Of the organisms with sequenced genomes, plants appear to possess the most rhomboid protease-encoding genes. However, our knowledge of processes in plants that involve Regulated Intramembrane Proteolysis (RIP) and rhomboid proteases remains low. As expressed recently by other researchers, finding a natural substrate for a rhomboid protease represents the biggest experimental challenge. Using yeast mitochondria-based assays, a potential link between the plastid translocon component Tic40 and organellar rhomboid proteases was recently uncovered. In this particular link, rhomboid proteases appear capable of influencing the pattern of imported Tic40 in yeast mitochondria. Tic40 may thus represent a natural plant target of organellar rhomboid proteases. Here, we obtained further motif-oriented evidence supporting Tic40 as a natural plant rhomboid substrate. A comparative analysis of sequences revealed that Tic40 may also possess similar TMD motifs found in the model substrate, Spitz. Rhomboid proteases often require these motifs to cleave substrates within intramembrane environments. Using site-directed mutagenesis and yeast mitochondria assays, the impact of mutations occurring in the motifs ASISS, GV, QP, and GVGVG of Tic40 was assessed. In terms of cleavage and changing the pattern of imported Tic40, some of the mutations showed decreased activities and a few showed enhancements. More importantly, the overall observed pattern associated with select Tic40 mutations resembled the characteristics reported for the model substrates. In particular, mutations in the Tic40 GV motif produced similar results as that observed with Spitz, by drastically decreasing or increasing cleavage as a function of amino acid sequence. / Thesis (Master, Biology) -- Queen's University, 2010-05-30 10:22:07.72

Glucansucrases são enzimas que atuam em reações de síntese de polissacarídeos e oligossacarídeos. Para que esses biocatalisadores sejam aplicados em escala industrial, é desejável ótimas estabilidades térmica e operacional, o que pode ser alcançado com a imobilização de enzimas. Como alternativa aos suportes sólidos amplamente estudados, está a quitosana, polímero que não apresenta toxicidade e possui alta biocompatibilidade e alta afinidade com proteínas. Outra possibilidade promissora na imobilização de enzimas, é a síntese dos agregados enzimáticos entrecruzados (CLEAs), os quais apresentam alta atividade catalítica e alta estabilidade. Contudo, uma peculiaridade das glucansucrases quando produzidas em meio contendo sacarose é a camada de polímero que as envolve, e que bloqueia o acesso aos grupos reativos na superfície da proteína. No caso da expressão heteróloga das glucansucrases em Escherichia coli essa dificuldade pode ser contornada. Além disso, o uso da mutagênese sítio-dirigida pode proporcionar modificações de aminoácidos na superfície da enzima, tais como os resíduos Lys, Cys, His, com o intuito de que melhorias na imobilização sejam alcançadas. Sendo assim, na primeira etapa desse trabalho, uma extensa discussão é apresentada em relação às metodologias de imobilização de dextransucrase encontradas na literatura. A seguir, estudos referentes à imobilização da dextransucrase de Leuconostoc mesenteroides B-512 F em esferas de quitosana ativadas com glutaraldeído foram realizados. Esse imobilizado apresentou alta atividade catalítica (197 U/g) quando utilizada a carga de proteína de 400 mg/g de suporte. Além disso, observou-se que a imobilização covalente e os açúcares maltose e glicose promoveram proteção à enzima em temperaturas de 40 ºC e 50 ºC. Na etapa seguinte, a produção e a caracterização de CLEAs de dextransucrase de L. mesenteroides B-512 F foram investigados. Demonstrou-se que o tratamento com a dextranase foi essencial para a imobilização da glucansucrase e que o isopropanol foi o melhor agente precipitante. Os CLEAs apresentaram pH e temperatura ótimos de 3,0 e 60 