Insights on Hsp90 chaperone interactions using transgenic and biophysical approaches

Orientadoesr: Carlos Henrique Inácio Ramos, Gonçalo Amarante Guimarães PereiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Chaperonas moleculares (Heat Shock proteins - HSPs) são componentes chave do sistema de controle de qualidade de proteínas (PQC - Protein Quality Control), que é essencial para a vida, sendo responsável por manter a homeostase proteica e a adequada função de diversas vias. Problemas no processo de enovelamento estão relacionados a doenças degenerativas, amilóides e câncer. Em plantas, as chaperonas moleculares desempenham um papel crucial na proteção contra estresses bióticos e abióticos, pois como organismos sésseis, as plantas devem ser capazes de responder rapidamente a mudanças na temperatura, salinidade, déficit hídrico, entre outros. A chaperona molecular Hsp90 (Heat Shock protein 90 kDa) compreende uma família ubíqua, considerada um 'hub' por interagir com chaperonas, co-chaperonas e ter como clientes proteínas regulatórias essenciais como fatores de transcrição, quinases, receptores de hormônios, entre outros. A Hsp90 age em conjunto com co-chaperonas, as quais modulam e direcionam sua função. Uma destas co-chaperonas é a Hop (Hsp70-Hsp90 organizing protein), capaz de interagir simultaneamente com a Hsp90 e Hsp70, mediando a transferência de substratos. A Hop é composta por três domínios com repetições de tetratricopeptídeos (TPR) (TPR1, TPR2A e TPR2B), responsáveis pela interação com as chaperonas, porém a dinâmica desta interação não está bem entendida, uma vez que ainda não há estrutura da Hop inteira e o estado oligomérico desta co-chaperona ainda é controverso na literatura. Neste trabalho apresentamos a classificação de um gene de Hsp90 de cana-de-açúcar, e o início de sua caracterização funcional através de transgenia em Arabidopsis thaliana. Apresentamos também a caracterização biofísica de uma importante co-chaperona da Hsp90, a Hop (Hsp70-Hsp90 organizing protein) humana. Através da análise de sequências a Hsp90 de cana-de-açúcar foi classificada como Hsp90-3, uma isoforma citosólica. Plantas transgênicas de A. thaliana, produzidas a partir da inserção do gene da Hsp90-3 de cana-de-açúcar, apresentaram níveis reduzidos de Hsp90. Tal perturbação nos níveis de Hsp90 parece ter afetado a expressão de outras proteínas da rede de interações, relacionadas com processos diversos como resposta imune e fotossíntese. As plantas transgênicas também exibiram germinação mais rápida e raízes mais longas em relação ao controle. Sob estresse térmico, linhagens transgênicas apresentaram maior suscetibilidade à alta temperatura em relação ao controle. Tais resultados sugerem que a Hsp90 tem um importante papel na fisiologia celular e no desenvolvimento, e que os níveis de Hsp90 são críticos para a resposta frente a estresses. A caracterização biofísica do mutante Hop D456G, uma mutação no domínio TPR2B, mostrou que esta proteína é uma mistura de monômeros, dímeros e oligômeros maiores, porém com prevalência do estado monomérico. O resíduo D456 pode ter uma participação na dinâmica de dimerização e é possível que o estado oligomérico da Hop seja regulado entre os estados monomérico e dimérico, com a finalidade de facilitar sua atividade adaptadoraAbstract: Molecular chaperones (heat shock proteins - HSPs) are key components of protein quality-control system (PQC - Protein Quality Control), which maintains protein homeostasis and the proper function of several pathways, being essential for life. Defects in folding processes are related to degenerative diseases, amyloidosis and cancer. In plants, which as sessile organisms must be able to respond rapidly to changes in temperature, salinity, water deficit, and others, molecular chaperones play a crucial role in protecting against such biotic and abiotic stresses. Molecular chaperone Hsp90 (Heat Shock Protein 90 kDa) comprise an ubiquitous family, considered a hub as it interacts with chaperones, co-chaperones, and have as clients key regulatory proteins such as transcription factors, kinases, hormone receptors, and others. The chaperone acts together with co-chaperones, which modulate and guide Hsp90 function. The co-chaperone Hop (Hsp70-Hsp90 organizing protein), interacts simultaneously with Hsp90 and Hsp70, mediating substrate transfer. Hop has three TPR domains (TPR1, and TPR2A TPR2B) responsible for interaction with the chaperones, but this interaction dynamics remains unclear, since there is no structure of full length Hop and its oligomeric state is controversial in literature reports. This work presents the classification of an Hsp90 gene from sugarcane, and primary functional characterization studies in Arabidopsis thaliana transgenic lines. We also present the biophysical characterization of the human Hsp90 co-chaperone Hop (Hsp70-Hsp90 organizing protein). Through sequence analysis the Hsp90 from sugarcane has been classified as Hsp90-3, a cytosolic isoform. Transgenic A. thaliana, produced by Hsp90-3 insertion, exhibited reduced transcript levels of Hsp90. This disruption in Hsp90 levels seems to affect the expression of other proteins from the interaction network, which are related to various processes such as immune response and photosynthesis. Transgenics also exhibited faster germination and longer roots than the control. Under heat stress, transgenic lines showed increased susceptibility to high temperature. These results suggest that Hsp90 has an important role in cellular physiology and development; in addition the levels of Hsp90 are critical for responses to stresses. The biophysical characterization of the mutant D456G Hop, a mutation in domain TPR2B showed that this protein is a mixture of monomers, dimers and higher oligomers, but the monomeric state is majoritary. The residue D456 may be involved in dimerization dynamics, and it is possible that Hop is regulated between monomeric and dimeric species, to enable its adaptor functionsMestradoBioquimicaMestre em Biologia Funcional e Molecula

