Dissertations / Theses: 'Chaperones'

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Gomes, Francisco Edvan Rodrigues. "Clonagem, expressão e estudo de 3 co-chaperonas de Leishmania braziliensis." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/75/75132/tde-16092011-160310/.

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A leishmaniose é uma enfermidade infecciosa causada por várias espécies de parasitas do gênero Leishmania e representa um dos principais problemas de saúde pública nos países subdesenvolvidos. No hospedeiro, a sobrevivência do protozoário causador dessa doença depende de uma classe especial de proteínas, as chaperonas moleculares ou proteínas de choque térmico como também são conhecidas. A função dessas proteínas é auxiliar no processo de enovelamento protéico, no transporte de proteínas entre as membranas e em muitas outras importantes funções celulares. Neste processo, as chaperonas moleculares são auxiliadas pelas suas co-chaperonas que desempenham função de destaque. Dentre as principais famílias de chaperonas moleculares temos as Hsp70 e as Hsp90 com suas respectivas co-chaperonas, as Hsp40 e a Aha1. O presente trabalho pretendeu inicialmente expressar e purificar as co-chaperonas moleculares Hsp40I e Hsp40II de L. braziliensis para realizar estudos de caracterização estrutural por meio das técnicas de dicroísmo circular e fluorescência. Contudo, a insolubilidade dessas proteínas, que pode ter sido causada pela presença de mutações nas sequências de DNA, motivou a caracterização de outra co-chaperona, a Aha1 de L. braziliensis (LbAha1). A LbAha1 foi expressa no sobrenadante celular e purificada por três etapas cromatográficas (troca aniônica, afinidade por íons cálcio e gel filtração). A análise da sequência de aminoácidos dessa proteína mostra que ela possui 9 resíduos de triptofano distribuídos nos dois domínios característicos da LbAha1. Estudos de desnaturação química por uréia, monitorados pelas técnicas de dicroísmo circular e fluorescência, mostram que os dois domínios da LbAha1 apresentam estabilidades diferentes. Os estudos estruturais realizados permitiram identificar as transições com o respectivo domínio.
Leishmaniasis is an infectious disease caused by several species of Leishmania species and represents major public health problems in developing countries. In the harborer, the survival of the parasite that cause this disease depends on a special class of proteins, molecular chaperones or heat shock proteins as they are also known. The function of these proteins is to assist in protein folding, transport of proteins and many other important cellular functions. In this process the molecular chaperones are helped by their co-chaperones that play a prominent role. Among the main families of molecular chaperones, there are Hsp70 and Hsp90 with their respective co-chaperones, Hsp40 and the Aha1. The present work, initially pretended to express and purify the molecular co-chaperones Hsp40I and Hsp40II of the L. braziliensis for structural characterization by spectroscopic techniques like fluorescence and circular dichroism. However, the insolubility of these proteins, possibly caused by the presence of mutations in their DNA sequences, led to the characterization of another co-chaperone, the Aha1 of the L. braziliensis. These proteins were expressed in the cell supernatant and purified by three chromatographic steps (anion exchange, affinity for calcium ions and gel filtration). The analysis of the DNA sequence of this protein shows that it has nine Trp residues distributed between the two domains and by urea denaturation studies monitored by fluorescence techniques and circular dichroism show that they have different stabilities.

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Moosavi, Behrooz. "The Role of Molecular Chaperone Hsp104 and its Co-chaperones in the Yeast [PSI+] Propagation." Thesis, University of Kent, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.499804.

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Gonçalves, Danieli Cristina 1986. "Estudos iniciais de ineraçãos da HSP90 através da caracterização funcioanl de um transgênico e biofísica de uma co-chaperona." [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314030.

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Orientadoesr: Carlos Henrique Inácio Ramos, Gonçalo Amarante Guimarães Pereira
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: 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 adaptadora
Abstract: 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 functions
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular

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Zahn, Ralph. "Prion propagation and molecular chaperones." Zürich : Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Zürich, 2002. http://e-collection.ethbib.ethz.ch/show?type=habil&nr=4.

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Pemberton, Samantha. "Molecular chaperones in the assembly of α-Synuclein and Parkinson’s Disease." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA114840/document.

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La formation et le dépôt de fibres d'α-Synucléine dans le cerveau humain sont à l‟origine de la maladie de Parkinson. Cette thèse documente le rôle de deux chaperons moléculaires dans l‟assemblage en fibres de l'α-Syn : Hsc70 (protéine de choc thermique constitutivement exprimée chez l‟Homme) et Ssa1p (son équivalent chez la levure). Le but était d'élargir le catalogue d'effets connus des chaperons moléculaires sur α-Syn, pour éventuellement ouvrir la voie à des applications thérapeutiques. Nous avons montré que Hsc70 inhibe l'assemblage de l'α-Syn en fibres, en se liant avec une forte affinité à la forme soluble de l'α-Syn. Hsc70 se lie préférentiellement aux fibres de l'α-Syn, et cette liaison a un effet cytoprotecteur puisqu'elle rend les fibres moins toxiques pour les cellules de mammifères en culture. Pareillement à Hsc70, Ssa1p inhibe l'assemblage de l'α-Syn en fibres, et a une plus forte affinité pour les fibres que pour la forme soluble de l'α-Syn. En revanche, la liaison de Ssa1p aux fibres de l'α-Syn n'a pas d'effet cytoprotecteur, sûrement due aux différences entre les séquences du site de liaison aux peptides des deux chaperons moléculaires, qui fait que Ssa1p a une affinité plus faible que Hsc70 pour les fibres d'α-Syn. Nous avons fixé le complexe entre Ssa1p et α-Syn avec des agents pontants, pour ensuite établir une carte du site d'interaction entre les deux protéines en utilisant la spectrométrie de masse. Ceci est indispensable si un « mini » Ssa1p, constitué des éléments nécessaires et suffisants sera utilisé comme agent thérapeutique pour réduire la toxicité des fibres d'α-Syn
The formation and deposition of α-Synuclein fibrils in the human brain is at the origin of Parkinson’s disease. The objective of my thesis was to document the role of two molecular chaperones on the assembly of α-Syn into fibrils: Hsc70, a constitutively expressed human heat shock protein, and Ssa1p, its yeast equivalent. The aim was to expand the catalogue of known effects of molecular chaperones on the PD implicated protein, which could have therapeutic significance. We showed that Hsc70 inhibits the assembly of α-Syn into fibrils, by binding with high affinity to the soluble form of α-Syn. We documented that Hsc70 binds preferentially to α-Syn fibrils and that this binding has a cytoprotective effect, as it renders the fibrils less toxic to cultured mammalian cells. Similarly to Hsc70, Ssa1p inhibits the assembly of α-Syn into fibrils, and has a higher affinity for fibrils than for the soluble form of α-Syn. On the other hand, binding of Ssa1p to α-Syn fibrils does not have a cytoprotective effect, almost certainly due to differences in the amino acid sequences of the peptide binding sites of the two molecular chaperones, which mean that Ssa1p has a lower affinity than Hsc70 for α-Syn fibrils. We stabilized the complex between Ssa1p and α-Syn using chemical cross-linkers, to then map the interaction site between the two proteins. This is indispensable if a “mini” Ssa1p, comprised of only what is necessary and sufficient of Ssa1p, is to be used as a therapeutic agent to decrease the toxicity of α-Syn fibrils. A therapeutic agent based on exogenous protein Ssa1p is less likely to trigger an autoimmune response than for example the endogenous protein Hsc70

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Beecham, Matthew Peter. "Supramolecular chaperones to assist protein folding." Thesis, University of Warwick, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422081.

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Gokhale, Kavita Chandan. "Interactions between endogenous prions, chaperones and polyglutamine proteins in the yeast model." Diss., Available online, Georgia Institute of Technology, 2005, 2005. http://etd.gatech.edu/theses/available/etd-02272005-193343/unrestricted/gokhale%5Fkavita%5Fc%5F200505%5Fphd.pdf.

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Thesis (Ph. D.)--Biology, Georgia Institute of Technology, 2005.
Dr Yury Chernoff, Committee Member ; Dr Jung Choi, Committee Member ; Dr Nick Hud, Committee Member ; Dr Roger Wartell, Committee Member ; Dr Harish Radhakrishna, Committee Member. Vita. Includes bibliographical references.

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Amin-Wetzel, Niko. "Regulation of mammalian IRE1α : co-chaperones and their importance." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/274869.