ºC, respectivamente, enquanto que a dextransucrase imobilizada nas esferas de quitosana funcionalizada com glutaraldeído apresentaram os valores de 4,5 e 20 ºC. Ambas formas imobilizadas apresentaram boa estabilidade operacional na síntese de oligossacarídeos uma vez que após 10 ciclos, 40 % de atividade residual foi observada. Por fim, estão apresentados estudos sobre a modelagem das estruturas tridimensionais e a mutagênese sítio-dirigida das glucansucrases DSR-S vardel Δ4N and ASR C-APY del. Os modelos preditos demonstraram boa qualidade e a mutagênese sítio-dirigida não promoveu perdas significativas na atividade enzimática dos mutantes. Somente o mutante DSR_S326C mostrouse inativo. Os resultados obtidos sugerem que a imobilização da dextransucrase foi satisfatória e que cada técnica possibilita diferentes características ao imobilizado. Além disso, os imobilizados foram adequados para síntese de dextrana e oligossacarídeos. / Glucansucrases are enzymes that catalyze the synthesis of polysaccharides and oligosaccharides. In order to assure continuous processing and reuse of the biocatalyst in industrial applications, enzyme immobilization techniques are required to promote good thermal and operational stabilities. Among the several solid supports for enzyme immobilization, chitosan shows interesting properties because it is non-toxic, it is biocompatible, and it has high protein affinity. Other possibility is the production of cross-linked enzyme aggregates (CLEAs), which presents high catalytic activity and good stability. However, glucansucrases have a particularity when produced in sucrose medium, since a polymer layer surrounds the protein, blocking the access to reactive groups on the enzyme surface. To overcome this problem, it is possible to make the heterologous production of glucansucrases in Escherichia coli. Likewise, the site-directed mutagenesis may promote changes in the amino acids located on the surface to improve immobilization parameters. Therefore, this work aimed to discuss the several techniques applied for dextransucrase immobilization, and to design new immobilized biocatalysts. In a first step, it is presented a review about the distinct immobilization methodologies for dextransucrase. In a second study, an investigation about dextransucrase from Leuconostoc mesenteroides B-512 F immobilized on glutaraldehyde-activated chitosan particles was carried out. The novel immobilized biocatalyst showed 197 U/g (400 mg/g dried support) of catalytic activity. The covalent immobilization promoted protection against enzyme damages at 40 ºC and 50 ºC, whereas maltose and glucose acted as stabilizers. Furthermore, it was studied the production and characterization of CLEAs dextransucrase from L. mesenteroides B-512 F. It was demonstrated that dextranase treatment was crucial for immobilization. Isopropanol was chosen as the best precipitant agent. CLEAs presented optimal pH and temperature of 3.0 and 60 ºC, respectively, whereas it was found values of 4.5 e 20 ºC for dextransucrase immobilized on glutaraldehyde-activated chitosan particles. Both immobilized biocatalysts showed good operational stability in the oligosaccharides synthesis, exhibiting 40 % of residual activity after 10 cycles. Finally, the study concerning the homology modeling and site-directed mutagenesis of glucansucrases DSR-S vardel Δ4N and ASR C-APY del is presented. The predicted models showed good quality and it has been demonstrated that the site-directed mutagenesis did not promote significant losses in the variant enzyme activities. Only one mutant (DSR_S326C) had shown no dextransucrase activity. The results obtained in this work suggest that the immobilization of dextransucrase was satisfactory, also showing that each technique promotes different characteristics to the immobilized biocatalyst. Besides, these immobilized enzymes were feasible for the synthesis of dextran and oligosaccharides.