Xylem Transcription Profiles Indicate Potential Metabolic Responses For Economically Relevant Characteristics Of Eucalyptus Species.

Eucalyptus is one of the most important sources of industrial cellulose. Three species of this botanical group are intensively used in breeding programs: E. globulus, E. grandis and E. urophylla. E. globulus is adapted to subtropical/temperate areas and is considered a source of high-quality cellulose; E. grandis grows rapidly and is adapted to tropical/subtropical climates; and E. urophylla, though less productive, is considered a source of genes related to robustness. Wood, or secondary xylem, results from cambium vascular differentiation and is mostly composed of cellulose, lignin and hemicelluloses. In this study, the xylem transcriptomes of the three Eucalyptus species were investigated in order to provide insights on the particularities presented by each of these species. Data analysis showed that (1) most Eucalyptus genes are expressed in xylem; (2) most genes expressed in species-specific way constitutes genes with unknown functions and are interesting targets for future studies; (3) relevant differences were observed in the phenylpropanoid pathway: E. grandis xylem presents higher expression of genes involved in lignin formation whereas E. urophylla seems to deviates the pathway towards flavonoid formation; (4) stress-related genes are considerably more expressed in E. urophylla, suggesting that these genes may contribute to its robustness. The comparison of these three transcriptomes indicates the molecular signatures underlying some of their distinct wood characteristics. This information may contribute to the understanding of xylogenesis, thus increasing the potential of genetic engineering approaches aiming at the improvement of Eucalyptus forest plantations productivity.1420

Xylem transcription profiles indicate potential metabolic responses for economically relevant characteristics of Eucalyptus species

Abstract Background Eucalyptus is one of the most important sources of industrial cellulose. Three species of this botanical group are intensively used in breeding programs: E. globulus, E. grandis and E. urophylla. E. globulus is adapted to subtropical/temperate areas and is considered a source of high-quality cellulose; E. grandis grows rapidly and is adapted to tropical/subtropical climates; and E. urophylla, though less productive, is considered a source of genes related to robustness. Wood, or secondary xylem, results from cambium vascular differentiation and is mostly composed of cellulose, lignin and hemicelluloses. In this study, the xylem transcriptomes of the three Eucalyptus species were investigated in order to provide insights on the particularities presented by each of these species. Results Data analysis showed that (1) most Eucalyptus genes are expressed in xylem; (2) most genes expressed in species-specific way constitutes genes with unknown functions and are interesting targets for future studies; (3) relevant differences were observed in the phenylpropanoid pathway: E. grandis xylem presents higher expression of genes involved in lignin formation whereas E. urophylla seems to deviates the pathway towards flavonoid formation; (4) stress-related genes are considerably more expressed in E. urophylla, suggesting that these genes may contribute to its robustness. Conclusions The comparison of these three transcriptomes indicates the molecular signatures underlying some of their distinct wood characteristics. This information may contribute to the understanding of xylogenesis, thus increasing the potential of genetic engineering approaches aiming at the improvement of Eucalyptus forest plantations productivity