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When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER protein folding capacity to restore protein folding homeostasis. Unfolded proteins activate UPR signalling across the ER membrane to the nucleus by promoting oligomerisation of IRE1, a conserved transmembrane ER stress receptor. Despite significant research, the mechanism of coupling ER stress to IRE1 oligomerisation and activation has remained contested. There are two proposed mechanisms by which IRE1 may sense accumulating unfolded proteins. In the direct binding mechanism, unfolded proteins are able to bind directly to IRE1 to drive its oligomerisation. In the chaperone inhibition mechanism, unfolded proteins compete for the repressive BiP bound to IRE1 leaving IRE1 free to oligomerise. Currently, these two mechanisms respectively lack compelling in vivo and in vitro evidence required to assess their validity. The work presented here first describes in vivo experiments that identify a role of the ER co-chaperone ERdj4 as an IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). This is then built on by a series of in vitro experiments showing that ERdj4 catalyses formation of a repressive BiP-IRE1LD complex and that this complex can be disrupted by the presence of competing unfolded protein substrates to restore IRE1LD to its default, dimeric, and active state. The identification of ERdj4 and the in vitro reconstitution of chaperone inhibition establish BiP and its J-domain co-chaperones as key regulators of the UPR. This thesis also utilises the power of Cas9-CRISPR technology to introduce specific mutations into the endogenous IRE1α locus and to screen for derepressing IRE1α mutations. Via this methodology, two predicted unstructured regions of IRE1 are found to be important for IRE1 repression. Finally, this thesis challenges recent in vitro findings concerning the direct binding mechanism.

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Seraphim, Thiago Vargas. "Estudos bioquímicos e biofísicos de proteínas de choque térmico da família Hsp40 de cana-de-açúcar e de levedura." [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314017.

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Orientador: Carlos Henrique Inacio Ramos
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O enovelamento protéico é essencial para a correta função biológica das proteínas. A existência de um ambiente com alta concentração dos mais diferentes tipos de moléculas, dentro da célula, e de diversos tipos de situações de estresse, podem agir induzindo a formação de espécies improdutivas na via de enovelamento, como proteínas mal enoveladas e/ou até mesmo agregados protéicos. Para controlar estes eventos, há a maquinaria de chaperonas moleculares, que tem por objetivo garantir a homeostase protéica celular. As chaperonas moleculares são capazes de ligar e estabilizar um polipeptídio, mas sem contribuir com informações para a sua conformação final. Dentro desta maquinaria, o sistema Hsp70 tem um papel central, sendo responsável por receber proteínas desenoveladas ou mal enoveladas de outras chaperonas, podendo auxiliar no reenovelamento e direcionamento para outras chaperonas moleculares ou para degradação. A Hsp70 é regulada por co-chaperonas, como a Hsp40, que é responsável pela entrega de proteínas clientes à Hsp70 e pelo estímulo da atividade ATPase, essencial para a funcionalidade da Hsp70. Este trabalho apresenta a caracterização de uma Hsp40 tipo I de cana-de-açúcar, nomeada SHsp40, e o estudo de uma Hsp40 tipo II de levedura e seus mutantes, a fim de entender a relação estrutura-função destas proteínas. A SHsp40 foi expressa em E. coli, purificada e obtida enovelada, como verificado por dicroísmo circular. Além disso, a SHsp40 apresentou atividade chaperona em experimentos de proteção ao substrato desenovelado e se comportou como um dímero alongado em solução, como mostrado por SEC-MALS e pela determinação do fator de Perrin. Experimentos de desenovelamento térmico monitorado pelo sinal de CD a 222 nm revelaram que a SHsp40 possui pelo menos um intermediário, e a fluorescência de tioflavina T e bis-ANS mostraram que este intermediário é rico em folhas ? e parcialmente desenovelado, características de espécies na via de formação de fibrilas. A SHsp40 agregada foi examinada por microscopia eletrônica de varredura, que comprovou sua capacidade de formar de fibrilas. Este trabalho também contribuiu para o estudo de uma Hsp40 tipo II de levedura, Sis1, e seus mutantes de deleção, Sis1?124-174 e Sis1?121-257. Ensaios de fluorescência estática do triptofano, fotoapagamento e anisotropia mostraram que a deleção do domínio G/M não afetou a estrutura e hidrodinâmica de Sis1?124-174 em relação à proteína selvagem. Estudos de estabilidade destas proteínas, realizado anteriormente em nosso grupo de pesquisa e complementado neste trabalho pelo uso da técnica de SEC-MALS, mostrou que Sis1 e Sis1?124-174 foram mais estáveis que Sis1?121-257, mutante que o domínio G/M e subdomínio CTDI estão ausentes
Abstract: Correct protein folding is essential for proper protein biological function. There is a crowded environment and many types of molecules inside the cell and a variety of external stresses can act inducing unproductive species, as unfolded and/or misfolded proteins and even protein aggregates. To control these undesired events and ensures the protein homeostasis there is a molecular chaperone machinery. Molecular chaperones are able to bind and stabilize polypeptides but with no contributions for their final conformations. Inside this machinery, the Hsp70 system has a central role and is responsible to receive unfolded or misfolded proteins from other chaperones, helping in protein refolding and delivering the clients to other chaperones and even protein targeting for degradation. Hsp70 is regulated by its co-chaperones, such as Hsp40, which is responsible to client proteins deliver to Hsp70 and stimulation of its ATPase activity, essential processes for Hsp70 function. This work presents a sugarcane type I Hsp40 characterization, named SHsp40, and studies of an yeast type II Hsp40 and its mutants in order to understand the structure-function relationship of these proteins. The SHsp40 was expressed in E. coli, purified and obtained folded, as verified by circular dichroism. Furthermore, SHsp40 presented chaperone activity in unfolded substrate protection experiments and behaved as an elongated dimer in solution, as shown by SEC-MALS and estimated by Perrin factor. Thermal-induced unfolding experiments monitored by CD signal at 222 nm revealed that SHsp40 has at least one intermediate which is populated and tioflavin T and bis-ANS fluorescence showed that this intermediate is ? sheet-rich and partially folded, such as intermediate species in the fibril formation pathway. The aggregated SHsp40 was examined by scanning electron microscopy, wich proved its ability to fibril formation. This work also contributed for the study of an yeast type II Hsp40, Sis1, and its deletion mutants, Sis1?124-174 and Sis1?121-257. Steady-state tryptophan fluorescence, quenching and anisotropy assays showed that the G/M domain deletion did not affect the structure and hydrodynamic properties of Sis1?124-174 in relation to the wild type protein. Stability studies of these proteins, previously performed in our research group and complemented in this work by using the SEC-MALS technique, showed that Sis1 and Sis1?124-174 were more stable than Sis1?121-257, a mutant with the G/M domain and CTDI subdomain absents
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular

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Coto, Amanda Laís de Souza. "Estudo estrutural e funcional da co-chaperona SGT de Leishmania braziliensis." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-17112016-135653/.