Retinoblastoma Binding Protein 6 (RBBP6) is a 200 KDa protein shown to play a role in 3'- polyadenylation of mRNA transcripts, as well as to function as an E3 ligase catalysing ubiquitination of cancer-associated proteins. RBBP6 has been previously reported to localise to nuclear speckles, which are thought to play a role in mRNA splicing, presumably as a result of its RS domain, which is known to target mRNA splicing factors to nuclear speckles. However recent studies in our laboratory have shown that isoform 3 of RBBP6, consisting mainly of the DWNN domain, also localises to speckles in resting cells, but more strongly in cells subjected to various stresses, suggesting that the DWNN domain may be the speckle-targeting domain. / Magister Scientiae - MSc (Biotechnology)

Glucansucrases são enzimas que atuam em reações de síntese de polissacarídeos e oligossacarídeos. Para que esses biocatalisadores sejam aplicados em escala industrial, é desejável ótimas estabilidades térmica e operacional, o que pode ser alcançado com a imobilização de enzimas. Como alternativa aos suportes sólidos amplamente estudados, está a quitosana, polímero que não apresenta toxicidade e possui alta biocompatibilidade e alta afinidade com proteínas. Outra possibilidade promissora na imobilização de enzimas, é a síntese dos agregados enzimáticos entrecruzados (CLEAs), os quais apresentam alta atividade catalítica e alta estabilidade. Contudo, uma peculiaridade das glucansucrases quando produzidas em meio contendo sacarose é a camada de polímero que as envolve, e que bloqueia o acesso aos grupos reativos na superfície da proteína. No caso da expressão heteróloga das glucansucrases em Escherichia coli essa dificuldade pode ser contornada. Além disso, o uso da mutagênese sítio-dirigida pode proporcionar modificações de aminoácidos na superfície da enzima, tais como os resíduos Lys, Cys, His, com o intuito de que melhorias na imobilização sejam alcançadas. Sendo assim, na primeira etapa desse trabalho, uma extensa discussão é apresentada em relação às metodologias de imobilização de dextransucrase encontradas na literatura. A seguir, estudos referentes à imobilização da dextransucrase de Leuconostoc mesenteroides B-512 F em esferas de quitosana ativadas com glutaraldeído foram realizados. Esse imobilizado apresentou alta atividade catalítica (197 U/g) quando utilizada a carga de proteína de 400 mg/g de suporte. Além disso, observou-se que a imobilização covalente e os açúcares maltose e glicose promoveram proteção à enzima em temperaturas de 40 ºC e 50 ºC. Na etapa seguinte, a produção e a caracterização de CLEAs de dextransucrase de L. mesenteroides B-512 F foram investigados. Demonstrou-se que o tratamento com a dextranase foi essencial para a imobilização da glucansucrase e que o isopropanol foi o melhor agente precipitante. Os CLEAs apresentaram pH e temperatura ótimos de 3,0 e 60 ºC, respectivamente, enquanto que a dextransucrase imobilizada nas esferas de quitosana funcionalizada com glutaraldeído apresentaram os valores de 4,5 e 20 ºC. Ambas formas imobilizadas apresentaram boa estabilidade operacional na síntese de oligossacarídeos uma vez que após 10 ciclos, 40 % de atividade residual foi observada. Por fim, estão apresentados estudos sobre a modelagem das estruturas tridimensionais e a mutagênese sítio-dirigida das glucansucrases DSR-S vardel Δ4N and ASR C-APY del. Os modelos preditos demonstraram boa qualidade e a mutagênese sítio-dirigida não promoveu perdas significativas na atividade enzimática dos mutantes. Somente o mutante DSR_S326C mostrouse inativo. Os resultados obtidos sugerem que a imobilização da dextransucrase foi satisfatória e que cada técnica possibilita diferentes características ao imobilizado. Além disso, os imobilizados foram adequados para síntese de dextrana e oligossacarídeos. / Glucansucrases are enzymes that catalyze the synthesis of polysaccharides and oligosaccharides. In order to assure continuous processing and reuse of the biocatalyst in industrial