Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A Bothrops leucurus (jararaca do rabo branco) à uma serpente peÃonhenta que habita a regiÃo Nordeste do Brasil. Os efeitos biolÃgicos devido ao envenenamento por B. leucurus tÃm perfil semelhante Ãqueles apresentados por outras serpentes do gÃnero Bothrops, tais como, importantes efeitos locais e sistÃmicos graves como a InsuficiÃncia Renal Aguda (IRA). O veneno de Bothrops leucurus induziu nefrotoxicidade em sistema de perfusÃo de rim isolado de rato, associado com citotoxicidade em cÃlulas tubulares epiteliais renais. Neste trabalho foi avaliado o efeito nefrotÃxico direto da enzima L-aminoÃcido oxidase isolada do veneno de Bothrops leucurus (LAAO-Bl) sobre cÃlulas epiteliais renais (MDCK e HK2) e o seu potencial nefrotÃxico em rim isolado de rato. O tratamento com LAAO-Bl, 1.56 â 100 Âg/mL induziu significativa morte celular de maneira concentraÃÃo dependente em ambas Ãs linhagens celulares apÃs 12 horas de tratamento. Nas cÃlulas MDCK nÃo foi observada liberaÃÃo de LDH apÃs 12 horas de exposiÃÃo à LAAO-Bl, enquanto nas cÃlulas HK2 LAAO-Bl induziu ruptura de membrana nas maiores concentraÃÃes estudadas quando comparado ao controle nÃo tratado. Nas cÃlulas MDCK o tratamento com LAAO-Bl aumentou significativamente a porcentagem de cÃlulas em apoptose (Anexina-V+, IP-), necrose (Anexina-V-, IP+) e necrose secundÃria (Anexina-V+, IP+). Nas cÃlulas HK2 LAAO-Bl induziu um aumento na porcentagem de cÃlulas em necrose (IP+) e necrose secundÃria (Anexina-V+, IP+) de maneira concentraÃÃo dependente. A induÃÃo de apoptose nas cÃlulas MDCK foi acompanhada de liberaÃÃo de Ca2+ do retÃculo endoplasmÃtico, aumento de espÃcies reativas de oxigÃnio, disfunÃÃo mitocondrial e aumento de expressÃo de Bax. O tratamento com LAAO-Bl induziu ativaÃÃo de caspase 3 e 7 em ambas as linhagens celulares. LAAO-Bl (10 Âg/mL) exerce efeitos significativos no rim isolado de rato aumentando a pressÃo de perfusÃo e o fluxo urinÃrio e diminuindo a taxa de filtraÃÃo glomerular e os transportes tubulares de sÃdio, potÃssio e cloreto. Os nossos resultados sugerem que LAAO-Bl contribui para nefrotoxicidade observada no envenenamento por Bothorps leucurus. AlÃm disso, os efeitos citotÃxicos de LAAO-Bl nas cÃlulas epiteliais renais podem ser responsÃveis, pelo menos em parte, pela nefrotoxicidade observada no rim isolado. / The pit viper Bothrops leucurus (White-tailed-jararaca) is a poisonous snake habituating area in the northeast of Brazil. The biological effects due envenomation have similar profile than those observed with other Bothrops, such as important severe local and systemic effects such as Acute Renal Failure (ARF). Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney of rats associated with cytotoxicity against renal tubular epithelia cells. In this study, it was evaluated the direct nefrotoxicity of L-aminoacid oxidase isolated of B. leucurus venom (LAAO-Bl) on renal epithelial cells (MDCK and HK2) and their potential nephrotoxic in isolated rat kidney. In these cells treated with LAAO-Bl, 1.56 â 100 Âg/mL for 12 h, there was a decrease in their viability in a concentration-dependent manner after 12 hours of treatment. In MDCK cells LDH release was not observed after 12 h of LAAO-Bl exposure while LAAO-Bl induced membrane rupture in HK-2 cells at the highest concentrations studied when compared with untreated cells. In MDCK cells, LAAO-Bl significantly increased the percentage of early apoptotic (Annexin-V+, PI-), necrotic (Annexin-V-, PI+) and secondary necrotic cells (Annexin-V+, PI+) when compared with control untreated cells. In HK-2 cells LAAO-Bl induced an increase in necrotic (PI+ cells) and secondary necrotic cells (Annexin-V+, PI+) in a concentration-dependent manner. MDCK apoptosis induction was accompanied with Ca2+ release from the endoplasmic reticulum, reactive oxygen species (ROS) generation, mitochondria dysfunction with enhanced expression of Bax protein levels. LAAO-Bl induced caspase-3 and caspase-7 activation in both cell lines. LAAO-Bl (10 Âg/mL) exerts significant effects on the isolated kidney perfusion increasing perfusion pressure and urinary flow and decreasing the glomerular filtration rate and sodium, potassium and chloride tubular transport. Taken together our results suggest that LAAO-Bl contributes for the nephrotoxicity observed in the envenomation by Bothrops leucurus. Moreover, the cytotoxic of LAAO-Bl to renal epithelial cells might be responsible, least in part for the nephrotoxicity observed in isolated kidney.