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As chaperonas moleculares são ativas em muitos processos celulares envolvendo o enovelamento e a homeostase de proteínas. Essas características fazem das chaperonas alvos potenciais para o tratamento de diversas doenças. As Hsp70 e as Hsp90, em especial, são proteínas ubíquas altamente conservadas biologicamente que atuam no enovelamento de proteínas nascentes, prevenção da agregação proteica, recuperação de proteínas de agregados, sinalização e crescimento celular, dentre outros. Contudo, para que essas proteínas cumpram eficientemente suas funções, elas devem ser moduladas por co-chaperonas moleculares. A SGT é uma co-chaperona que pode ser dividida em três regiões: domínio N-terminal, domínio TPR e domínio C-terminal, sendo que a região do domínio TPR é a responsável pela interação com o motivo EEVD no C-terminal das Hsp90 e Hsp70 citoplasmáticas. A SGT é encontrada em vários organismos, dentre eles os protozoários do gênero Leishmania spp.. Estes organismos são responsáveis pela leishmaniose, uma doença negligenciada que afeta milhares de pessoas todos os anos, principalmente em países subdesenvolvidos. Evidências indicam que a SGT em protozoários é essencial para o crescimento e viabilidade da forma promastigota. Diante disso, nesse trabalho foi feito o estudo estrutural e funcional da co-chaperona SGT de Leishmania braziliensis (LbSGT). A LbSGT recombinante foi produzida e purificada. A caracterização estrutural indica que a LbSGT é uma proteína rica em estrutura secundária do tipo hélice α que se comporta como um dímero alongado em solução. Dados de estabilidade térmica e química indicam que a LbSGT é uma proteína formada por domínios com diferentes estabilidades. A LbSGT foi identificada in vivo e o western blotting indicou sua presença cognata nas formas promastigotas do protozoário. Os ensaios de interação indicam que as interações entre a LbSGT e a Hsp90 de L. braziliensis (LbHsp90) e a LbSGT e Hsp70-1A humana (usada como proteína modelo) são diferentes da interação da LbSGT com o peptídeo MEEVD. Sendo assim, esses dados sugerem que a interação da LbSGT com a Hsp70-1A e LbHsp90 envolvem mais regiões das proteínas do que somente o motivo de interação da Hsp70-1A e da LbHsp90 com o domínio TPR da LbSGT. Em conjunto, as propriedades estruturais e funcionais da LbSGT observadas estão de acordo com a possível função da SGT como proteína adaptadora entre os sistemas Hsp70 e Hsp90 no foldossoma.
The molecular chaperones are active in many cellular processes involving protein folding and homeostasis. These characteristics make the chaperones potential targets to the treatment of many diseases. Hsp70 and Hsp90, in special, are highly conserved ubiquitous proteins that act in the folding of nascent proteins, protein aggregation prevention, aggregate recovering, signaling and cellular growth, among others. However, for these proteins to effectively fulfill their function, they must be modulated by molecular co-chaperones. SGT is a co-chaperone that can be divided into three domains: a N-terminal domain, a TPR domain and a C-terminal domain, being the TPR domain responsible for the interaction with the EEVD motif at the C-terminus of cytoplasmic Hsp90 and Hsp70. SGT is found in various organisms; among they are the protozoans of Leishmania spp.. These organisms are responsible for leishmaniasis, a neglected disease that affects thousands people every year, mainly at underdeveloped countries. Evidences indicate that SGT in protozoans are essential to the growth and viability of promastigote form. Therefore, the structural and functional study of the Leishmania braziliensis SGT (LbSGT) is presented. Recombinant LbSGT was produced and purified. The structural characterization points that LbSGT is rich in α-helix secondary structure and behaves as an elongated dimer in solution. Chemical and thermal stability data suggest that LbSGT is formed by domains of different stabilities. LbSGT was identified in vivo and the western blotting indicates its cognate presence in the protozoan promastigote forms. The interaction assays show that the interaction between LbSGT and Hsp90 of L. braziliensis (LbHsp90) or human Hsp70-1A (used as model protein) were different from the interaction between LbSGT with MEEVD peptide. Moreover, these data suggests that the interaction between LbSGT and Hsp70-1A and LbHsp90 involves additional protein regions besides the Hsp70-1A and LbHsp90 interaction motif. Altogether, the observed functional and structural proprieties of LbSGT accord to the SGT possible function as an adapter protein between the Hsp70 and Hsp90 systems in the foldossome.

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Silva, Kelly Pereira da. "Estudos estruturais e funcionais da Hsp90 de Leishmania braziliensis e suas co-chaperonas p23." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-24072012-172313/.

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As chaperonas moleculares são proteínas que auxiliam no enovelamento correto de outras proteínas, entre outras funções importantes para as células, motivo pelo qual elas têm sido alvo para o combate de várias doenças. As Hsp90 (82-96 kDa) são chaperonas abundantes que interagem com diversas proteínas-cliente. São constituídas por três domínios: N-terminal, intermediário ou central (M) e C-terminal, o qual é responsável pela dimerização da proteína. A atividade da Hsp90 está diretamente relacionada à sua atividade ATPásica. Durante o ciclo funcional, as Hsp90 podem interagir com inúmeras co-chaperonas. Uma delas é a co-chaperona p23 (18-22 kDa) que interage com o dímero da Hsp90 e algumas das suas funções são a inibição da atividade ATPásica e atividade chaperona. O objetivo do trabalho foi obter a proteína recombinante Hsp90 de Leishmania braziliensis e os domínios N e N+M, determinar fatores importantes que relacionam mudanças conformacionais e função da Hsp90 e as bases moleculares da inibição por GA. Também obter as co-chaperonas Lbp23A e Lbp23B e investigar a interação com a LbHsp90 e suas funções. As proteínas produzidas foram purificadas e caracterizadas por técnicas biofísicas. Em solução, a LbHsp90 foi caracterizada como dímero assimétrico e as demais proteínas como monômeros assimétricos.A interação da LbHsp90 e domínios com nucleotídeos foi analisada por fluorescência e as constantes de dissociação ficaram em torno de 150 µM. A afinidade por GA foi maior que a verificada para ATP e em ordem crescente para LbHsp90, LbHsp90_NM e LbHsp90_N. A LbHsp90 apresentou grande atividade chaperona em relação à citrato sintase, de maneira independente de ATP. A LbHsp90 mostrou baixa atividade ATPásica, a qual foi inibida pela GA com IC50 de 0,7 µM. Tanto a Lbp23A quanto a Lbp23B inibiram a atividade ATPásica da LbHsp90, porém a Lbp23A aproximou-se de 100% de inibição e a Lbp23B apenas 30%. A interação in vitro entre a LbHsp90 e a Lbp23B foi observada por pull-down na presença/ausência de nucleotídeos e essa técnica não se mostrou adequada para a Lbp23A.O pioneirismo do trabalho com a Hsp90/p23 de L. braziliensis oferece uma grande contribuição para futuros trabalhos que visam o entendimento das relações funcionais entre essas proteínas e o contexto das Hsp90 no desenvolvimento da leishmaniose.
Molecular chaperones are proteins involved in proper folding of other proteins, and others important cellular functions, why they have been targeted for combating various diseases. The Hsp90 (82-96 kDa) are ubiquitous chaperones that interact with a wide range of client proteins. They are formed by three domains: N-terminal, central or middle (M), and C-terminal, which is responsible by its dimerization. The Hsp90 activity is related to its ATPase activity. During the Hsp90 functional cycle, diverse co-chaperones. One of them is the p23 (18 kDa), that interacts with one Hsp90 dimer, and some p23 functions are the inhibition of Hsp90 ATPase activity and chaperone activity. The aim of this work was obtain the Hsp90 recombinant Leishmania braziliensis Hsp90, the N and N+M domains, to determine the important factors related to conformational changes and Hsp90 function, and the molecular basis of GA inhibition. Also, to obtain the Lbp23A and Lbp23B co-chaperones in order to establish relevant aspects for LbHsp90 interaction and its co-chaperones functions. The recombinant proteins were produced, purified and characterized by biophysics techniques. The LbHsp90 was identified as an asymmetric dimer for whereas the others were identified as asymmetric monomers. The interactions between LbHsp90 and domains with nucleotides were determined by fluorescence and the dissociation constants were about 150 µM. The GA-affinity was greater than ATP one, in increasing order for LbHsp90, LbHsp90_NM, and LbHsp90_N. The LbHsp90 showed large chaperone activity related to citrate synthase independently of ATP. The LbHsp90 presented low ATPase activity, which was inhibited by GA with a IC50 of 0,7. The Lbp23A and Lbp23B inhibited the ATPase activity with different values, the Lbp23A inhibition was closed to 100% whereas the Lbp23B one was 30%. The in vitro interaction between the LbHsp90 and Lbp23B was observed by pull-down, in the absence or presence of nucleotides, and for Lbp23A this technique was not appropriated. The pioneering work with Hsp90/p23 from L. braziliensis offers an important contribution to future studies aimed at understanding the functional relationships between these proteins and the context of Hsp90 in the development of leishmaniasis.

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Reis, Dayane Eliara Bertolino. "Caracterização estrutural da Hsp70/Hsp90 organizing protein (Hop) de Plasmodium falciparum." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-28022018-095723/.

