A leishmaniose é uma doença tropical negligenciada que afeta milhares de pessoas podendo até levar a óbito em sua forma visceral. Durante o seu ciclo de vida, o parasita passa por diversas mudanças ambientais como mudança de temperatura e pH, principalmente quando da transfecção do inseto vetor para o hospedeiro mamífero. Tais mudanças geram estresse celular que pode levar proteínas ao enovelamento incorreto assim como a processos agregativos, sendo necessários sistemas de controle de qualidade proteico para manter a homeostase celular, do qual fazem parte as chaperonas moleculares. Chaperonas como a Hsp100, ajudam a manter a homeostase celular e a adaptação desempenhando um papel importante para protozoários como a Leishmania braziliensis, causador da leishmaniose. A Hsp100 tem papel desagregase, atuando com outras chaperonas moleculares para a extração de polipeptídios de agregados proteicos possibilitando seu desenovelamento e posterior reenovelamento, evitando seu efeito tóxico sobre a célula. A Hsp100 parece ser essencial para esses microrganismos, no entanto não há muito dados disponíveis para Hsp100 em Leishmania sp. e Plasmodium sp. Neste trabalho está descrito o protocolo para expressão e purificação da Hsp100 recombinante de L. braziliensis (rLbHsp100), assim como sua caracterização estrutural e funcional inicial in vitro. A proteína foi analisada por espectropolarimetria de dicroísmo circular, apresentando estrutura típica de proteínas ricas em hélices α, a fluorescência estática de triptofano demonstrou que a proteína possui estrutura terciária local com seus triptofanos parcialmente expostos ao solvente. Por cromatografia de exclusão molecular analítica, observou-se que a LbHsp100 se comporta como um oligômero cujo estado é influenciado tanto pela concentração proteica como pela presença de nucleotídeos adenosina. Análises por ultracentrifugação analítica evidenciaram que a rLbHsp100 em solução apresenta um equilíbrio de diversas espécies havendo deslocamento para um hexâmero de maneira concentração dependente. Análises de SAXS confirmaram a estrutura hexamérica e proporcionaram a obtenção de um modelo ab initio da proteína. Através de microscopia eletrônica de transmissão pode-se observar a forma toróide e a dispersividade do sistema. Por fim, atestou-se que a proteína foi obtida funcional com fraca atividade ATPásica, apresentando também interações com nucleotídeos adenosina (ATP e ADP) assim como com a suramina. / Leishmaniasis is a neglected tropical disease that affects thousands of people and may even lead to death in its visceral form. During its life cycle, the parasite undergoes several environmental changes such as temperature and pH changes, especially when transfecting from the vector insect into the mammalian host. Such changes generate a cellular stress that can lead to misfolding as well as to aggregative processes, therefore a protein quality control system is necessary to maintain cell homeostasis, which includes molecular chaperones. Chaperones such as Hsp100 can help maintain cellular homeostasis and adaptation playing an important role for protozoa such as Leishmania braziliensis, which causes leishmaniasis. The Hsp100 has a disaggregase action, acting with other proteins of the chaperone system to extract polypeptides from protein aggregates, allowing their unfolding and subsequent refolding, avoiding their toxic effect on the cell. Hsp100 appears to be essential for these microorganisms, however there is not much data available for Hsp100 in Leishmania sp. and Plasmodium sp. This work describes the protocol for expression and purification of the recombinant Hsp100 of Leishmania braziliensis (rLbHsp100), as well as its initial in vitro characterization. The protein was analyzed by circular dichroism spectropolarimetry, presenting a typical structure of ?-helix rich protein as well as a concentration-dependent structure gain, static fluorescence of tryptophan demonstrated that the protein has local tertiary structure with its tryptophans partially exposed to the solvent. Analytical size exclusion chromatography showed that LbHsp100 behaves as an oligomer whose state is influenced by both the concentration and the presence of adenosine nucleotides. Analysis by analytical ultracentrifugation has shown that the rLbHsp100 in solution exhibits an equilibrium of several species shifting towards a hexamer in a concentration dependent manner. SAXS analyzes confirm the hexameric structure and had provide an ab initio model for the protein. Transmission electron microscopy shows the toroidal form and dispersivity of the system. Finally, the obtained protein had showed catalytic function, and also interacted with adenosine nucleotides (ATP and ADP) as well as suramine.

