Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves [120]-134).

As doenças tropicais negligenciadas, que atingem as populações mais carentes do mundo, representam em termos humanitários e socioeconômicos uma grande preocupação global. As tripanossomíases estão entre as doenças parasitárias mais importantes, e, particularmente, a tripanossomíase africana, ou doença do sono, destaca-se como uma grave condição de saúde, causada pelo parasita unicelular Trypanosoma brucei. Dentre os principais alvos metabólicos considerados para o desenvolvimento de novos fármacos para o tratamento das tripanossomíases, a glicólise recebe especial atenção em função de seu papel vital no processo de produção de ATP para o parasita que vive na corrente sanguínea. Esta tese de doutorado tem como objetivo identificar novos candidatos a inibidores da enzima aldolase (EC 4.1.2.13) da via glicolítica de T. brucei. Considerando-se que o alvo macromolecular em questão é validado para o planejamento de fármacos, inibidores desta enzima são candidatos a novos agentes quimioterápicos. Este trabalho explora a integração de métodos experimentais e computacionais através de estratégias de planejamento de fármacos baseado na estrutura do receptor (SBDD, na sigla inglesa para structure-based drug design) e na estrutura do ligante (LBDD, na sigla inglesa para ligand-based drug design) para a identificação de inibidores da enzima alvo. Foram produzidos resultados significativos, tais como a identificação através de triagens virtuais em larga escala de novas moléculas capazes de inibir a atividade da aldolase. Adicionalmente, destaca-se a obtenção de protocolos de expressão, purificação e cristalização para a enzima alvo. Como parte da estratégia de identificação de novos inibidores da aldolase, foram desenvolvidos modelos de QSAR 2D e 3D e estudos de dinâmica molecular. / Neglected tropical diseases, which affect the poorest populations across the developing world, are a major global concern. The trypanosomiases are amongst the most serious neglected tropical diseases, and particularly, African trypanosomiasis (sleeping sickness), caused by the unicellular parasite Trypanosoma brucei, appears as a fatal condition. The glycolytic pathway emerges as a promising target among the metabolic pathways for the development of new drugs, due to its essential role in the ATP generating process in the bloodstream form of the parasite. The goal of this work is to identify new inhibitors for the glycolytic enzyme aldolase (EC 4.1.2.13) from Trypanosoma brucei. Inhibitors of this enzyme are drug candidates with high potential for clinical development, as the respective target enzyme was validated as a molecular target for the therapy of trypanosomiasis. The strategy employed in this study includes the integration of SBDD (structure-based drug design) and LBDD, (ligand-based drug design) for the identification of inhibitors of the target enzyme, through the combination of computational and experimental methodologies. Significant results were obtained, such as the identification of new small molecule inhibitors of the aldolase enzyme through high-throughput virtual screening. Additionally, it is highlighted the standardization of expression, purification and crystallization protocols for the target enzyme. As a component of the strategy for the identification of novel aldolase inhibitors, 2D and 3D QSAR models were developed, as well as molecular dynamics studies.

Mutationen im Gen der Aldolase B auf Chromosom 9 führen zur „hereditären“ Fructoseintoleranz (HFI), die nach einer längeren Fructoseexposition zu schweren Leber- und Nierenschäden bis hin zum völligen Versagen dieser Organe führen kann. Zum Ausschluß bzw. zur Bestätigung einer HFI werden seit ca. 20 Jahren hauptsächlich molekularbiologische Verfahren eingesetzt. Dabei zeigte es sich, dass wenige, regional unterschiedlich häufig vorkommende Mutationen (in Deutschland A149P und A174D) bei dem Großteil der Patienten für diese Erkrankung verantwortlich sind. Viele Laboratorien beschränken sich daher auf den Nachweis bzw. den Ausschluß dieser Mutationen. Ziel der vorliegenden Arbeit war es zu zeigen, dass auch bei den Patienten mit der Verdachtsdiagnose „hereditäre Fruktoseintoleranz“, bei denen die häufig vorkommen-den Mutationen A149P und A174D nur auf einem oder auf keinem der zwei Allele nachweisbar waren, die Diagnose mit molekularbiologischen Methoden durch den Nachweis seltener oder bisher nicht beschriebener Mutationen gesichert werden kann. Untersucht wurden 8 Patienten aus 5 Familien mit dieser Verdachtsdiagnose. Dabei handelte sich um Patienten mit klinischen Symptomen und in einem Fall enzymatisch gesicherter Diagnose. In allen Fällen mit Ausnahme von Patient III3 (Genotyp A174T/Wildtyp) konnten zwei Mutationen im Aldolase B-Gen nachgewiesen werden. Von den insgesamt identifizierten 8 Mutationen waren 3 zum Zeitpunkt der Durchführung der experimentellen Arbeiten in der Fachliteratur noch nicht beschrieben und erhöhen die Zahl der bekannten Mutationen im Aldolase B – Gen auf insgesamt 45.