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A malária é uma doença tropical negligenciada causada por protozoários do gênero Plasmodium spp, afeta populações em mais de 100 países ao redor do globo, apresentando 219 milhões de novos casos por ano sendo, portanto, um grave problema de saúde pública. Apresenta um ciclo complexo e digenético, necessitando do mosquito vetor e do hospedeiro vertebrado para se completar - ciclo este que envolve etapas de transformação e adaptação, já que o patógeno passa por 28 formas diferentes ao longo do ciclo, além de enfrentar situações de stress térmico, no momento do contágio e durante os picos febris. Sendo assim, é necessário que o protozoário garanta sua sobrevivência e possibilite a infecção do hospedeiro. Isso é realizado com a assistência de chaperonas moleculares, proteínas estas que são superexpressas no estágio intra-eritrocitário. Uma dessas proteínas é a Hsp90, uma Heat shock protein com diferentes funções, entre elas, maturação de proteínas clientes, encaminhamento de proteínas para translocação por membranas e marcação de proteínas para degradação. Para cumprir adequadamente as diversas funções, as Hsp90 contam com o auxílio de co-chaperonas, como a Hsp70/Hsp90 Organizing Protein (Hop) que modulam sua função. A Hop é uma co-chaperona do sistema foldossoma formado pelas Hsp70 e Hsp90 citoplasmáticas e que atua como proteína adaptadora transferindo proteínas clientes da primeira para a segunda chaperona molecular. A interação da Hop com Hsp70 e Hsp90 ocorre via domínios TPR, que se ligam ao motivo EEVD presente na extremidade C-terminal de ambas as chaperonas citoplasmáticas. É encontrada em diversos organismos, incluindo Plasmodium falciparum, o agente etiológico da malária. Sendo assim, conhecer a Hop de P. falciparum (PfHop), estrutural e funcionalmente, é importante para o entendimento do funcionamento das Hsp90 e Hsp70, proteínas essenciais para a sobrevivência do patógeno e, portanto, possíveis alvos terapêuticos. A PfHop recombinante foi obtida com pureza superior a 95%. A caracterização biofísica da mesma foi feita através de diferentes técnicas. Como outras Hops, a PfHop é majoritariamente constituída por hélices alfa. Os parâmetros hidrodinâmicos determinados sugerem que a PfHop se comporta como um equilíbrio monômero-dímero quando em solução. Dados de espalhamento de raios X a baixo ângulo mostram a PfHop como uma proteína dimérica e alongada. Este trabalho de dissertação de mestrado permitiu alcançar a caracterização estrutural da PfHop e com este conhecimento, espera-se avançar na caracterização funcional da mesma sobre a Hsp70 e Hsp90.
Malaria is a neglected tropical disease caused by protozoa of the genus Plasmodium spp, affects populations in more than 100 countries around the globe, presenting 219 million new cases per year and is therefore a serious public health problem. It presents a complex and digenetic cycle, necessitating the vector mosquito and the vertebrate host to complete - this cycle involves transformation and adaptation stages, since the pathogen goes through 28 different forms along the cycle, besides facing situations of thermal stress , At the time of the contagion and during the feverish peaks. Thus, it is necessary that the protozoan guarantees its survival and makes possible a host infection. This is accomplished with the assistance of molecular chaperones, proteins that are overexpressed in the intra-erythrocyte stage. A life of proteins and Hsp90, a protection of thermal shock with different functions, among them, maturation of client proteins, routing of proteins for membrane translocation and labeling of proteins for degradation. To comply properly, for example, as Hsp90 rely on the help of co-chaperones, such as Hsp70 / Hsp90 Organizing Protein (Hop) that modulate their function. The Hop is a co-chaperone system folded by Hsp70 and Hsp90 cytoplasmic and which acts as an adapter protein transferring client proteins from the first to the second molecular chaperone. The interaction of Hop with Hsp70 and Hsp90 occurs via TPR domains, which bind to the EEVD motif present at the C-terminus of both as cytoplasmic chaperones. It is found in several organisms, including Plasmodium falciparum, the etiologic agent of malaria. Therefore, knowing a Hop of P. falciparum (PfHop), structurally and functionally, is important for the understanding of the functioning of Hsp90 and Hsp70, essential proteins for a pathogen survival and, therefore, in all the therapeutic aspects. A recombinant PfHop was obtained in greater than 95% purity. The biophysical characterization by the same brand made through different techniques. As there is Hops, a PfHop is mostly constituted by alpha helices. The indicated parameters are a PfHop behaves as a monomer-dimer balance when in solution. Higher low-angle X-ray scattering data on PfHop as a dimeric and elongated protein. This work of master\'s dissertation allowed to reach a structural characterization of the PfHop and with this knowledge, it is expected to advance in the functional characterization of the same in Hsp70 and Hsp90.

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Gaiser, Andreas M. [Verfasser]. "Studies on the molecular chaperone Hsp90 and its regulation by co-chaperones in Caenorhabditis elegans / Andreas M. Gaiser." München : Verlag Dr. Hut, 2011. http://d-nb.info/1011441659/34.

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Cardeal, Isabel Cristina Mendonça de Azevedo. "Uso terapêutico de chaperones em doenças conformacionais." Master's thesis, [s.n.], 2013. http://hdl.handle.net/10284/4093.

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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
Os chaperones são proteínas que têm por função principal assistir e promover o enrolamento adequado de cadeias polipeptídicas, quer as cadeias recém-sintetizadas nos ribossomas do retículo endoplasmático quer pós-traducionalmente durante o seu processo de translocação através das membranas intracelulares. No ambiente celular existem várias classes de chaperones não relacionadas estruturalmente que se organizam formando redes cooperativas de vigilância e manutenção da conformação nativa de proteínas ou de indução da destruição de proteínas misfolded através da formação de corpos de inclusão e posterior degradação pelas proteases do sistema lisosomal ou proteossomal. As doenças conformacionais, como por exemplo as doenças amiloides, são caracterizadas pela redução do nível de proteína nativa e pela acumulação da respetiva proteína misfolded, resultando na sua aglomeração e deposição em tecidos específicos que está associada a um aumento de morbilidade e mortalidade. A investigação ao nível terapêutico sugere que o tratamento com chaperones farmacológicos pode ser preventivo, ao reduzir o stress oxidativo que é um agente causador comum a estas doenças, ou curativo, seja pela aplicação/administração de chaperones farmacológicos ou pelo meio de indução de produção destes chaperones pelo próprio organismo. No entanto, ainda existe um longo caminho para percorrer até que seja identificado um fármaco que consiga devolver a estes doentes a qualidade de vida que eles merecem, facto que torna fundamental a continuidade da investigação sobre chaperones, desde a elucidação do seu funcionamento à sua aplicação farmacológica. Chaperones are proteins whose function is to assist and promote the correct folding of proteins, either newly proteins synthesized at ribosomes of the endoplasmic reticulum or post-translationally during the process of translocation across intracellular membranes. In the cellular environment, there are several classes of structurally unrelated chaperones. These molecules are organized in cooperative networks involved in surveillance and maintenance of the native conformation of proteins, or in the destruction of misfolded proteins through the formation of inclusion bodies that are subsequently degraded by lysosomal or proteosomal systems. Protein conformational diseases, such as amyloid disorders, are characterized by a reduction in the level of native protein and, simultaneously, by the accumulation of misfolded proteins. These alterations result in the agglomeration of misfolded proteins and their accumulation at toxic levels in a specific tissue is associated with disorders with an increased morbidity and mortality. Data from investigation of therapeutic options suggest that pharmacological chaperons may act preventively, by reducing oxidative stress which is a common causative agent of these diseases or correctively by either the application/administration of these molecules or the induction of its production by the body itself. However, there is still a long way until the identification of a drug that can return to these patients the quality of life they deserve, thus underline the importance of future research on chaperones, not only to better elucidate its molecular mechanism in the cell but also to identify more effective drugs for the treatment of conformational diseases.

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Gava, Lisandra Marques 1982. "Caracterização e interação do domínio C-terminal da chaperona Hsp90 humana e das co-chaperonas Tom 70 e Hop." [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314027.