A leishmaniose é uma doença tropical negligenciada que afeta milhares de pessoas podendo até levar a óbito em sua forma visceral. Durante o seu ciclo de vida, o parasita passa por diversas mudanças ambientais como mudança de temperatura e pH, principalmente quando da transfecção do inseto vetor para o hospedeiro mamífero. Tais mudanças geram estresse celular que pode levar proteínas ao enovelamento incorreto assim como a processos agregativos, sendo necessários sistemas de controle de qualidade proteico para manter a homeostase celular, do qual fazem parte as chaperonas moleculares. Chaperonas como a Hsp100, ajudam a manter a homeostase celular e a adaptação desempenhando um papel importante para protozoários como a Leishmania braziliensis, causador da leishmaniose. A Hsp100 tem papel desagregase, atuando com outras chaperonas moleculares para a extração de polipeptídios de agregados proteicos possibilitando seu desenovelamento e posterior reenovelamento, evitando seu efeito tóxico sobre a célula. A Hsp100 parece ser essencial para esses microrganismos, no entanto não há muito dados disponíveis para Hsp100 em Leishmania sp. e Plasmodium sp. Neste trabalho está descrito o protocolo para expressão e purificação da Hsp100 recombinante de L. braziliensis (rLbHsp100), assim como sua caracterização estrutural e funcional inicial in vitro. A proteína foi analisada por espectropolarimetria de dicroísmo circular, apresentando estrutura típica de proteínas ricas em hélices α, a fluorescência estática de triptofano demonstrou que a proteína possui estrutura terciária local com seus triptofanos parcialmente expostos ao solvente. Por cromatografia de exclusão molecular analítica, observou-se que a LbHsp100 se comporta como um oligômero cujo estado é influenciado tanto pela concentração proteica como pela presença de nucleotídeos adenosina. Análises por ultracentrifugação analítica evidenciaram que a rLbHsp100 em solução apresenta um equilíbrio de diversas espécies havendo deslocamento para um hexâmero de maneira concentração dependente. Análises de SAXS confirmaram a estrutura hexamérica e proporcionaram a obtenção de um modelo ab initio da proteína. Através de microscopia eletrônica de transmissão pode-se observar a forma toróide e a dispersividade do sistema. Por fim, atestou-se que a proteína foi obtida funcional com fraca atividade ATPásica, apresentando também interações com nucleotídeos adenosina (ATP e ADP) assim como com a suramina. / Leishmaniasis is a neglected tropical disease that affects thousands of people and may even lead to death in its visceral form. During its life cycle, the parasite undergoes several environmental changes such as temperature and pH changes, especially when transfecting from the vector insect into the mammalian host. Such changes generate a cellular stress that can lead to misfolding as well as to aggregative processes, therefore a protein quality control system is necessary to maintain cell homeostasis, which includes molecular chaperones. Chaperones such as Hsp100 can help maintain cellular homeostasis and adaptation playing an important role for protozoa such as Leishmania braziliensis, which causes leishmaniasis. The Hsp100 has a disaggregase action, acting with other proteins of the chaperone system to extract polypeptides from protein aggregates, allowing their unfolding and subsequent refolding, avoiding their toxic effect on the cell. Hsp100 appears to be essential for these microorganisms, however there is not much data available for Hsp100 in Leishmania sp. and Plasmodium sp. This work describes the protocol for expression and purification of the recombinant Hsp100 of Leishmania braziliensis (rLbHsp100), as well as its initial in vitro characterization. The protein was analyzed by circular dichroism spectropolarimetry, presenting a typical structure of ?-helix rich protein as well as a concentration-dependent structure gain, static fluorescence of tryptophan demonstrated that the protein has local tertiary structure with its tryptophans partially exposed to the solvent. Analytical size exclusion chromatography showed that LbHsp100 behaves as an oligomer whose state is influenced by both the concentration and the presence of adenosine nucleotides. Analysis by analytical ultracentrifugation has shown that the rLbHsp100 in solution exhibits an equilibrium of several species shifting towards a hexamer in a concentration dependent manner. SAXS analyzes confirm the hexameric structure and had provide an ab initio model for the protein. Transmission electron microscopy shows the toroidal form and dispersivity of the system. Finally, the obtained protein had showed catalytic function, and also interacted with adenosine nucleotides (ATP and ADP) as well as suramine.