applications, enzyme immobilization techniques are required to promote good thermal and operational stabilities. Among the several solid supports for enzyme immobilization, chitosan shows interesting properties because it is non-toxic, it is biocompatible, and it has high protein affinity. Other possibility is the production of cross-linked enzyme aggregates (CLEAs), which presents high catalytic activity and good stability. However, glucansucrases have a particularity when produced in sucrose medium, since a polymer layer surrounds the protein, blocking the access to reactive groups on the enzyme surface. To overcome this problem, it is possible to make the heterologous production of glucansucrases in Escherichia coli. Likewise, the site-directed mutagenesis may promote changes in the amino acids located on the surface to improve immobilization parameters. Therefore, this work aimed to discuss the several techniques applied for dextransucrase immobilization, and to design new immobilized biocatalysts. In a first step, it is presented a review about the distinct immobilization methodologies for dextransucrase. In a second study, an investigation about dextransucrase from Leuconostoc mesenteroides B-512 F immobilized on glutaraldehyde-activated chitosan particles was carried out. The novel immobilized biocatalyst showed 197 U/g (400 mg/g dried support) of catalytic activity. The covalent immobilization promoted protection against enzyme damages at 40 ºC and 50 ºC, whereas maltose and glucose acted as stabilizers. Furthermore, it was studied the production and characterization of CLEAs dextransucrase from L. mesenteroides B-512 F. It was demonstrated that dextranase treatment was crucial for immobilization. Isopropanol was chosen as the best precipitant agent. CLEAs presented optimal pH and temperature of 3.0 and 60 ºC, respectively, whereas it was found values of 4.5 e 20 ºC for dextransucrase immobilized on glutaraldehyde-activated chitosan particles. Both immobilized biocatalysts showed good operational stability in the oligosaccharides synthesis, exhibiting 40 % of residual activity after 10 cycles. Finally, the study concerning the homology modeling and site-directed mutagenesis of glucansucrases DSR-S vardel Δ4N and ASR C-APY del is presented. The predicted models showed good quality and it has been demonstrated that the site-directed mutagenesis did not promote significant losses in the variant enzyme activities. Only one mutant (DSR_S326C) had shown no dextransucrase activity. The results obtained in this work suggest that the immobilization of dextransucrase was satisfactory, also showing that each technique promotes different characteristics to the immobilized biocatalyst. Besides, these immobilized enzymes were feasible for the synthesis of dextran and oligosaccharides.

No description available.

<p>Antibodies are proteins capable of specific interactions to a wide range of molecules. These interactions are facilitated by the complementary determining regions (CDR).</p><p>Carcinomas are the most common of human cancers and they release significant amount of cytokeratins (CK) in the necrotic areas of the tumors. The CKs stay in the tumor, since they have low solubility. The antibody studied in this thesis, the anti-CK 8 antibody TS1, has shown to be effective in tumor targeting and is proposed to be useful in therapy.</p><p>Single-chain antibodies (scFv) are recombinant antibodies which are much smaller than the intact IgG. This is an advantage when used in tumor therapy, since they can penetrate the tumors more easily than the larger IgG. Moreover, they are expressed by one single gene which make them easy to modify, for example by site-directed mutagenesis.