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Orientador: Carlos Henrique Inácio Ramos
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-18T21:37:15Z (GMT). No. of bitstreams: 1 Gava_LisandraMarques_D.pdf: 9573403 bytes, checksum: 4d69a29d08ffc20e4544b876f131fb0d (MD5) Previous issue date: 2011
Resumo: A função biológica das proteínas está relacionada à sua estrutura tridimensional adquirida pelo processo de enovelamento protéico. Neste contexto, proteínas denominadas, genericamente, de chaperonas moleculares exercem papel fundamental atuando no auxílio do enovelamento correto, no reenovelamento e na dissociação de agregados protéicos. A Hsp90 é uma das chaperonas moleculares mais importantes, é essencial para a viabilidade celular em eucariotos e está normalmente associada a proteínas atuantes no ciclo e sinalização celular, o que torna essa chaperona um alvo bastante interessante para abordagens terapêuticas de diversas doenças. A Hsp90 pode ser modulada por co-chaperonas diversas. Nesse trabalho foram caracterizadas as proteínas CHsp90 (domínio C-terminal da Hsp90 humana), e as co-chaperonas Hop e Tom70, além da interação entre C-Hsp90 e Tom70. Foram aplicadas técnicas de dicroísmo circular e emissão de fluorescência do triptofano; seguidas pela caracterização por ultracentrifugação analítica, gel filtração analítica, espalhamento dinâmico de luz, cromatografia de gel filtração acoplada a espalhamento de luz em multi-ângulos (SEC-MALS) e gel nativo. Para os ensaios de interação foram aplicadas técnicas de pull-down, SEC-MALS e calorimetria de titulação isotérmica. As proteínas foram produzidas puras e enoveladas, com estado oligomérico determinado como dímero para C-Hsp90 e monômero para Hop e Tom70, sendo que essas também foram encontradas como espécies diméricas. A estequiometria de interação entre a C-Hsp90 e Tom70 foi determinada em 1 monômero da Tom70 para 1 dímero da C-Hsp90, com KD de 360 ± 30 nM, ?Happ = -2,6 ± 0,1 kcal/mol e ?S = 21 ± 1 cal/mol.K, sugerindo que a interação é dirigida por entalpia e entropia. Os resultados obtidos nesse trabalho contribuem para uma melhor compreensão do sistema Hsp90, que está envolvido em diversos processos celulares essenciais e patológicos, como doenças neurodegenerativas, processos inflamatórios, infecções e câncer
Abstract: The biological function of proteins is related to its three dimensional structure acquired via protein folding process. In this context, the molecular chaperones play a key role acting as auxiliary protein on protein folding, refolding and dissociation of protein aggregates. Hsp90 is one of the most important molecular chaperones, is essential for cell viability in eukaryotes and is usually associated with proteins involved in cell cycling and cell signaling, which makes these chaperone a very interesting targeting for therapeutic approaches for several diseases. The chaperone activity of Hsp90 can be modulated by other proteins, called co-chaperones. In this work, we characterized the protein C-Hsp90 (Cterminal domain of human Hsp90) and the co-chaperones Hop and Tom70, and also the interaction between C-Hsp90 and Tom70. Circular dichroism and fluorescence emission of tryptophan was first applied for initial characterization of the proteins, followed by analytical ultracentrifugation, analytical gel filtration, dynamic light scattering, size exclusion chromatography - multi angle light scattering (SEC-MALS) and native gel. The interaction between C-Hsp90 and Tom70 were measured by techniques like pull-down, SEC-MALS and isothermal titration calorimetry. The proteins were produced pure and soluble and their oligomeric state were determined as dimer for C-Hsp90, and monomer for Hop and Tom70, these two co-chaperones were also found as dimeric species. The stoichiometry of interaction between C-Hsp90 and Tom70 was determined by SEC-MALS and ITC as been 1 dimer of C-Hsp90 to 1 monomer of Tom70, with a KD of 360 ± 30 nM, ?Happ = -2.6 ± 0.1 kcal/mol and ?S = 21 ± 1 cal/mol.K, suggesting that these interaction is driven by both, enthalpy and entropy. The results contribute to a better understanding of the important Hsp90 machinery, which is involved in many essential cellular and pathological processes, such as neurodegenerative diseases, inflammation, infection and cancer
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular

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Ayala, Mariscal Sara Maria. "Modulation of Alzheimer's disease amyloid beta peptide aggregation by molecular chaperones, polyphosphates and metal ions, and their interplay." Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30108.

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La maladie d'Alzheimer est la démence la plus répandue dans le monde. Le nombre de cas augmente de manière exponentielle et il est donc important de comprendre les mécanismes moléculaires donnant lieu à cette terrible maladie. Une des hypothèses les plus supportées est celle suggérant que la production et dégradation déséquilibrées de l'amyloïde-beta (Aß), un peptide de 42 acides aminés trouvé dans tous les individus sains, est un événement clé dans le déroulement de la maladie d'Alzheimer. En effet, une production accrue ou une dégradation faible du peptide ont pour conséquence son agrégation et accumulation dans des plaques de fibres entre les neurones des régions spécifiques du cerveau. C'est pourquoi la modulation de l'agrégation du peptide Aß est une des approches envisageables pour modifier l'évolution de la maladie d'Alzheimer. Les protéines chaperons dont une des fonctions est d'assister d'autres protéines dans leur repliement, sont parmi les molécules les plus étudiées pour leur capacité modulatrice de l'agrégation des protéines (inclus le peptide Aß). Plusieurs chaperons ont montré la capacité d'inhiber la formation des fibres par l'Aß. Cependant, du fait que les chaperons sont des molécules conservées et peu spécifiques, leur surexpression ou administration directe peut avoir des conséquences négatives si les chaperons interagissent avec des protéines autres que la protéine cible. Dans ce travail, nous nous sommes intéressés à une protéine chaperon bactérienne possédant une forte activité " holdase " (i.e., elle empêche le repliement précoce des protéines) comme possible modulateur de l'agrégation du peptide Aß. Le chaperon sauvage a une très faible capacité d'inhibition de la formation de fibres par le peptide Aß. Cependant, nous avons démontré qu'en modifiant légèrement la surface de liaison du chaperon, la protéine devient un puissant inhibiteur de l'agrégation d'Aß. En parallèle, nous nous sommes intéressés à l'influence des ions métalliques sur l'agrégation du peptide Aß. [...]
Alzheimer's disease is the most frequent type of dementia. With an exponentially growing number of cases, understanding the underlying molecular events leading to this devastating condition is of crucial importance. Much evidence points to a disequilibrium in the production and degradation of amyloid beta (Aß), a normally physiological 42 amino acid peptide, as an early key event in Alzheimer's etiology. Whether Aß is overproduced or poorly degraded, the overall result is an abnormally large pool of peptide that gradually aggregates forming extracellular deposits of fibrils, called amyloid plaques, in specific brain regions. Hence, modulation of Aß aggregation process is one of the suggested approaches to control the evolution of Alzheimer's disease. Universally conserved molecular chaperones have been intensively studied for their capacity to prevent aggregation of disease-related proteins, and many of them have proven to efficiently modulate Alzheimer's Aß aggregation. In a scenario where chaperones are overexpressed or directly administered into the affected tissue, the universal conservation and the relatively poor client-specificity of generic chaperones can become a downside because of the risk of interaction with proteins other than the targeted one is not dismissible, and thus the consequences unpredictable. In the first part of this work, we looked upon a bacterial chaperone call SecB with an unusually robust holdase activity (i.e. it prevents early protein folding) as a promising modulator of Alzheimer's Aß peptide aggregation. [...]

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Dhavale, Madhura Vinayak. "Role of Molecular Chaperonin CCT and Its Co-Chaperone PhLP1 in the Assembly of mTOR Complexes." BYU ScholarsArchive, 2017. https://scholarsarchive.byu.edu/etd/6942.

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mTOR is the central kinase in biochemical pathways that regulate cellular growth, protein synthesis and cell survival. Deregulation of mTOR signaling results in uncontrolled cell proliferation and hence is implicated in various cancers and autoimmune diseases. mTOR functions through two distinct signaling complexes, called mTORC1 and mTORC2. CCT is a cytosolic chaperonin that assists in folding of several protein substrates. In these studies, we have identified two components of the mTOR complexes, mLST8 and Raptor, as substrates of CCT. We have performed biochemical and signaling studies which indicate that CCT is involved in assembly and signaling of mTOR complexes by folding β-propeller domains of mLST8 and Raptor. We have also obtained high resolution structural information of the mLST8-CCT complex by cryo-EM and mass spectrometric cross-linking. Moreover, we have explored the role of PhLP1 as a co-chaperone for CCT in the assembly of mTOR complexes. Interestingly, we found that PhLP1 plays very different roles in the case of mLST8 and Raptor. While PhLP1 participate in assembly of mLST8 into mTOR complexes, it facilitates degradation of Raptor. These biochemical data, combined with structural information, can be used to design small molecules that modulate mTOR signaling by affecting the formation of intact mTOR complexes.

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Kota, Jhansi. "Membrane chaperones : protein folding in the ER membrane /." Stockholm : Karolinska institutet, 2007. http://diss.kib.ki.se/2007/978-91-7357-102-9/.

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Bennett, J. C. Q. "Substrate-specific export chaperones mediating bacterial flagellum assembly." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596575.

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During flagellum assembly by motile enterobacteria, the highly helical flagellar proteins destined for polymerization into the growing cell surface structure are exported through a small channel at the core of the flagellum, but the mechanism preventing their premature folding and oligomerization in the cytosol remained unknown. This thesis addresses this issue by characterizing the roles of FlgN and FliT, two flagellar proteins of previously undetermined function. Affinity blotting of radiolabelled FlgN to Proteus mirabilis or Salmonella typhimurium cell extracts indicated that it binds to two flagellar proteins. Comparative blotting of proteins secreted by wild-type S. typhimurium and specific flagellar mutants suggested that FlgN binds the flagellar hook-associated proteins (HAPs) FlgK and FlgL. This was confirmed by FlgN binding to each HAP, following their artificial expression in Escherichia coli. Export of FlgK and FlgL was specifically reduced in a S. typhimurium flgN mutant, concomitant with increased release of FliC and FliD, the immediately distal components of the flagellum. Comparable experiments showed that FliT binds specifically to the third HAP, FliD. Removal of HAP C-terminal domains abolished binding to FlgN and FliT, and polypeptides comprising each of the HAP C-termini were specifically bound by FlgN and FliT. The data suggest that FlgN and FliT are specialized substrate-specific chaperones that act as 'bodyguards' to shield interactive surfaces of their targets prior to export.