The addition of Mn(II) to an early stationary-phase Deinococcus radiodurans RI culture could induce a new round of cell division (MnCD effect). The addition of Mn(II) could also stimulate the utilization of glucose and fructose in this bacterium. Class II fructose-1,6-bisphosphate aldolase (FBA) is an Mn-dependent key enzyme in pentose phosphate pathway. Therefore, in this research, we focused on the studies of the fba gene. Base on the gene sequence, FBA protein was composed of 306 amino acids, (M.W., 32.4 kDa¡F pI, 5.4). The expected PCR product size of the fba gene is 9.3 kbp. We had amplified the fba gene by using both Taq DNA polymerase and pfu turbo DNA polymerase. The sequence of the pfu turbo DNA polymerase products showed a higher homology with the fba gene than those of using Taq DNA polymerase. These amplified fba gene was cloned into three expression vectors, pGEX-4T-2, pQE30, and pET28a, and then further expressed in E. coli BL21(DE3)RIL and JM109. The recombinant GST-FBA protein could be overproduced in pTDA2/BL21(DE3)RIL. However, the expressed insoluble protein accumulated as inclusion bodies in the cells and exhibited no enzyme activity. After partial purification, and processing by thrombin protease cleavage, urea treatment, and the addition of Mn(II), this enzyme still showed no activity. The recombinant pEDA2/BL21(DE3)RIL strain cells grew in 18¢J and induced by 0.1mM IPTG could produced a soluble form His-Thrombin-T7-FBA protein which performed a 50X higher activities than those cells grew in 30¢J. This result indicated that decreasing the indicatioin temperature could improve the protein solubility and activity.

Universiẗat, Diss., 2000--Freiburg (Breisgau).

No description available.