Neste trabalho descrevemos o isolamento e a caracterização bioquímica, estrutural e funcional de uma L-aminoácido oxidase isolada do veneno da serpente Bothrops jararacussu, proteína esta denominada BjussuLAAO. O isolamento foi realizado pela combinação de três etapas cromatográficas, utilizando filtração molecular em Sephadex G-75, seguida de cromatografia de afinidade em Benzamidina-Sepharose, seguida pela última etapa cromatográfica, realizada com interação hidrofóbica em Fenil-Sepharose. A BjussuLAAO é uma proteína com massa molecular de 61 kDa e pI 5,8, e apresentou alta similaridade sequencial com as LAAOs já isoladas dos venenos de serpentes. A enzima BjussuLAAO induziu edema apenas na maior dose (100 ?g/animal), não apresentou atividade miotóxica, também não apresentou atividade hemorrágica, mesmo nas doses mais elevadas. A BjussuLAAO, demonstrou ter efeito coagulante e ação fibrinogenolítica e, mesmo utilizando inibidores de metaloproteases e serino proteases, continuou apresentando tal atividade, demonstrando assim não existir contaminantes destas classes de proteínas na amostra. As atividades mais interessantes do ponto de vista farmacológico, onde podemos visualizar a BjussuLAAO como um futuro fármaco, foram as atividades antitumoral, bactericida e antiparasitária. Esta apresentou, em baixas concentrações, citotoxicidade sobre células tumorais, ação leishmanicida e tripanocida, demonstrando assim ser uma substância candidata a fármaco multifuncional. / In this research we described the isolation and biochemical, structural and functional characterization of an L-amino acid oxidase from Bothrops jararacussu venom, named BjussuLAAO. The purification was carried out through three chromatography steps, using a molecular filtration on Sephadex G-75, followed by an affinity chromatography on Benzamidine-Sepharose, and finally a hydrophobic chromatography on Phenyl-Sepharose. BjussuLAAO is a protein with a molecular mass of 61 kDa and pI 5.8. This protein showed high sequencial similarity with other LAAOs from snake venoms. BjussuLAAO induced edema only in the highest dose (100 ?g/animal), did not show any myotoxic or hemorrhagic activity. BjussuLAAO, showed clotting and fibrinogenolitic activity, even in the presence of metalloproteases and serino-proteases inhibitors. The more interesting pharmacological activities, where from we can look a future application, were the anti-tumoral, bactericide, leishmanicidal and tripanocidal actions. The enzyme displayed a bactericide effect too, showing to be a multifunctional drug.

Neste trabalho descrevemos o isolamento e a caracterização bioquímica, estrutural e funcional de uma L-aminoácido oxidase isolada do veneno da serpente Bothrops jararacussu, proteína esta denominada BjussuLAAO. O isolamento foi realizado pela combinação de três etapas cromatográficas, utilizando filtração molecular em Sephadex G-75, seguida de cromatografia de afinidade em Benzamidina-Sepharose, seguida pela última etapa cromatográfica, realizada com interação hidrofóbica em Fenil-Sepharose. A BjussuLAAO é uma proteína com massa molecular de 61 kDa e pI 5,8, e apresentou alta similaridade sequencial com as LAAOs já isoladas dos venenos de serpentes. A enzima BjussuLAAO induziu edema apenas na maior dose (100 ?g/animal), não apresentou atividade miotóxica, também não apresentou atividade hemorrágica, mesmo nas doses mais elevadas. A BjussuLAAO, demonstrou ter efeito coagulante e ação fibrinogenolítica e, mesmo utilizando inibidores de metaloproteases e serino proteases, continuou apresentando tal atividade, demonstrando assim não existir contaminantes destas classes de proteínas na amostra. As atividades mais interessantes do ponto de vista farmacológico, onde podemos visualizar a BjussuLAAO como um futuro fármaco, foram as atividades antitumoral, bactericida e antiparasitária. Esta apresentou, em baixas concentrações, citotoxicidade sobre células tumorais, ação leishmanicida e tripanocida, demonstrando assim ser uma substância candidata a fármaco multifuncional. / In this research we described the isolation and biochemical, structural and functional characterization of an L-amino acid oxidase from Bothrops jararacussu venom, named BjussuLAAO. The purification was carried out through three chromatography steps, using a molecular filtration on Sephadex G-75, followed by an affinity chromatography on Benzamidine-Sepharose, and finally a hydrophobic chromatography on Phenyl-Sepharose. BjussuLAAO is a protein with a molecular mass of 61 kDa and pI 5.8. This protein showed high sequencial similarity with other LAAOs from snake venoms. BjussuLAAO induced edema only in the highest dose (100 ?g/animal), did not show any myotoxic or hemorrhagic activity. BjussuLAAO, showed clotting and fibrinogenolitic activity, even in the presence of metalloproteases and serino-proteases inhibitors. The more interesting pharmacological activities, where from we can look a future application, were the anti-tumoral, bactericide, leishmanicidal and tripanocidal actions. The enzyme displayed a bactericide effect too, showing to be a multifunctional drug.