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Coward, Christopher. "Chaperones and ATP-dependent proteases of Lactococcus lactis." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392771.

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Kosmaoglou, M. "Using molecular chaperones to manipulate rhodopsin retinitis pigmentosa." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18766/.

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The experiments described in this thesis were designed in order to test the hypothesis that molecular chaperones are involved in the biogenesis of rhodopsin and may be used in the treatment of rhodopsin retinitis pigmentosa. Rhodopsin is the prototypical G-protein coupled receptor found at high concentration in the outer segments of rod photoreceptor cells. Rhodopsin is made up of the rod opsin apoprotein and 11-cis-retinal, the photoactive ligand. Rhodopsin initiates the phototransduction cascade under dim light conditions and mutations in its primary sequence have been linked to the neurodegenerative blinding disease, retinitis pigmentosa. Mutations such as P23H, cause the misfolding of the protein, resulting in its retention in the endoplasmic reticulum of heterologous expression systems and the inner segment of photoreceptor cells. Whilst selecting suitable modifiers, the subcellular compartments occupied by rhodopsin during its biogenesis and the chaperones resident in these, were considered. Calnexin is a central component of the quality control machinery in the endoplasmic reticulum. As calnexin has been widely documented to assist in the maturation of nascent glycoproteins, mouse embryonic fibroblast cells were used, which expressed a truncated version of calnexin, unable to bind client glycoproteins. The expression of rod opsin was compared in cells expressing truncated calnexin and in their wild-type counterparts, assessing the contribution of calnexin in the subcellular localization and biochemical profile of rod opsin. Calnexin was found to be dispensable for the maturation and folding of rod opsin. EDEM1, the ER-degradation enhancing mannosidase α-like 1 protein, has been shown to accelerate the degradation of misfolded glycoproteins, extracting these from futile folding attempts in the calnexin cycle. EDEM1 was found to enhance the degradation of P23H rod opsin and importantly, promoted the cell surface expression of any remaining P23H molecules which escaped degradation. The localization of EDEM1 in murine retina was determined to be within a subset of the inner segment and rhodopsin was found to form a physiological immune complex in porcine retina. The binding protein, BiP, associates with nascent proteins as these are translated and translocated in the ER lumen. A toxin that efficiently cleaves BiP in two fragments was used in order to probe the effects of BiP deletion on the biogenesis of wild-type and mutant rod opsin. Wild-type rod opsin was found retained in the endoplasmic reticulum in the absence of functional BiP and was misfolded as ubiquitin was recruited to the endoplasmic reticulum surface from a previous diffuse localization. Therefore BiP appears to be critical for maintaining rod opsin in a folding competent state. A chaperone on the cytoplasmic face of the endoplasmic reticulum, namely HSJ1b, has previously been shown to result in the stalling of wild-type and mutant rod opsin folding. We have investigated the effects of coexpressing CHIP, the carboxy-terminus of Hsp70-interacting protein. CHIP is an E3 ligase, which has been shown to present misfolded proteins to the proteasome for degradation, via an association with the Hsp70 machinery and HSJ1b initiates the process by stimulating ATP hydrolysis by Hsp70. In the presence of HSJ1b, expression of CHIP resulted in the degradation of rod opsin by the proteasome. Hence chaperones in the endoplasmic reticulum lumen and the cytoplasm can be used to manipulate mutant P23H rod opsin and may be used in the treatment of rhodopsin RP.

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MENON, SHEKAR. "INTERACTION BETWEEN REDOX CHAPERONES AND RDW MUTANT THYROGLOBULIN." University of Cincinnati / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1074086301.

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Shrestha, Pooja. "Mechanism of substrate protein remodeling by molecular chaperones." University of Cincinnati / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1378113185.

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Beliakoff, Jason Allyn. "Regulation of estrogen receptor function by molecular chaperones." Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/289969.

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The estrogen receptor (ER) plays a major role in breast cancer progression, and ER+ tumors respond favorably to hormonal manipulation. The selective estrogen receptor modulator (SERM) tamoxifen (Tam) induces remissions in most ER+ patients. However, acquired resistance is often observed. Tam-resistant breast cancer is sensitive to other antiestrogenic compounds, but resistance to these agents has also been described, illustrating a major limitation to antiestrogen therapy. Therefore, we investigated a ligand-independent approach for treating Tam-resistant breast cancer by targeting the molecular chaperone Hsp90. The ER exists in a multi-protein complex containing Hsp90, which regulates the activity and stability of the receptor. Hsp90 regulates the stability of other proteins relevant to breast cancer, including Akt and Raf-1. The benzoquinone ansamycin antibiotic geldanamycin (GA) and its clinically relevant analog, 17-demethoxy-17-allylaminogeldanamycin (17AAG), bind to Hsp90 and induce the degradation of Hsp90 clients. In these studies, we show that GA depletes ER levels in Tam-resistant cell lines, and the Hsp90 clients Akt and Raf-1. Unexpectedly, Tam inhibited GA-induced degradation of the ER, but not Akt and Raf-1. This effect was consistent in vivo, where ER levels were measured in tumor xenografts growing in Tam-supplemented mice. However, Tam-stimulated tumor growth was inhibited by 17AAG, and tumor Akt and Raf-1 levels were downregulated. Immunoprecipitation experiments showed that Tam does not inhibit GA-induced changes in the ER-chaperone complex, suggesting an alternate mechanism for the inhibition of GA-mediatied ER degradation. Through cell fractionation, immunostaining, and chromatin immunoprecipitation experiments, we have found that the mechanism involves prolonged association of the ER with the DNA in the presence of Tam, which leads to nuclear accumulation of the ER and sequestration from the proteasome. Furthermore, inhibition of GA-induced ER degradation was inhibited by another SERM, Raloxifene, indicating that the effect is not Tam-specific. Based on its ability to downregulate critical signaling proteins involved in breast cancer, including the ER, 17AAG may provide a useful alternative for patients that have failed hormonal therapy. Because SERMs inhibit the degradation of ER protein induced by GA, they may compromise the efficacy of GA on ER activity, and combined therapy should be approached with caution.

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Tennant, Esther Paula. "Interactions of the chaperones and components of UB system in the formation and propagation of the yeast prion [PSI+]." Thesis, Available online, Georgia Institute of Technology, 2005, 2005. http://etd.gatech.edu/theses/available/etd-05292005-220155/.

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Fellerer, Christine. "Molecular chaperones involved in preprotein targeting to plant organelles." Diss., Ludwig-Maximilians-Universität München, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-163801.

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Taylor, David M. 1977 Nov 23. "Understanding the regulation of molecular chaperones in motor neurons." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111857.

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Cells are constantly challenged by acute and chronic stresses that must be counteracted by upregulation of protective pathways. The premise of this thesis is that motor neurons have an impaired ability to trigger these protective mechanisms, which may contribute to their preferential vulnerability in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). The first objective was to study the involvement of metallochaperones in motor neuronal stress response, including their potential for rescuing motor neurons from toxicity conferred by a mutant Cu/Zn-superoxide dismutase (SOD1G93A ) that causes a form of familial ALS. Motor neurons in dissociated spinal cord cultures failed to induce the metallochaperone, metallothionein (MT), in response to classical MT inducers, although overexpression of MT in motor neurons failed to protect them from SOD1G93A. A second response system, involving protein chaperones called heat shock proteins (Hsp), was more therapeutically promising, but was also impaired in motor neurons due to an inability to activate the regulatory protein heat shock transcription factor 1 (Hsf1). The remaining objectives were to examine if activation of Hsf1 in motor neurons would protect against SOD1G93A and to understand the mechanisms responsible for its impaired activation. A constitutively active form of Hsf1 induced multiple Hsps in motor neurons and nearly eliminated SOD1G93A toxicity and aggregation. Experiments also demonstrated that failure of stressed motor neurons to activate endogenous Hsf1 is not a result of inappropriate or insufficient activity of kinases that phosphorylate key residues of Hsf1 in nonneuronal cell lines with a competent heat shock response. Disruption of inhibitory Hsp90/multichaperone complexes is another important step in Hsf1 activation. Four different pharmacological inhibitors of Hsp90 induced multiple Hsps in motor neurons, although failure to observe the same response by targeting inhibitory complexes with activator of Hsp90 ATPase 1 (Aha1) or Daxx suggested other mechanisms were involved. A constitutively active form of calcium/calmodulin-dependent kinase N induced Hsp70 in motor neurons, but not in fibroblasts and likely through an Hsf1-independent mechanism. These results provide further evidence for disparity between the stress response of motor neurons and other cells and suggest the possibility of a unique Hsp regulatory system in neurons.