Cellular growth and proliferation are among the most important processes for cells and organisms. One of the major determinants of these processes is the amount of proteins and consequently also the amount of ribosomes. Their synthesis involves several hundred proteins and four different ribosomal RNA species, is highly coordinated and very energy-demanding. However, the molecular mechanims of transcriptional regulation of the protein-coding genes involved, is only poorly understood in mammals. In this thesis, unbiased genome-wide knockout reporter screens were performed, aiming to identify previously unknown transcriptional regulators of ribosome biogenesis factors (RiBis), which are important for the assembly and maturation of ribosomes, and ribosomal proteins (RPs), which are ribosomal components themself. With that approach and follow-up (validation) experiments, ALDOA and RBM8A among others, could be identified as regulators of ribosome biogenesis. Depletion of the glycolytic enzyme ALDOA led to a downregulation of RiBi- and RPpromoter driven reporters on protein and transcript level, as well as to a downregulation of ribosome biogenesis gene transcripts and of mRNAs of other genes important for proliferation. Reducing the amount of the exon junction complex protein RBM8A, led to a more prominent downregulation of one of the fluorescent reporters, but this regulation was independent of the promoter driving the expression of the reporter. However, acute protein depletion experiments in combination with nascent RNA sequencing (4sU-Seq) revealed, that mainly cytosolic ribosomal proteins (CRPs) were downregulated upon acute RBM8A withdrawal. ChIP experiments showed RBM8A binding to promoters of RP genes, but also to other chromatin regions. Total POL II or elongating and initiating POL II levels were not altered upon acute RBM8A depletion. These data provide a starting point for further research on the mechanisms of transcriptional regulation of RP and RiBi genes in mammals. / Zelluläres Wachstum und Proliferation zählen zu den wichtigsten Prozessen für Zellen und Organismen. Eine der größten Determinanten dieser Prozesse ist die Menge an Proteinen und in der Konsequenz auch die Menge an Ribosomen. Deren Synthese erfordert mehrere hundert Proteine und vier verschiedene ribosomale RNA-Spezies, ist stark koordiniert und sehr energiefordernd. Dennoch sind die molekularen Mechanismen der transkriptionellen Regulation der beteiligten protein-kodierenden Gene in Säugetieren nur schlecht verstanden. In dieser Arbeit wurden hypothesenfreie genomweite Knockout-Reporterscreens mit dem Ziel durchgeführt, bisher unbekannte transkriptionelle Regulatoren von ribosomalen Biogenesefaktoren (RiBis), welche wichtig für den Zusammenbau und die Reifung der Ribosomen sind, und ribosomalen Proteinen (RPs), welche selbst ribosomale Bestandteile sind, zu identifizieren. Durch diesen Ansatz und nachfolgende (Validierungs- )Experimente, konnten unter anderem ALDOA und RBM8A als Regulatoren ribosomaler Biogenese identifiziert werden. Eine Depletion des glykolytischen Enzyms ALDOA führte sowohl zu einer Herunterregulation von RiBi- und RP-Promotor-gesteuerten Reportern auf Protein- und Transkriptebene, als auch zu einer Herunterregulation von ribosomalen Biogenesegentranskripten und von mRNAs anderer für die Proliferation wichtiger Gene. Eine Reduktion der Menge des Exon-Junction-Komplexproteins RBM8A führte zu einer deutlicheren Herunterregulation eines der beiden fluoreszierenden Reporter, aber diese Regulation war unabhängig vom Promotor, der die Expression des Reporters steuert. Akute Proteinabbauexperimente in Verbindung mit einer Sequenzierung naszenter RNA (4sU-Seq) zeigten allerdings, dass hauptsächlich zytosolische ribosomale Proteine (CRPs) nach akuter RBM8A-Depletion herunterreguliert waren. ChIP-Experimente zeigten RBM8A-Bindung an Promotoren von RP-Genen, aber auch an andere Chromatinregionen. Gesamt-POL II- oder elongierende und initiierende POL II-Mengen waren nach akuter RBM8A-Depletion nicht verändert. Diese Daten stellen einen Ausgangspunkt für weitere Forschung zu den Mechanismen transkriptioneller Regulation von RP- und RiBi-Genen in Säugetieren dar.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The enzyme, deoxyribophosphoaldolase, reversibly cleaves deoxyribose- 5-P to glyceraldehyde-3-P and acetaldehyde. Until this exposition, it was not known to exist in human erythrocytes. However, the enzyme was postulated to occur in erythrocytes in order to account for the synthesis of glyceraldehyde-3-P observed in ghosts when deoxynucleosides were metabolized. Therefore, initial experiments were designed to detect acetaldehyde as an intermediate of deoxynucleoside metabolism by ghosts. Indeed, it was found in incubation mixtures containing deoxyinosine or deoxyadenosine as substrate. Accumulation of acetaldehyde was found to depend upon substrate concentration and no acetaldehyde was detected in the absence of substrate or in the presence of inosine or ribose-5-P. Acetaldehyde was identified spectrophotometrically by its reaction with yeast alcohol dehydrogenase and colorimetrically by its reaction with buffered semicarbazide solution. Confirmation of its identity was obtained by isolation of the 2,4 dinitrophenylhydrazone derivative of acetaldehyde aerated from incubation extracts. A direct relationship between acetaldehyde production and triosephosphate production to deoxyribose-5-P utilization by hemolysates prepared from human erythrocytes was shown. Conclusive evidence for the existence of deoxyribophosphoaldolase was obtained by isolating the enzyme from human erythrocytes. Two procedures were developed to isolate the enzyme. The first involved the use of Sephadex G-100 and DEAE-cellulose columns. The second procedure proved more fruitful and employed ammonium sulfate fractionation followed by elution from calcium phosphate gels. It was found that 26% of the original enzyme activity was recovered and purification was approximately 3,000 fold. Several characteristic properties of partially purified and purified preparations of the enzyme were studied. It was found that: 1. Enzyme activity decayed rapidly upon storage. Magnesium ion, cysteine HCl, beta-mercaptoethanol or reduced glutathione increased enzyme stability. In addition, the sulfhydryl containing compounds were able to partially reactivate previously inactivated enzyme. 2. The molecular weight of deoxyribophosphoaldolase was estimated by Sephadex gel fractionation to be slightly greater than 68,000, the molecular weight of hemoglobin. Because of its low affinity for DEAE-cellulose and high affinity for carboxy methyl cellulose, the enzyme appeared to be a very basic protein. 3. The reaction catalyzed by deoxyribophosphoaldolase favored cleavage of deoxyribose-5-P. At equilibrium, 60% of deoxyribose-5-P was converted to products. When acetaldehyde and glyceraldehyde-3-P were used as substrates, 40% of each was converted to deoxypentose product. 4. The enzyme showed a high specificity for each of its three substrates. No reaction of the enzyme occurred with ethyl alcohol, pyruvate, ribose-5-P, deoxyribose, lactate and dihydroxyacetone phosphate. 5. The enzyme reacted optimally at pH 6.5. 6. Deoxyribophosphoaldolase was activated by several carboxylic acids. The degree of activation was greater in the presence of citric acid than any dicarboxylic acid tested. An optimal activation of enzyme occurred when the concentration of citrate was varied between 3-15 umoles/ml. Citrate activation did not appear to reside in its ability to act as a chelator. Comparison of enzyme elution from Sephadex G-100 columns in the presence and absence of citrate suggested that citrate causes enzyme aggregation by a still unexplained mectanism. 7. The apparent Michaelis constants for deoxyribose-5-P cleavage were determined in the presence and absence of citrate and were found to be 6.4 x 10^-4 moles/liter and 24.0 x 10^-4 moles/liter respectively. The presence of deoxyribophosphoaldolase in human erythrocytes can clearly explain the production of triosephosphate and acetaldehyde from deoxynucleoside substrates and may, in fact, provide a major catabolic pathway for the deoxypentose moiety of these deoxynucleosides. The enzyme provides a simple mechanism for triosephosphate formation from deoxypentose and may be part of a pathway that converts deoxypentose phosphate to pentose phosphate. / 2999-01-01