</p><p>The anti-idiotypic antibody αTS1 can be used to clear the TS1 form the circulation and thereby clear the body from non-tumor bound TS1 in therapy. To be able to modify the binding of an antibody to its antigen and or anti-idiotype, these interactions must be studied. In this study this is accomplished by chemical modifications of the IgGs TS1 and αTS1 and the antigen CK 8. Guided by these results, amino acid residues were mutated by using site-directed mutagenesis in the TS1-218 scFv and the effects were studied. From mutational study results, the functional epitope could be mapped and it was found that there are mainly tyrosines, but also charged residues, serine and a tryptophan that are important for both interactions. The binding of TS1-218 to both αTS1 and CK 8 could be improved by changing the negatively charged side-chains by mutations to their corresponding amide or alanine.</p><p>Both the IgG and scFv versions of TS1 were administered in vivo. The IgG αTS1 was used to clear the TS1 from the circulation by forming immune complexes. The immune complexes, consisting of four or more antibodies, were mainly metabolized by the liver. The scFv TS1-218 could localize to the tumor in a tumor xenograft mouse model, although a higher uptake would be desired in a therapeutic strategy. The scFv was cleared rapidly by the kidneys, but the clearance could be slowed by pre-formed immune complexes with anti-TS1 scFv in vitro, prior to administration in vivo.</p>

Levels of the enzyme α-methylacyl-CoA racemase (AMACR) are increased ca. 9-fold in prostate cancer cells. AMACR is a very promising novel drug target as reducing AMACR levels converts castrate-resistant prostate cancer cells to androgen-dependent cells which will respond to androgen-deprivation. Despite the importance of AMACR in prostate and other cancers, there are very limited numbers of AMACR inhibitors described to-date. This is mainly due to the absence of a high-throughput assay for the screening of inhibitors against AMACR. The active-site residues and catalytic mechanism of human AMACR are still unknown, which make the rational design of drugs targeting AMACR very difficult. A range of novel potential inhibitors were synthesised using a rational drug design approach to explore the structure-activity relationship (SAR) on the side-chains of AMACR inhibitors. Their potencies were assessed using the fluoride elimination assay based on 1H and 19F NMR. Potency, mode of binding and kinetic parameters of these inhibitors were assessed using the multi-well colorimetric assay, which is the first AMACR high-throughput continuous assay reported to-date. A site-directed mutagenesis study was carried out to identify the active-site residues and catalytic mechanism of human AMACR. His-122, Asp-152, Met-184 and Glu-237 were identified as potential active-site residues, so the cDNA was mutated and expressed. The activity of wild-type and mutant AMACR enzymes were assessed using the deuterium wash-in, fluoride elimination and multi-well colorimetric assays. Results from these assays showed that human AMACR does not operate using a ‘two-base’ mechanism. Instead, it operates using a ‘one-base’ mechanism, most likely via water molecules acting as intermediaries within the hydrogen-bondings network in the active site. The knowledge obtained from this research informs rational drug design for this castrate-resistant prostate cancer target.

Les lipoxygénases (LOX, EC 1.13.11.12) sont des dioxygénases à fer non héminique très répandues. Chez les végétaux, ces enzymes sont à l’origine d’une voie métabolique impliquée dans de nombreux processus physiologiques, mais aussi dans la réponse à un stress environnemental. La LOX initie la voie en catalysant l’incorporation régiospécifique et stéréospécifique de dioxygène sur le système pentadiénique d’un acide gras libre polyinsaturé (préférentiellement l’acide linoléique ou l’acide linolénique) pour générer un hydroperoxyde d’acide gras.Une lipoxygénase d’olive appelée LOX1, clonée au laboratoire, a été exprimée chez E. coli et purifiée. Elle produit à partir d’acide linoléique des hydroperoxydes de configuration 9S et 13R dans des proportions 2:1. Elle est la seule lipoxygénase végétale décrite à ce jour produisant des hydroperoxydes de configuration R. Les modèles proposés pour expliquer