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Bhangoo, Melanie. "Multiple Hsp40 chaperones function in Tom70-dependent mitochondrial import." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=18268.

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Mitochondrial preproteins with internal targeting signals are imported via the Tom70 receptor, which is located on the outer mitochondrial membrane. However, before being targeted to the mitochondria, preproteins are complexed with cytosolic chaperones, maintaining them in an import competent state. The Adenine Nucleotide Transporter (ANT), a carrier protein of the mitochondrial inner membrane, follows this Tom70 pathway and its purified mature form is identical to that of the preprotein. Purified ANT was reconstituted in reticulocyte lysate, and bound proteins were identified by mass spectrometry. In addition to Hsc70 and Hsp90, a specific subset of co-chaperones were found, but no mitochondria specific targeting factors. Interestingly, three different Hsp40-related J-domain proteins were identified: DJA1, DJA2 and DJA4. The DJAs were found to bind preproteins to different extents through their C-terminal regions. DJAs lacking the N-terminal J-domains interfered with mitochondrial import, and blocked the binding of Hsc70 to preprotein, but with varying efficiency. Moreover, the DJAs showed significant differences in their ability to activate the Hsc70 ATPase as well as promote Hsc70-dependent protein folding. No single DJA was superior to the others in all characteristics, but each had an individual profile of properties. The Hsp90 co-chaperones p23 and Aha1 also regulated Hsp90-preprotein interactions. Multiple co-chaperones with similar yet distinct properties may cooperate in optimal chaperone-preprotein complexes.
Les pré-protéines nucléaires, avec une séquence de ciblage interne destinées à la mitochondrie, sont importées via le récepteur Tom70, situé sur la membrane externe de la mitochondrie. Cependant, avant d’être amenées vers la mitochondrie, les pré-protéines sont maintenues dans un état permettant l’importation par un complexe de chaperons dans le cytosol. Le Transporteur du Nucléotide Adénine (ANT), une protéine porteuse de la membrane interne de la mitochondrie suit le cheminement via Tom70 et sa forme mature purifiée est semblable à celle de la pré-protéine. L’ANT purifiée reconstituée dans du lysat de réticulocyte a permit d’identifier par spectroscopie de masse les protéines avec lesquelles elle interagit. A part Hsc70 et Hsp90, un sous-groupe spécifique de co-chaperons moléculaires furent identifiés mais aucun facteur de ciblage spécifique pour la mitochondrie. Trois protéines voisines de Hsp40 avec un domaine J furent identifiées : DJA1, DJA2 et DJA4. Les trois protéines DJA accrochent les pré-protéines via leur région C-terminale, mais avec une force variable. Des protéines DJA, avec un domaine J manquant dans la région N-terminale, interféraient avec l’importation mitochondriale et bloquaient l’interaction de Hsc70 avec les pré-protéines, tout ceci avec une efficacité variable. De plus les protéines DJA montrent une habileté différente à stimuler l’hydrolyse d’ATP par Hsc70 de même que son activité de repliement de protéines. Pas une des protéines DJA n’était supérieure aux autres dans tous les domaines, chacune ayant plutôt un profile caractéristique. Les co-chaperons moléculaires de Hsp90, p23 et Aha1, régulent les interactions de ce dernier avec les pré-protéines. Ainsi, de multiples co-chaperons moléculaires avec des caractéristiques de même ordre mais différentes peuvent coopérer pour des complexes chaperons/pré-protéines optimaux.

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Thomas, Joanne. "Functional analysis of flagellum-specific type III export chaperones." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615655.

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30

Luo, Wen-I. "The Role of Chaperones in Iron-Sulfur Cluster Biogenesis." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1325168796.

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31

Zhang, Zhehao, and 張哲豪. "Triptolide inhibits Hsp90β atpase and chaperone activity to promote cell cycle arrest and programmed cell death through multiple regulations." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2015. http://hdl.handle.net/10722/209519.

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32

Benoit, Matthias. "Histone H3 variants and chaperones in Arabidopsis thaliana heterochromatin dynamics." Thesis, Clermont-Ferrand 2, 2014. http://www.theses.fr/2014CLF22497/document.

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Afin d’étudier la prise en charge des histones H3 jusqu’à l’ADN et pour comprendre l’influence de leur dynamique dans l’organisation d’ordre supérieur de la chromatine, une analyse des chaperonnes d’histones a été menée. Nous avons identifié et caractérisé les sous-unités du complexe HIR, impliqué dans l’assemblage de la chromatine réplication-indépendante chez Arabidopsis. La perte d’AtHIRA, la sous-unité centrale du complexe, affecte le niveau d’histone soluble, l’occupation nucléosomale des régions euchromatiniennes et héterochromatiniennes ainsi que la mise sous silence transcriptionnel des séquences d’ADN répétées. Alors que le complexe HIR ne participe pas à l’organisation d’ordre supérieur de la chromatine, j’ai montré que CAF-1, impliqué dans l’assemblage de la chromatine au cours de la réplication, joue un rôle central dans la formation des chromocentres. Lors du développement post-germinatif des cotylédons, les séquences d’ADN répétées centromériques et péricentromériques se concentrent dans les chromocentres et s’enrichissent en histone H3.1 de manière CAF-1 dépendante. Cet enrichissement, associé à des modifications post-traductionnelles d’histones associées à un état répressif de la transcription, participe à la formation des chromocentres et met en évidence l’importance de l’assemblage de la chromatine par CAF-1 dans la structure et le maintien du génome. Alors que la perte individuelle de HIR ou de CAF-1 n’affecte pas la viabilité, l’absence des deux complexes altère fortement l’occupation nucléosomale et le développement des plantes. Ceci suggère que la compensation fonctionnelle entre ces complexes de chaperonnes ainsi que la plasticité des voies de dépôt des histones restent limitées
To understand how histones H3 are handled and how histone dynamics impact higher-order chromatin organization such as chromocenter formation in Arabidopsis, a comprehensive analysis of the different histone chaperone complexes is required. We identified and characterized the different subunits of the Arabidopsis HIR complex. AtHIRA is the central subunit and its loss affects non-nucleosomal histone levels, reduces nucleosomal occupancy not only at euchromatic but also at heterochromatic targets and alleviates transcriptional gene silencing. While the HIR complex-mediated histone deposition is dispensable for higher-order organization of Arabidopsis heterochromatin, I show that CAF-1 plays a central role in chromocenter formation. During postgermination development in cotyledons when centromeric and pericentromeric repeats cluster progressively into chromocenter structures, these repetitive elements but not euchromatic loci become enriched in H3.1 in a CAF-1- dependent manner. This enrichment, together with the appropriate setting of repressive histone post-translational marks, contributes to chromocenter formation, identifying chromatin assembly by CAF-1 as driving force in formation and maintenance of genome structure. Finally, while absence of HIR or CAF-1 complexes sustains viability, only the simultaneous loss of both severely impairs nucleosomal occupancy and plant development, suggesting a limited functional compensation between the different histone chaperone complexes and plasticity in histone variant interaction and deposition in plants

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33

Bruel, Nicolas. "Hsp33 controls elongation factor-tu stability and allows escherichia coli growth in the absence of the major dnak and triggerfactor chaperones." Toulouse 3, 2013. http://thesesups.ups-tlse.fr/2098/.