Orientador: Carlos Henrique Inacio Ramos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T20:53:19Z (GMT). No. of bitstreams: 1 Cagliari_ThiagoCarlos_D.pdf: 4482929 bytes, checksum: a1439ac0cca9a21c77eb47d2e163c224 (MD5) Previous issue date: 2009 / Resumo: As proteinas sao macromoleculas que possuem importancia vital para o funcionamento celular, participando da maioria das reacoes biologicas e tambem como componentes estruturais. Para que uma proteina possa exercer sua funcao, precisa atingir sua estrutura nativa atraves de um processo denominado enovelamento proteico. Neste contexto, as chaperonas moleculares sao proteinas capazes de auxiliar no enovelamento de outras proteinas, atuando na prevencao de agregados, desagregacao, translocacao, ativacao, entre outros. Dentre os muitos tipos de chaperonas existentes, neste trabalho foram abordadas as chaperonas das familias Hsp100 e Hsp90, as quais estao relacionadas aos processos de desagregacao e auxilio do enovelamento de proteinas-substrato, respectivamente. O presente trabalho pretendeu produzir as proteinas recombinantes Hsp100 e Hsp82 de cana-de-acucar para a caracterizacao de suas respectivas relacoes estrutura-funcao. Para isto foram empregadas tecnicas como: dicroismo circular, fluorescencia, espalhamento dinamico de luz e ultracentrifugacao analitica. Assim, foi observado que a forca ionica do meio e capaz de influenciar a estrutura quaternaria da proteina Hsp100, a qual se apresenta hexamerica em menores concentracoes de sal. Alem disto, e capaz de reconhecer agregados proteicos formados pelas proteinas luciferase e citrato sintase em ensaios in vitro. Ja a proteina Hsp82 apresentou uma estrutura dimerica, a qual nao e influenciada pela presenca de nucleotideos e apresenta grande estabilidade termica. Finalmente, a proteina p23 humana, a qual e responsavel por auxiliar a proteina Hsp90 no enovelamento de muitas proteinas/complexos proteicos, tambem foi caracterizada. Foram observados indicios de que a regiao C-terminal, rica em residuos de aminoacidos carregados, pode possuir algum grau de estruturacao, apesar de alguns estudos na literatura indicarem o contrario. O estudo das chaperonas de cana-de-acucar foi direcionado por um trabalho previo de anotacao de sequencias relacionadas as chaperonas moleculares no banco de dados do projeto SUCEST (Sugarcane EST Genome Project), o qual foi realizado por nosso grupo de pesquisa. Alem disto, sao apresentados os resultados da anotacao das sequencias relacionadas as chaperonas de eucalipto no banco de dados FORESTs (Eucalyptus Genome Sequencing Project Consortium), possibilitando futuros estudos com estas proteinas. / Abstract: Proteins are macromolecules that are vital to the functioning cell, participating in most of the biological reactions as well as structural components. To perform its function, a protein need to achieve its native structure through a process called protein folding. In this context, the molecular chaperone proteins are able to assist in the folding of other proteins, acting in the prevention of aggregation, disaggregation, translocation, activation, among others. From all types of existing chaperones, here were highlight the Hsp100 and Hsp90 families, which are related to processes of disaggregation and assistance of substrateprotein folding, respectively. This study sought to produce the recombinant proteins Hsp100 and Hsp82 from sugar cane for the characterization of their structure-function relationships. In order to do this, some techniques were employed such as: circular dichroism, fluorescence, dynamic light scattering and analytical ultracentrifugation. As a result, it was observed that the ionic strength of the solvent is capable of influencing the quaternary structure of protein Hsp100, which presents as a hexamer in lower salt concentrations. Furthermore, it is capable of recognizing protein aggregates formed by luciferase protein and citrate synthase in in vitro essays. The Hsp82 protein showed a dimeric structure, which was not influenced by the presence of nucleotides and presented a great thermal stability. Finally, the human protein p23, which is responsible for assisting in the Hsp90 protein folding of many proteins/protein complexes, was also characterized. In spite of some studies indicating the contrary, we observed evidence that the C-terminal region, which is rich in charged amino acid residues, can possible have some structure. The sugarcane chaperones study was guided by a previous chaperone sequence annotation work in the SUCEST (Sugarcane EST Genome Project) databank performed by our research group. In addition, results regarding chaperone sequences annotation in the eucalyptus databank (FORESTs - Eucalyptus Genome Sequencing Project Consortium) were presented here as well, which can also lead to future chaperone proteins function and structure studies. / Doutorado