le contrôle de la spécificité réactionnelle des LOX ne s’appliquent pas à la LOX1 d’olive. Afin de mieux comprendre son mécanisme de fonctionnement, un modèle tridimensionnel de la LOX1 d’olive a été construit. La modification par mutagénèse dirigée de deux résidus particuliers, la phénylalanine 277 et la tyrosine 280, a permis d’identifier l’entrée du site actif de la LOX1 d’olive. D’autres résidus particuliers ont été modifiés par mutagénèse dirigée afin d’étudier leur rôle dans le mécanisme catalytique et le contrôle de la spécificité réactionnelle de la LOX1 d’olive. L’analyse globale des résultats obtenus a permis de proposer une première hypothèse quant au fonctionnement de cette enzyme : le substrat pénètrerait dans le site actif de la LOX1 d’olive par son extrémité carboxylate, et serait stabilisé dans le site actif par plusieurs résidus hydrophobes. Un canal pourrait cibler l’oxygène dans le site actif par l’intermédiaire du résidu L579 sur le système pentadiénique du substrat, contrôlant de cette manière la spécificité réactionnelle de la LOX1 d’olive.Par ailleurs, des oxylipines retrouvées chez Arabidopsis, appelées arabidopsides, pourraient être formées par action directe d’une 13-LOX sur des acides gras estérifiés des galactolipides. L’action de la 13-LOX1 de soja, la 9/13-LOX1 d’olive et la 9-LOX de pomme de terre a été testée avec des galactolipides. Une faible activité a été mesurée avec la 13-LOX1 de soja et la 9/13-LOX1 d’olive. Une activité plus importante a été mesurée avec la 9-LOX de pomme de terre. Ces résultats suggèrent que l’action des LOX est possible sur des acides gras estérifiés des galactolipides. / Lipoxygenases (LOXs, EC 1.13.11.12) are widespread dioxygenases containing a non heminic iron atom. In plants, LOXs are at the beginning of a metabolic pathway involved in several physiological processes and in the response to environmental stress. A LOX initiates the pathway, catalyzing a regiospecific and stereospecific insertion of oxygen on the pentadiene system of a free polyunsaturated fatty acid (linoleic or linolenic acid) to form fatty acid hydroperoxides.An olive lipoxygenase called olive LOX1, cloned at laboratory, has been expressed in E. coli strain and purified. Olive LOX1 produces 9S-hydroperoxides of and 13R-hydroperoxides from linoleic acid, in a ratio of 2:1, being the only plant LOX to produce R-hydroperoxides described to date. From the currently known models explaining the control of reactional specificity, none can be applied to olive LOX1. A three-dimensional model has been built by homology modeling to understand the catalytic mechanism of olive LOX1. Site-directed mutagenesis experiments have been used to modify two residues of particular interest, the phenylalanine 277 and the tyrosine 280, allowing us to point the active site entrance near these two residues. Other residues of interest have been modified to study their role in the catalytic mechanism and the reactional specificity of olive LOX1. The results have led us to propose a first hypothesis for the reactional mechanism of this enzyme: the substrate could enter into the active site with its carboxylate-end first, and could be stabilized in the active site by hydrophobic side chains of several residues. A channel could bring oxygen into the active site at a position near the side chain of the leucine 579 residue, this one targeting oxygen onto the pentadiene system of the substrate, controlling by this way the reactional specificity of olive LOX1.LOX are involved in oxylipins synthesis. Arabidopsides are a class of oxylipins found in Arabidopsis that could be produced by action of a 13-LOX on galactolipids, which carry esterified fatty acids. Activity of soybean 13-LOX, olive 9/13-LOX1 and potato 9-LOX has been investigated with galactolipids. A low activity was measured when soybean and olive LOXs were used. Activity was far more important when potato LOX was used. These results suggest that LOX can act on esterified fatty acids, especially galactolipids.