La transcétolase thermostable de Geobacillus stearothermophilus (TKgst, EC 2.2.1.1) permet de synthétiser efficacement à haute température des cétoses à 4, 5 et 6 atomes de carbone de configuration d-thréo (3S, 4R), par formation stéréosélective d’une liaison C-C, à partir d’aldéhydes α-hydroxylés (2R) à courte chaîne. L’objectif de ces travaux est d’utiliser la TKgst à 60°C pour gagner en efficacité et étendre son spectre de substrats à de nouveaux donneurs et accepteurs par Evolution dirigée, selon une approche semi-rationnelle, basée sur la mutagenèse par saturation de site. Ainsi, à l’issue du criblage des banques générées, les TKgst mutées les plus performantes (L382D/D470S, L191I, L382F/F435Y, R521Y/H462N et R521V/S385D/H462S) ont été sélectionnées pour leurs activités spécifiques supérieures à celle de la TKgst sauvage (gain de 3,3 à 5) vis-à-vis d’aldéhydes α-hydroxylés (2S) et d’aldéhydes α-hydroxylés (2R) à longue chaîne polyhydroxylée (C5-C6). La TKgst sauvage, ainsi que ces TKgst mutées performantes ont permis d’obtenir, à 60°C, onze cétoses, dont neuf de configuration l-érythro (3S, 4S) à 5 à 6 atomes de carbone et de configuration d-thréo (3S, 4R) de 4 à 8 atomes de carbone d’intérêt biologique, avec de très bons rendements, quatre étant inaccessibles avec les TKs microbiennes utilisées jusqu’alors. D’autres TKgst mutées ont par ailleurs conduit à une amélioration significative de l’activité de la TKgst vis-à-vis d’aldéhydes aliphatiques et aromatiques, mais également vis-à-vis d’un nouveau substrat donneur, l’acide pyruvique et d’analogues, ouvrant le champs des applications aux 1-désoxycétoses. De plus, ces travaux ont permis de développer un procédé multi-enzymatique innovant et éco-comptatible, dans lequel les substrats donneurs et accepteurs de la TKgst sont générés par voie enzymatique, via l’utilisation d’une transaminase ou d’une d-aminoacide oxydase et d’une aldolase, à partir de composés naturels et peu coûteux. Cette stratégie pourra être appliquée aux TKgst mutées, afin d’accéder efficacement et à moindre coût, à d’autres cétoses rares hautement valorisables. / Thermostable transketolase from Geobacillus stearothermophilus (TKgst, EC 2.2.1.1) catalyzes efficiently the synthesis of d-threo (3S, 4R) ketoses having 4, 5 and 6 carbon atoms, by the stereoselective formation of a new C-C bond, from short chain (2R)-α-hydroxylated aldehydes. The aim of this work is to use TKgst at 60°C, in order to increase reaction rates and to broad its substrate scope to new donors and acceptors by Directed Evolution, according to a semi-rationnal approach, based on site saturation mutagenesis. Thus, the screening of the libraries led to TKgst variants (L382D/D470S, L191I, L382F/F435Y, R521Y/H462N and R521V/S385D/H462S) having significantly improved specific activities towards (2S)-α-hydroxylated aldehydes and (2R)-α-hydroxylated aldehydes having a long polyhydroxylated chain (C5-C6), compared to wild type TKgst (3,3 to 5-fold increased activity). Wild-type TKgst as well as these TKgst variants were used, at 60°C, to obtain eleven, including nine l-erythro (3S, 4S) ketoses having 5 and 6 carbon atoms and d-threo (3S, 4R) ketoses having from 4 to 8 carbon atoms of biological interest, with good yields, four being inaccessible using common TK sources. Besides, other TKgst variants led to significantly improved activities towards hydrophobic aldehydes and towards a new donor substrate, pyruvic acid and derivatives, extending TKgst product scope to 1-deoxyketoses. In addition, a multienzymatic innovative and environmentally friendly process, in which TKgst substrates are generated through enzymatic pathways, using a transaminase or a d-aminoacid oxidase and an aldolase, from non-expensive and natural compounds was developed, in the presence of wild-type TKgst and will be able to be applied to TKgst variants, in order to synthesize efficiently and at lower cost, other highly valuable rare ketoses.