Structural and functional characterization of the interaction between the chaperone alphaB-crystallin and the tyrosine-phosphatase Shp2

Orientador: Kleber Gomes FranchiniTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O estudo de circuitos de sinalização celular ativados frente a estímulos fisiopatológicos constitui uma importante fronteira na pesquisa da patogênese da hipertrofia e insuficiência cardíacas. Estudos anteriores do nosso laboratório investigaram a participação de proteínas como Shp2, uma tirosino-fosfatase que é crítica para a sinalização hipertrófica de míocitos cardíacos, e CryAB (?B-Crystalina), uma chaperona altamente expressa no coração, que protege FAK contra degradação e promove a sobrevivência celular. O presente estudo demonstrou que a CryAB se associa com Shp2 e reduz sua atividade catalítica em miócitos cardíacos estirados. CryAB interage diretamente com Shp2 in vitro e em células vivas, como mostrado pelas técnicas de imunoprecipitação, pull-down, FLIM-FRET e cross-linking acoplado a espectrometria de massas. Técnicas de docking e modelagem molecular demonstraram que os domínios PTP e NSH2 da Shp2 fazem contatos no domínio alfa-cristalino da CryAB oligomérica, mantendo a estrutura autoinibida da Shp2. Somente o oligômero de CryAB, mas não o dímero, é capaz de reduzir a atividade fosfatase da Shp2 de maneira dose dependente, como mostrado por meio da quantificação da atividade tirosina fosfatase da Shp2 na presença destas proteínas. Miócitos ventriculares de ratos neonatos (MVRNs) em cultura apresentam, mediante estiramento, uma robusta associação CryAB/Shp2 e reduzida atividade tirosina fosfatase de Shp2. Adicionalmente, MVRNs submetidos a silenciamento de CryAB seguido de estiramento exibiram uma atividade de Shp2 aumentada. Os resultados indicam que a CryAB pode atuar como modulador da atividade da Shp2 em miócitos cardíacos submetidos a estresse mecânicoAbstract: The study of cell signaling circuits activated during pathophysiological stimuli is an important challenge in cardiac hypertrophy and failure pathogenesis research. Previous studies from our group have investigated the role of proteins such as Shp2 (SH2 domain-containing tyrosine phosphatase 2), a tyrosine phosphatase critical in cardiac myocytes hypertrophic signaling, and CryAB (?B-Crystallin), a molecular chaperone highly expressed in heart, which interacts with and protects FAK (Focal Adhesion Kinase) from degradation, promoting cell survival. Here we show that CryAB interacts with and regulates the tyrosine-phosphatase Shp2 catalytic activity in stretched cardiomyocytes. We used a combination of pull-down, immunoprecipitation, FRET-FLIM and chemical cross-linking coupled to mass spectrometry to show that CryAB and Shp2 directly bind in vitro and in living cells. Docking and molecular modeling showed that the PTP and NSH2 domains of Shp2 bind ACD (alpha-crystallin domain) of oligomeric CryAB, which holds Shp2 in closed and inhibited conformation. Protein tyrosine-phosphatase assays showed that the binding of CryAB oligomer, but not the dimer, is important for Shp2 stability and function, and in addition, it attenuates Shp2 activity in a dose dependent mode. Stretched Neonate Rat Ventricular Myocytes (NRVMs) show a robust CryAB/Shp2 association and reduced Shp2 phosphatase activity. In addition, NRVMs subjected to CryAB knockdown followed by mechanical stress exhibited enhanced activity of Shp2. These results indicates that CryAB might act as a modulator of Shp2 phosphatase activity in cardiomyocytes under mechanical stressDoutoradoBioquimicaDoutora em Biologia Funcional e Molecular13/05877-8FAPES