Nesse projeto trabalhamos com uma &#946;-glicosidase digestiva da larva da lagarta Spodoptera frugiperda (Sf&#946;gli50, 50 kD - AF052729), expressa na forma de proteína recombinante em E.colli. O nosso objetivo foi estudar o papel de dois resíduos de aminoácidos envolvidos na atividade catalítica da Sf&#946;gli50. O primeiro resíduo estudado foi o Y456, envolvido na afinidade pela porção redutora do substrato (aglicone), o segundo resíduo foi o N329 envolvido na modulação do pH ótimo. Estudo do papel do resíduo Y456 na afinidade pelo aglicone do substrato. O sítio-ativo da Sf&#946;gli50 é formado por quatros subsítios (-1, +1, +2, e +3). O subsítio que acomoda a porção não-redutora do substrato (glicone) recebe numeração negativa (-1), já os subsítios que acomodam a porção redutora do substrato (aglicone) recebem números positivos (+1, +2 e +3). Trabalhando com duas &#946;-glicosidases de plantas (milho e sorgo), Cicek et al. (2000) demonstraram que uma pequena porção da extremidade C-terminal destas &#946;-glicosidases (462SSGYTERF469 - numeração da enzima do sorgo) está envolvida na especificidade pelo aglicone do substrato, sendo que muitos desses aminoácidos são conservados em outras &#946;-glicosidases da família 1. O alinhamento das sequências destas duas enzimas com a Sf&#946;gli50 sugere que Y456 pode fazer parte do sítio de ligação do aglicone nesta &#946;-glicosidase de inseto. Utilizando experimentos de mutação sítio-dirigida, o Y456 foi substituído por uma alanina (mutante Y456A) sendo que este foi expresso na forma de proteína recombinante em bactérias BL21 DE3 utilizando o vetor pT7-7. O mutante Y456A foi parcialmente purificado através de uma cromatografia hidrofóbica em sistema de FPLC, e caracterizado utilizando diversos inibidores competitivos (glucono &#948;-lactona, celobiose, celotriose, pentilbglicosídeo e octilbtioglicosídeo). Comparando os Kis obtidos para a Sf&#946;gli50 selvagem e mutante Y456A com os inibidores glucono &#948;-lactona, celobiose e celotriose, foi proposto que Y456 encontra-se no subsítio +1 do sítio ativo da Sf&#946;gli50. Já através da comparação entre os inibidores octil&#946;tioglicosídeo e pentil&#946;glicosídeos constatou-se que Y456 interage com uma porção polar do aglicone do substrato, talvez através de uma ligação de hidrogênio. Baseando-se nestes Kis foi calculada a energia de associação de resíduos de glicose e grupos alquila nos subsítios +1 e +2, indicando que o subsítio +1 do mutante Y456A tem uma especificidade mais ampla frente à ligantes polares (glicose) e apolares (grupos butil) do que a enzima selvagem. Sabendo que este resultado foi obtido removendo-se um resíduo com um grupo polar na cadeia lateral (Y456), estes dados estão de acordo com a hipótese de que a especificidade dos subsítios da região de ligação do aglicone é determinada por um balanço entre resíduos polares e apolares (Marana et al., 2001). Estudo do papel do resíduo N329 na modulação do pH ótimo. O mecanismo de catálise da Sf&#946;gli50 é dependente de dois resíduos de ácido glutâmico: um doador de prótons (E187 - pKa= 7,5) e um nucleófilo (E399 - pKa = 5,0). Sendo o pH ótimo da Sf&#946;gli50 (6,2) uma média aritmética dos pKas destes dois resíduos catalíticos. Uma análise estrutural do sítio ativo da Sf&#946;gli50 mostra que o resíduo N329 forma ligações de hidrogênio com o resíduo E187 (doador de prótons), talvez atuando na modulação do seu pKa. Para estudar o papel do resíduo N329 na atividade da Sf&#946;gli50 foram construídos 3 mutantes, nos quais tal resíduo foi substituído por alanina (N329A), ácido aspártico (N329D) e uma glutamina (N329Q). Os mutantes foram expressos na forma de proteína recombinante em bactérias BL21 DE3 utilizando os vetores pT7-7 e pCal-n-Flag. Entretanto, tentativas de purificação das Sf&#914;gli50 mutantes através de cromatografia hidrofóbica foram infrutíferas, sugerindo uma possível inativação destas enzimas. Esta hipótese foi reforçada pela purificação das Sf&#946;gli50 mutantes e selvagem contendo o peptídeo de fusão CBP (calmodulin binding peptide) através de cromatografia de afinidade. Este experimento demonstrou que as enzimas mutantes eram de fato inativas. Frente à