Ca2+, Mg2+ and pH integrate novel regulatory mechanisms of structure and function of mitochondrial peroxiredoxins from Leishmania contributing to parasite survival

Orientador: Mário Tyago MurakamiTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Peroxirredoxinas (Prxs) 2-Cys da subfamília Prx1 são peroxidases, com duas cisteínas catalíticas, que regulam os níveis intracelulares de H2O2, além de estarem envolvidas na sinalização celular e de assumirem atividade chaperona em certas condições. Todas as suas funções são reguladas por mudanças na estrutura quaternária induzidas por múltiplos fatores, tais como estado redox, modificações pós-traducionais e pH. Entretanto, as bases moleculares que regulam esses fenômenos ainda não são totalmente compreendidas. As Prxs mitocondriais (Prx1m) de Leishmania, por exemplo, exibem a dupla função de peroxidases e chaperonas moleculares, porém somente a última é crucial para a sobrevivência do parasita no hospedeiro humano. Estudos recentes in vitro mostraram que, quando decamérica e reduzida, Prx1m de Leishmania infantum constitui uma chaperona ativa, enquanto dímeros oxidados são inativos. Neste trabalho foram resolvidas as estruturas cristalográficas da Prx1m de Leishmania braziliensis (LbPrx1m) nas formas dimérica e decamérica, mostrando que alterações conformacionais na região I do loop catalítico estão associadas ao processo de decamerização. Pela primeira vez, foram obtidos dados estruturais de um membro da subfamília Prx1 em dois estados oligoméricos, induzidos por mudanças de pH. Com base nesses dados, desvendamos o mecanismo molecular pelo qual o pH modula a conversão entre dímeros e decâmeros, o qual parece ser fisiologicamente relevante e altamente conservado na subfamília Prx1. Além disso, foi demonstrado que os cátions divalentes Ca2+ e Mg2+ - os quais desempenham papéis importantes no metabolismo na mitocôndria - ativam a função chaperona de Prx1m e estimulam a atividade peroxidase, através de um mecanismo de estabilização de decâmeros inédito e exclusivo de Prxs mitocondriais de Leishmania. Utilizando um mutante dimérico constitutivo de LbPrx1m, cuja oligomerização não é influenciada pelo pH, cátions divalentes ou estado redox, demonstramos que a formação de decâmeros é um pré-requisito não somente para a função peroxidase, mas também para a função chaperona de Prx1m, sendo necessária para conferir resistência à Leishmania infantum frente ao estresse térmico sofrido durante a transição do inseto para o hospedeiro humano. Em conjunto, nossos resultados mostram que concentrações basais de Ca2+ e Mg2+ encontrados na matriz mitocondrial dão suporte à função dupla de Prx1m em Leishmania, enquanto pequenas quedas de pH e altos níveis de Ca2+ podem aumentar o reservatório mitocondrial de chaperonas de Prx1m. Esses dados implicam que a busca de compostos que previnam a decamerização da Prx1m de Leishmania representa uma boa estratégia para inibir a função chaperona desse atrativo alvo terapêuticoAbstract: 2-Cys peroxiredoxins (Prxs) from Prx1 subfamily are Cys-based peroxidases that control intracellular levels of H2O2 besides being involved in cell signaling pathways and assuming chaperone function under specific conditions. The regulation of their functions involves changes in their quaternary structure which are induced by multiple factors, such as redox state, posttranslational modifications and pH. However, there is still a lack of information about the molecular basis for these phenomena. The mitochondrial 2-Cys peroxiredoxin (Prx1m) from Leishmania, for example, exhibits dual peroxidase and chaperone functions, but only the latter is crucial for the parasite survival in the mammalian host. Recent in vitro studies indicated that reduced Leishmania infantum Prx1m forms chaperone-active decamers, whereas the oxidized protein dissociates into chaperone-inactive dimers. Herein, we solved the crystal structures of Leishmania braziliensis Prx1m (LbPrx1m) in both dimeric and decameric forms, showing that conformational changes of the region I from the catalytic loop are associated with the decamerization process. We provided, for the first time to our knowledge, structural data of a Prx1 member in two different oligomeric states induced by pH variations. Based on these data, we unveiled the molecular mechanism of pH modulation in dimers/decamers conversion. This mechanism might be extended to other peroxiredoxins and seems to be physiologically relevant. Moreover, we demonstrated that the divalent cations Ca2+ and Mg2+ - which play key roles in mitochondrial metabolism ¿ activate the chaperone function of oxidized Prx1m and stimulate the catalytic activity via a novel and exclusive mechanism of decamer stabilization of mitochondrial Prxs from Leshmania. Using a dimeric mutant of LbPrx1m unable to decamerize by pH, cation influence, or redox state, we also demonstrated that decamer formation is a pre-requisite not only for the peroxidase function of Prx1m but also for the chaperone activity, being essential to provide to Leishmania infantum resistance against heat stress during the transition from insect to the mammalian hosts. Together, our results showed that basal concentrations of Ca2+ and Mg2+, found in the mitochondrial matrix, support the dual function of Prx1m from Leishmania, whereas small decreases in pH or increased Ca2+ levels regulate the mitochondrial reservoir of chaperone-active Prx1m. Our findings imply that the search for compounds that prevent Leishmania Prx1m decamerization represents a promising strategy to inhibit the crucial chaperone function of this attractive therapeutic targetDoutoradoBioquimicaDoutora em Biologia Funcional e Molecular2012/24134-3, 2015/05851-4FAPES