Orientador: Carlos Henrique Inácio Ramos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T11:29:26Z (GMT). No. of bitstreams: 1 Silva_VivianeCristinaHeinzenda_D.pdf: 5558657 bytes, checksum: 719a2c54c3d42be8642a0beb9014221c (MD5) Previous issue date: 2013 / Resumo: As chaperonas moleculares estão envolvidas na manutenção da homeostase celular, auxiliando no correto enovelamento de proteínas, e consequentemente em sua funcionalidade. Duas famílias de chaperonas moleculares participam de pontos-chave neste sistema. Uma delas é a Hsp100 que tem papel importante na desagregação de proteínas; a outra é a Hsp90 que tem o papel de auxiliar no enovelamento, ativação, e na translocação de proteínas regulatórias e sinalizadoras. Neste trabalho foram caracterizadas as chaperonas Hsp100 e Hsp90 de cana-de-açúcar, denominadas SHsp101 e SsHsp90, respectivamente, cuja expressão em níveis basais foi detectada em tecido foliar. As proteínas recombinantes foram produzidas em Escherichia coli, de maneira solúvel, e após purificação apresentaram-se enoveladas. A SHsp101 foi obtida como um hexâmero em solução, apresentando capacidade de ligar nucleotídeos ATP e ADP, e de hidrolisar o ATP de maneira alostérica com cooperatividade positiva; mas não foi capaz de hidrolisar o ADP, que por sua vez mostrou-se inibidor da atividade ATPásica. A SHsp101 exibiu atividades de proteção do substrato luciferase contra agregação induzida por alta temperatura e de desagregação e reenovelamento da proteína-modelo GFPuv, na presença de ATP e ATP?S. Análises de complementação in vivo revelaram que a superexpressão heteróloga de SHsp101 em cepas de levedura mutantes nulo de hsp104, aumentou a termotolerância a 53°C, proporcionando um aumento de 80 vezes na sobrevivência das leveduras. A SsHsp90 apresentou-se dimérica em solução, com características estruturais e conformacionais (modelo tridimensional gerado por modelagem comparativa e validado por meio de análises de ligação cruzada acoplada à espectometria de massas) semelhantes às homólogas de outros organismos. A SsHsp90 apresentou atividade chaperona de proteção contra agregação da proteína-modelo citrato sintase desnaturada por choque térmico. As informações acerca da expressão, estrutura, e função de SHsp101 e SsHsp90 obtidas neste trabalho, contribuem para um melhor entendimento destas famílias de chaperonas moleculares, particularmente em plantas, que por serem organismos sésseis, estão mais expostos às condições adversas do ambiente / Abstract: Molecular chaperones are involved in the maintenance of cellular homeostasis by promoting the correct folding of proteins, and consequently, ensuring their functionality. Two families of molecular chaperones participate at key points in this system. The first is Hsp90, which assists in protein refolding, activation, and the trafficking of regulatory and signaling proteins, while the second is Hsp100, which has an important role in protein disaggregation. In this study, the Hsp90 and Hsp100 proteins from sugarcane were characterized, denoted as SsHsp90 and SHsp101, respectively, and their basal level of expression was detected in leaf tissue. In addition, both were produced by Escherichia coli as soluble form and then they were purified in a folded state. The SHsp101 was obtained folded as hexamer in solution and showed capacity of bind both ATP and ADP, but could only hydrolyze ATP in an allosteric manner with positive cooperativity. In fact, the presence of ADP had an inhibitory effect on the ATPase activity. SHsp101 exhibited protection against aggregation of luciferase, and showed a disaggregation and refolding activity of GFPuv in the presence ATP and ATP?S. In vivo complementation analysis revealed