns along the molecular dynamics simulation. This procedure was used to take in into account the flexibility of the enzyme. The results from these calculations indicate that the best way to develop a drug molecule is to consider both enzyme forms (holo and apo forms). In this way, one drug will inhibit the apo form and other, the holo form. To characterize the enzymatic mechanism, the glyceraldehyde 3-phosphate (G3P) was placed in the active site (via docking calculations) of the holo form conformation obtained in the end of the 20.0 ns trajectory. A 1.0 ns molecular dynamic simulation was performed in the (gGAPDH-NAD+-G3P) system. Quantum chemical calculations were performed to study reactive process in the enzyme catalytic site. A convenient model was built using about 698 atoms carefully chosen to represent the active site and its surroundings. The calculations were performed using a combination of MOZYME and the usual SCF procedure implemented in the MOPAC2009 program. The calculations were performed at the PM6 level. One of the reaction mechanisms proposed in the literature was characterized xix calculating the energy profile along the reaction path to transfer a proton from Cys166 to His194. Using a second structure obtained from molecular dynamics, a new mechanism for the gGAPDH enzyme was proposed and characterized by a similar MOPAC2009 calculation. In this mechanism, the hydroxyl group of the G3P acts as a bridge to transfer the proton from the Cys166 to His194 residue. / Métodos de química computacional foram utilizados para estudar a enzima gGAPDH do Trypanosoma cruzi, protozoário responsável pela doença de Chagas. Simulações por dinâmica molecular foram realizados para obter a evolução temporal da enzima gGAPDH na forma holo (com o cofator NAD+) e apo (sem o cofator) em solução. Os cálculos foram efetuados no ensemble NpT a T = 300K e p = 1bar. Nessas simulações ficaram evidenciadas pequenas mudanças conformacionais entre as formas holo e apo no tempo de simulação de 20,0 ns. Para posicionar ou encaixar algumas moléculas candidatas a fármacos no sítio ativo da enzima, cálculos de docking foram realizados utilizando a metodologia de ensemble docking, utilizando múltiplas conformações obtidas a cada intervalo de 2,0 ns de simulação da enzima na forma holo e apo. Essa metodologia foi adotada para levar em consideração a flexibilidade da enzima. Os resultados desses cálculos mostraram que a melhor estratégia para elaborar compostos candidatos a fármacos é considerar as duas formas da enzima (holo e apo). Com esta estratégia, um composto inibiria a forma apo e outro, a forma holo. Para caracterizar o mecanismo enzimático, o gliceraldeído 3-fosfato (G3P) foi colocado no sítio ativo (via cálculos de docking) da enzima gGAPDH na forma holo, a qual foi obtida a 20,0 ns de simulação. Nesse sistema (gGAPDHNAD+- G3P), foi realizado 1,0 ns de simulação por dinâmica molecular para a relaxação e equilibração do sistema. Ao longo dessa simulação, duas estruturas foram escolhidas para a realização de cálculos de química quântica relacionados com a atividade enzimática. Em uma dessas estruturas, um dos mecanismos propostos na literatura foi caracterizado a partir da coordenada de reação de transferência do próton da Cys166 para a His194. Estes cálculos foram efetuados utilizando um procedimento que combina a utilização da metodologia xvii MOZYME com o cálculo SCF usual implementado no programa MOPAC2009. Nestes cálculos, a parametrização PM6 foi utilizada. A partir de uma segunda estrutura obtida da dinâmica molecular, um novo mecanismo para a enzima gGAPDH foi proposto e caracterizado via cálculos quânticos. Nesse mecanismo, o grupo hidroxila da G3P age como uma ponte para a transferência do próton da Cys166 para a His194.

Made available in DSpace on 2016-06-02T20:34:28Z (GMT). No. of bitstreams: 1 3861.pdf: 2836276 bytes, checksum: bc721126635c9ba884c4ab4a6d99384d (MD5) Previous issue date: 2009-05-29 / Universidade Federal de Minas Gerais / The flavonoids are an important class of polyphenolic compounds present in relative abundance among the secondary metabolites of plants and are found in fruits, vegetables, nuts, seeds, flowers, tea, honey and propolis. They show several biological activities such as anti-inflammatory, antimicrobial, antioxidant, anticancer, antifungal, etc.. The present work aimed to synthetize and to evaluate the biological activity of a library of flavones and chalcones. The flavones and chalcones were prepared following classical organic synthesis and combinatorial chemistry. Among the flavones and chalcones prepared and tested against enzyme of T. cruzi gGAPDH, the best results were obtained with the flavone 56{1,6}, 85% of inhibition, and the chalcone 55{1,3}, 97% of inhibition, both at concentration of 100 M. In bioassay against human cathepsin V, a chalcone which showed the best inhibitory activity was chalcone 18 with 64% of inhibition and 9 flavones (40{1,1}, 40{2,1}, 40{3,1}, 56{1,1}, 56{1,5}, 56{2,1}, 56{3,1}, 56{3,2}, 56{4,1}) showed 100% of inhibition, both at concentration of 25 M. The flavones and chalcones prepared will contribute for the study of structure-activity relationship, which could let to the design and synthesis of more active flavones and chalcones. / Os flavonóides constituem uma importante classe de compostos polifenólicos presentes em relativa abundância entre os metabólitos secundários de plantas e são encontrados em frutas, vegetais, nozes, grãos, flores, chás, própolis e mel. Apresentam diversas atividades biológicas como, anti-inflamatória, antimicrobiana, anti-oxidante, anti-câncer, anti-fúngica, etc. O presente trabalho teve como objetivos síntetizar e avaliar a atividade biológica de uma coleção de flavonas e chalconas. A preparação das flavonas e chalconas foi realizada empregando-se metodologias de síntese orgânica clássica e também química combinatória. Dentre as flavonas e chalconas preparadas e testadas frente à enzima gGAPDH do T. cruzi, os melhores resultados foram obtidos com a flavona 56{1,6}, 85% de inibição, e a chalcona 55{1,3}, com 97% de inibição, ambas na concentração de 100 M. Nos testes realizados frente a catepsina humana V, a chalcona que apresentou maior atividade inibitória foi a chalcona 18 com 64% de inibição e 9 flavonas (40{1,1}, 40{2,1}, 40{3,1}, 56{1,1}, 56{1,5}, 56{2,1}, 56{3,1}, 56{3,2}, 56{4,1}) apresentaram 100% de inibição, ambas na concentração de 25 M. As flavonas e chalconas preparadas irão contribuir para o estudo de estrutura-atividade, visando o planejamento e síntese flavonas e chalconas mais ativas.