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the mitochondrial tryparedoxin peroxidase from Leishmania braziliensis

Submitted by Luciane Willcox ([email protected]) on 2016-09-02T17:49:47Z No. of bitstreams: 1 Cloning, expression, purification, crystallization.pdf: 350581 bytes, checksum: 0d688db5291c27edd0bfbdaf3b7372c5 (MD5)Approved for entry into archive by Luciane Willcox ([email protected]) on 2016-09-02T17:57:41Z (GMT) No. of bitstreams: 1 Cloning, expression, purification, crystallization.pdf: 350581 bytes, checksum: 0d688db5291c27edd0bfbdaf3b7372c5 (MD5)Made available in DSpace on 2016-09-02T17:57:41Z (GMT). No. of bitstreams: 1 Cloning, expression, purification, crystallization.pdf: 350581 bytes, checksum: 0d688db5291c27edd0bfbdaf3b7372c5 (MD5) Previous issue date: 2013-03-28This research was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Biociências. Campinas, SP, Brasil. / Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Luz Síncrotron. Campinas, SP, Brasil.Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Biociências. Campinas, SP, Brasil. / Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Luz Síncrotron. Campinas, SP, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Biociências. Campinas, SP, Brasil. / Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Luz Síncrotron. Campinas, SP, Brasil.Tryparedoxin peroxidase (TXNPx) is an essential constituent of the main enzymatic scavenger system for reactive oxygen species (ROS) in trypanosomatids. Genetic studies have demonstrated the importance of this system for the development and virulence of these parasites, representing a potential target for the discovery of new trypanocidal drugs. In this work, the mitochondrial TXNPx from Leishmania braziliensis was cloned, overexpressed, purified and crystallized. The crystals diffracted to 3.3 Å resolution and belonged to space group P4(2)2(1)2, with unit-cell parameters a = b = 131.8, c = 44.4 Å. These studies will contribute to a better understanding of the molecular mechanisms involved in ROS detoxification by trypanosomatids

The mechanism by which a distinguishing arabinofuranosidase can cope with internal di-substitutions in arabinoxylans

Abstract Background Arabinoxylan is an abundant polysaccharide in industrially relevant biomasses such as sugarcane, corn stover and grasses. However, the arabinofuranosyl di-substitutions that decorate the xylan backbone are recalcitrant to most known arabinofuranosidases (Abfs). Results In this work, we identified a novel GH51 Abf (XacAbf51) that forms trimers in solution and can cope efficiently with both mono- and di-substitutions at terminal or internal xylopyranosyl units of arabinoxylan. Using mass spectrometry, the kinetic parameters of the hydrolysis of 33-α-l-arabinofuranosyl-xylotetraose and 23,33-di-α-l-arabinofuranosyl-xylotetraose by XacAbf51 were determined, demonstrating the capacity of this enzyme to cleave arabinofuranosyl linkages of internal mono- and di-substituted xylopyranosyl units. Complementation studies of fungal enzyme cocktails with XacAbf51 revealed an increase of up to 20% in the release of reducing sugars from pretreated sugarcane bagasse, showing the biotechnological potential of a generalist GH51 in biomass saccharification. To elucidate the structural basis for the recognition of internal di-substitutions, the crystal structure of XacAbf51 was determined unveiling the existence of a pocket strategically arranged near to the − 1 subsite that can accommodate a second arabinofuranosyl decoration, a feature not described for any other GH51 Abf structurally characterized so far. Conclusions In summary, this study reports the first kinetic characterization of internal di-substitution release by a GH51 Abf, provides the structural basis for this activity and reveals a promising candidate for industrial processes involving plant cell wall depolymerization

NEOTROPICAL ALIEN MAMMALS: a data set of occurrence and abundance of alien mammals in the Neotropics

Biological invasion is one of the main threats to native biodiversity. For a species to become invasive, it must be voluntarily or involuntarily introduced by humans into a nonnative habitat. Mammals were among first taxa to be introduced worldwide for game, meat, and labor, yet the number of species introduced in the Neotropics remains unknown. In this data set, we make available occurrence and abundance data on mammal species that (1) transposed a geographical barrier and (2) were voluntarily or involuntarily introduced by humans into the Neotropics. Our data set is composed of 73,738 historical and current georeferenced records on alien mammal species of which around 96% correspond to occurrence data on 77 species belonging to eight orders and 26 families. Data cover 26 continental countries in the Neotropics, ranging from Mexico and its frontier regions (southern Florida and coastal-central Florida in the southeast United States) to Argentina, Paraguay, Chile, and Uruguay, and the 13 countries of Caribbean islands. Our data set also includes neotropical species (e.g., Callithrix sp., Myocastor coypus, Nasua nasua) considered alien in particular areas of Neotropics. The most numerous species in terms of records are from Bos sp. (n = 37,782), Sus scrofa (n = 6,730), and Canis familiaris (n = 10,084); 17 species were represented by only one record (e.g., Syncerus caffer, Cervus timorensis, Cervus unicolor, Canis latrans). Primates have the highest number of species in the data set (n = 20 species), partly because of uncertainties regarding taxonomic identification of the genera Callithrix, which includes the species Callithrix aurita, Callithrix flaviceps, Callithrix geoffroyi, Callithrix jacchus, Callithrix kuhlii, Callithrix penicillata, and their hybrids. This unique data set will be a valuable source of information on invasion risk assessments, biodiversity redistribution and conservation-related research. There are no copyright restrictions. Please cite this data paper when using the data in publications. We also request that researchers and teachers inform us on how they are using the data