that heterologous overexpression of SHsp101 in a null hsp104 yeast strain correlated with an 80 fold increase in yeast survival at 53°C. The dimer obtained for SsHsp90 had similar structural and conformational characteristics compared to other Hsp90 homologues, and was compatible with a three-dimensional model generated by comparative modeling, which was validated by cross-linking coupled to mass spectrometry. The SsHsp90 protected against thermal aggregation of citrate synthase. Taken together, the information about the expression, structure, and function of SHsp101 and SsHsp90 obtained in this study contribute to a better understanding of these molecular chaperone protein families, particularly in plants, which are sessile organisms and more exposed to adverse environmental conditions / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

<p>Unlike in the greenhouse, plants need to cope with many environmental stresses under natural conditions. Among these conditions are drought, waterlogging, excessive or too little light, high or low temperatures, UV irradiation, high soil salinity, and nutrient deficiency. These stress factors can affect many biological processes, and severely retard the growth and development of higher plants, resulting in massive losses of crop yield and wood production. Plants have developed many protective mechanisms to survive and acclimate to stresses, such as the rapid induction of specific molecular chaperones and proteases at the molecular level. Molecular chaperones mediate the correct folding and assembly of polypeptides, as well as repair damaged protein structures caused by stress, while proteases remove otherwise non-functional and potentially cytotoxic proteins. </p><p>The Clp/Hsp100 family is a new group of chaperones that consists of both constitutive and stress-inducible members. Besides being important chaperones, many Clp/Hsp100 also participate in protein degradation by associating with the proteolytic subunit ClpP to form the Clp protease complex. Higher plants have the greatest number and complexity of Clp proteins than any other group of organisms, and more than 20 different Clp isomers in plants have been identified (Paper I). Because of this diversity, we have adopted a functional genomics approach to characterise all Clp proteins in the model plant Arabidopsis thaliana. Our ongoing research strategy combines genetic, biochemical and molecular approaches. Central to these has been the preparation of transgenic lines for each of the chloroplast Clp isomers. These transgenic lines will be analysed to understand the function and regulation of each chloroplast Clp protein for plant growth and development.</p><p>In Paper II, an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX. Specific polyclonal antibodies were made and used to localise the ClpX homologue to plant mitochondria, consistent with that predicted by computer analysis of the putative transit peptide. In addition to ClpX, a nuclear-encoded ClpP protein, termed ClpP2, was identified from the numerous ClpP isomers in Arabidopsis and was also located in mitochondria. Relatively unchanged levels of transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions. Using β-casein as a substrate, plant mitochondria possessed an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease.</p><p>In Paper III, four nuclear-encoded Clp isomers were identified in Arabidopsis thaliana: ClpC1 and ClpP3-5. All four proteins are localized within the stroma of chloroplasts, along with the previously identified ClpD, ClpP1 and ClpP6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves. The increases in protein were mirrored at the mRNA level for most ClpP isomers but not for ClpC1, ClpC2 and ClpD and ClpP5, which exhibited little change in transcript levels. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. The results reveal that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions. </p><p>In Paper IV, antisense repression transgenic lines of clpP4 were prepared and then later characterised. Within the various lines screened, up to 90% of ClpP4 protein content was specifically repressed, which also led to the down-regulation of ClpP3 and ClpP5 protein contents. The repression of clpP4 mRNA retarded the development of chloroplasts and the differentiation of leaf mesophyll cells, resulting in chlorotic phenotypes. The chlorosis was more severe in young than in mature leaves due likely to the developmental expression pattern of the ClpP4 protein. Chlorotic plants eventually turned green upon aging, accompanied by a recovery in the amount of the ClpP4 protein. The greening process could be affected by the light quantity, either by altering the photoperiod or light intensity.</p>