Foi levado a efeito estudo sobre formas múltiplas de aldolase de músculo de coelho. A enzima foi purificada a pH 7,5 por eluição com substrato a partir de coluna de fosfocelulose. A enzima foi ainda cristalizada por diálise dessas preparações contra solução saturada de sulfato de amônio. Formas múltiplas de aldolase foram obtidas por fracionamento a diferentes pI por eletrofocalização em gradiente de Ampholine na faixa de pH entre 7,0 a 10,0. Nessas condições foram separados cinco híbridos resultantes da associação ao acaso das sub-unidades α e β, os quais foram analisados em estado de dissociação a partir de proteínas carboximetiladas e separadas por eletroforese em gel de poliacrilamida na presença de uréia 8M. Foi também estudado o aparecimento de sub-unidade α e β em músculo de coelhos de idades que variavam de 1 a 240 dias, verificando-se que em coelhos de 1 dia existia apenas a proteína α4, surgindo sub-unidades β já em animais de 10 dias. / Not available.

Foi levado a efeito estudo sobre formas múltiplas de aldolase de músculo de coelho. A enzima foi purificada a pH 7,5 por eluição com substrato a partir de coluna de fosfocelulose. A enzima foi ainda cristalizada por diálise dessas preparações contra solução saturada de sulfato de amônio. Formas múltiplas de aldolase foram obtidas por fracionamento a diferentes pI por eletrofocalização em gradiente de Ampholine na faixa de pH entre 7,0 a 10,0. Nessas condições foram separados cinco híbridos resultantes da associação ao acaso das sub-unidades α e β, os quais foram analisados em estado de dissociação a partir de proteínas carboximetiladas e separadas por eletroforese em gel de poliacrilamida na presença de uréia 8M. Foi também estudado o aparecimento de sub-unidade α e β em músculo de coelhos de idades que variavam de 1 a 240 dias, verificando-se que em coelhos de 1 dia existia apenas a proteína α4, surgindo sub-unidades β já em animais de 10 dias. / Not available.