estes resultados não foi possível concluir a caracterização do efeito do pH na atividade catalítica das Sf&#946;gli50 mutantes N329A, N329D e N329Q. Por fim, foi proposto que a inativação da Sf&#946;gli50 devido à mutações na posição N329 pode resultar de uma desnaturação das enzimas mutantes ou do reposicionamento do ácido catalítico devido à perda ou alteração da interação com o resíduo 329. / In this project it was studied the role of two residues (N329 and Y456) in the catalytic activity of a digestive &#946;-glycosidase from Spodoptera frugiperda (Sf&#914;gli50 - AF052729). N329 is believed to modulate the enzyme pH optimum, whereas Y456 may participate in the binding of the substrate aglycone. Role of Y456 The peptide 462SSGYTERF469 of the sorghum &#946;-glycosidase is proposed to be part of the aglycone binding site in that enzyme. Some of those residues are conserved in Sf&#946;gli50, among them Y456. Using site-directed mutagenesis Y456 was replaced by A and this mutant (Y456A) expressed in bacteria. Following that, this mutant enzyme was partially purified using hydrophobic chromatography. Inhibition experiments showed that binding of &#948;-gluconolactone, which occupies subsite -1, is not affected by that mutation. In contrast, Ki values for cellobiose (that binds to subsites -1 and +1) and cellotriose (that binds to subsites -1, +1 and +2) are two-fold higher than those of wild-type enzyme, indicating that mutation Y456A decrease the interaction with these oligocellodextrins. Moreover, binding of pentyl and octyl&#946;glucosides is not affected by mutation Y456A, suggesting that Y456 interacts with aglycone polar groups. Finally, evaluation of glucose and butyl binding energies in subsite +1 revealed that mutant Y456A specificity is broader than that of wild-type enzyme. Role of N329 A structural model of Sf&#946;gli50 active site revealed that catalytic proton donor (E187) may interact with N329. In order to study the role of this interaction in the activity of Sf&#946;gli50, N329 was replaced by A, D and Q (mutants N329A, N329D and N329Q, respectively). These mutants were expressed as recombinant proteins in bacteria and purified through affinity chromatography, revealing that Sf&#946;gli50 was inactivated by those mutations. It was proposed that this inactivation may be due to protein desnaturation or a wrong positioning of the catalytic proton donor.

Resistance to chemotherapeutic drugs is a major obstacle in the treatment of leukemia and multiple myeloma. We have previously found that myeloma and leukemic cells in transition from low-density log phase conditions to high-density plateau phase conditions exhibit a substantial export of endogenous topoisomerase II alpha from the nucleus to the cytoplasm. In order for topoisomerase-targeted chemotherapy to function, the topoisomerase target must have access to the nuclear DNA. Therefore, the nuclear export of topoisomerase II alpha may contribute to drug resistance, and defining this mechanism may lead to methods to preclude this avenue of resistance. In the current report, we have defined nuclear export signals for topoisomerase II alpha at amino acids 1017-1028 and 1054-1066, using FITC labeled BSA-export signal peptide conjugates microinjected into the nuclei of HeLa cells. Functional confirmation of both signals (1017-1028 and 1054-1066) was provided by transfection of human myeloma cells with plasmids containing the gene for a full-length human FLAG-topoisomerase fusion protein, mutated at hydrophobic amino acid residues in the export signals. Of the six putative export signals tested, the two sites above were found to induce export into the cytoplasm. Export by both signals was blocked by treatment of the cells with leptomycin B, indicating that a CRM-1 dependent pathway mediates export. Site-directed mutagenesis of two central hydrophobic residues in either export signal in full-length human topoisomerase blocked export of recombinant FLAG-topoisomerase II alpha, indicating that both signals may be required for export. Interestingly, this pair of nuclear export signals (1017-1028 and 1054-1066) also defines a dimerization domain of the topoisomerase II alpha molecule.