Unlike in the greenhouse, plants need to cope with many environmental stresses under natural conditions. Among these conditions are drought, waterlogging, excessive or too little light, high or low temperatures, UV irradiation, high soil salinity, and nutrient deficiency. These stress factors can affect many biological processes, and severely retard the growth and development of higher plants, resulting in massive losses of crop yield and wood production. Plants have developed many protective mechanisms to survive and acclimate to stresses, such as the rapid induction of specific molecular chaperones and proteases at the molecular level. Molecular chaperones mediate the correct folding and assembly of polypeptides, as well as repair damaged protein structures caused by stress, while proteases remove otherwise non-functional and potentially cytotoxic proteins. The Clp/Hsp100 family is a new group of chaperones that consists of both constitutive and stress-inducible members. Besides being important chaperones, many Clp/Hsp100 also participate in protein degradation by associating with the proteolytic subunit ClpP to form the Clp protease complex. Higher plants have the greatest number and complexity of Clp proteins than any other group of organisms, and more than 20 different Clp isomers in plants have been identified (Paper I). Because of this diversity, we have adopted a functional genomics approach to characterise all Clp proteins in the model plant Arabidopsis thaliana. Our ongoing research strategy combines genetic, biochemical and molecular approaches. Central to these has been the preparation of transgenic lines for each of the chloroplast Clp isomers. These transgenic lines will be analysed to understand the function and regulation of each chloroplast Clp protein for plant growth and development. In Paper II, an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX. Specific polyclonal antibodies were made and used to localise the ClpX homologue to plant mitochondria, consistent with that predicted by computer analysis of the putative transit peptide. In addition to ClpX, a nuclear-encoded ClpP protein, termed ClpP2, was identified from the numerous ClpP isomers in Arabidopsis and was also located in mitochondria. Relatively unchanged levels of transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions. Using β-casein as a substrate, plant mitochondria possessed an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease. In Paper III, four nuclear-encoded Clp isomers were identified in Arabidopsis thaliana: ClpC1 and ClpP3-5. All four proteins are localized within the stroma of chloroplasts, along with the previously identified ClpD, ClpP1 and ClpP6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves. The increases in protein were mirrored at the mRNA level for most ClpP isomers but not for ClpC1, ClpC2 and ClpD and ClpP5, which exhibited little change in transcript levels. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. The results reveal that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions. In Paper IV, antisense repression transgenic lines of clpP4 were prepared and then later characterised. Within the various lines screened, up to 90% of ClpP4 protein content was specifically repressed, which also led to the down-regulation of ClpP3 and ClpP5 protein contents. The repression of clpP4 mRNA retarded the development of chloroplasts and the differentiation of leaf mesophyll cells, resulting in chlorotic phenotypes. The chlorosis was more severe in young than in mature leaves due likely to the developmental expression pattern of the ClpP4 protein. Chlorotic plants eventually turned green upon aging, accompanied by a recovery in the amount of the ClpP4 protein. The greening process could be affected by the light quantity, either by altering the photoperiod or light intensity.