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Le repliement intracellulaire des protéines nouvellement synthétisées est assisté par des réseaux cellulaires de protéines chaperons. Chez Escherichia coli, la coopération entre les protéines chaperons Trigger Factor (TF) et DnaK est prédominante dans ce processus. En accord avec ceci, la délétion simultanée des gènes codants pour ces deux protéines chaperons conduit à une croissance bactérienne très réduite et à l'accumulation d'un grand nombre de protéines cytoplasmiques sous forme d'agrégats. Au cours de cette étude, nous avons utilisé ces phénotypes afin de mettre en évidence des interactions potentielles au sein du réseau de protéines chaperons in vivo. Nous avons montré que la perte des protéines chaperons TF et DnaK, et donc des voies de repliements dans lesquelles elles sont impliquées, pouvait être secourue de façon efficace par la surexpression du chaperon Hsp33, connu pour être activable en réponse à un stress oxydatif sévère. En outre, la délétion du gène hslO, codant pour Hsp33, n'était plus tolérée en l'absence de TF et DnaK. Cependant, en comparaison avec d'autres protéines chaperons comme GroESL ou SecB, la suppression de ces phénotypes par Hsp33 n'a pas pu être attribuée à un éventuel chevauchement de fonctions avec DnaK et TF. Au contraire, nos résultats montraient qu' Hsp33 surexprimée fixait de façon spécifique le facteur d'élongation-Tu (EF-Tu) et favorisait sa dégradation par la protéase Lon. Cette action synergétique entre Hsp33 et Lon était responsable du rétablissement de la croissance bactérienne en l'absence de TF et DnaK, possiblement via le rétablissement du couplage entre la vitesse de traduction et les capacités de repliement des protéines nouvellement synthétisées du double mutant. Afin de soutenir cette hypothèse, nous avons ensuite montré que la surexpression de la toxine HipA qui inhibe EF-Tu, était aussi capable de supprimer le phénotype de thermosensibilité et de réduire significativement l'agrégation des protéines en l'absence de TF et DnaK
Intracellular de novo protein folding is assisted by cellular networks of molecular chaperones. In Escherichia coli, cooperation between the chaperones Trigger Factor (TF) and DnaK is central to this process. Accordingly, the simultaneous deletion of both chaperone-encoding genes leads to severe growth and protein folding defects. Herein, we took advantage of such defective phenotypes to further elucidate the interactions of chaperone networks in vivo. We show that disruption of the TF/DnaK chaperone pathway is efficiently rescued by over-expression of the redox-regulated chaperone Hsp33. Consistent with this observation, the deletion of hslO, the Hsp33 structural gene, is no longer tolerated in the absence of the TF/DnaK pathway. However, in contrast with other chaperones like GroESL orSecB, suppression by Hsp33 was not attributed to its potential overlapping general chaperone function(s). Instead, we show that over-expressed Hsp33 specifically binds to elongation factor-Tu (EF-Tu) and targets it for degradation by the protease Lon. This synergistic action of Hsp33 and Lon was responsible for the rescue of bacterial growth in the absence of TF and DnaK, by presumably restoring the coupling between translation and the downstream folding capacity of the cell. In support of this hypothesis, we show that over-expression of the stress-responsive toxin HipA, which inhibits EF-Tu, also rescues bacterial growth and protein folding in the absence of TF and DnaK

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34

Wattin, Marion. "Modulation des mécanismes de Contrôle Qualité des Protéines dans la dystrophie musculaire de Duchenne." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1323/document.

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De nombreuses études ont mis en évidence l’importance du contrôle qualité des protéines, c’est à dire des mécanismes de reconformation (chaperons moléculaires) et de dégradation (autophagie, proteasome) des protéines dans différentes pathologies musculaires telles que la dystrophie musculaire d’Ullrich (UCMD), de Duchenne (DMD) ou d’Emery-Dreifuss (EDMD) ; cependant, à l’heure actuelle, aucune n’a été menée sur l’ensemble de ces mécanismes dans un seul et même modèle et sur des cellules musculaires avant leur différenciation en muscles. Nous nous sommes donc intéressés à la fonctionnalité des mécanismes de Contrôle Qualité des Protéines et à leurs interconnexions dans des myoblastes immortalisés de donneurs sains ou de patients atteints de DMD. Nous avons observé une augmentation de l’agrégation protéique dans les cellules DMD. Ce phénomène s’accompagne d’une dérégulation des mécanismes de séquestration par les chaperons moléculaires, conséquence d’une modulation de l’expression des protéines HSPB5 et HSPB8. Les mécanismes de dégradation sont également dérégulés; en effet, nous avons observé d’une part, une diminution de l’activité enzymatique du protéasome ainsi que des molécules d’adressage des protéines multiubiquitinées au protéasome et d’autre part, une augmentation de l’activité du facteur de transcription NF?B, de l’expression de protéines intervenant dans l’autophagie et des complexes BAG3/HspB8 conduisant à une augmentation du flux autophagique. L’ensemble de ces dérégulations reflète l’existence d’un stress d’agrégation protéique dans les myoblastes issus de patients DMD. Dans ce contexte, la modulation pharmacologique du PQC dans ces cellules pourrait représenter une nouvelle stratégie thérapeutique pour la Dystrophie Musculaire de Duchenne
Various studies have highlighted the importance of Protein Quality Control (PQC), including protein refolding (molecular chaperones) and degradation (autophagy, proteasome) mechanisms in inherited muscle disorders such as Ullrich Congenital Muscular Dystrophy (UCMD), Duchenne Muscular Dystrophy (DMD) or Emery-Dreifuss Muscular Dystrophy (EDMD); however, to date, no extensive study has been conducted on these mechanisms in a same model, in muscle cells before muscle differentiation. Thus, we were interested in PQC mechanisms functionality and their interconnection in human immortalized myoblasts from healthy donors or patients suffering from DMD. We observed an increase of protein aggregation in DMD cells. This phenomenon is accompanied by a deregulation of sequestration mechanisms by molecular chaperones, reflected by the modulation of HSPB5 and HSPB8 expression. Degradation mechanisms are also deregulated; indeed, we observed on one hand a decrease of proteasome enzymatic activity and multiubiquitinated proteins UPS-adressing molecules and on the other hand, an increase of NF?B transcription factor’s activity, involved in autophagy, and of BAG3/HSPB8 complexes, leading to an increase of the autophagic flux. These PQC defects reflect the existence of a protein aggregation stress in myoblasts coming from DMD patients. In this context, pharmacological modulation of PQC in these cells could represent a new therapeutic strategy for Duchenne Muscular Dystrophy

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35

Nickels, Christina Utta [Verfasser], Johannes [Akademischer Betreuer] Buchner, Johannes [Gutachter] Buchner, and Matthias J. [Gutachter] Feige. "Regulation of the molecular chaperone BiP by its co-chaperones / Christina Utta Nickels ; Gutachter: Johannes Buchner, Matthias J. Feige ; Betreuer: Johannes Buchner." München : Universitätsbibliothek der TU München, 2019. http://d-nb.info/1190285258/34.

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36

Sikor, Martin. "Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-138521.

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37

Novoselova, T. V. "Investigation of the role of putative chaperones in retinal degeneration." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/16134/.

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38

Huanyu, Wang. "Characterization of N1/N2 Family Histone Chaperones: Hif1p and NASP." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1279815431.

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39

Burger, Adélle. "The E.coli RNA degradosome analysis of molecular chaperones and enolase." Thesis, Rhodes University, 2010. http://hdl.handle.net/10962/d1004009.

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Normal mRNA turnover is essential for genetic regulation within cells. The E. coli RNA degradosome, a large multi-component protein complex which originates through specific protein interactions, has been referred to as the “RNA decay machine” and is responsible for mRNA turnover. The degradosome functions to process RNA and its key components have been identified. The scaffold protein is RNase E and it tethers the degradosome to the cytoplasmic membrane. Polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlB helicase) and the glycolytic enzyme enolase associate with RNase E to form the degradosome. Polyphosphate kinase associates with the degradosome in substoichiometric amounts, as do the molecular chaperones DnaK and GroEL. The role of DnaK as well as that of enolase in the RNA degradosome is unknown. Very limited research has been conducted on the components of the RNA degradosome under conditions of stress. The aim of this study was to understand the role played by enolase in the assembly of the degradosome under conditions of stress, as well as investigating the protein levels of molecular chaperones under these conditions. The RNA degradosome was successfully purified through its scaffold protein using nickel-affinity chromatography. In vivo studies were performed to investigate the protein levels of DnaK and GroEL present in the degradosome under conditions of heat stress, and whether GroEL could functionally replace DnaK in the degradosome. To investigate the recruitment of enolase to the degradosome under heat stress, a subcellular fractionation was performed to determine the localization of enolase upon heat shock in vivo. The elevated temperature resulted in an increased concentration of enolase in the membrane fraction. To determine whether there is an interaction between enolase and DnaK, enolase activity assays were conducted in vitro. The effect of DnaK on enolase activity was measured upon quantifying DnaK and adding it to the enolase assays. For the first time it was observed that the activity of enolase increased with the addition of substoichiometric amounts of DnaK. This indicates that DnaK may be interacting with the RNA degradosome via enolase.

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40

Chromy, Laura R. "The role of HSP70 chaperones in papovavirus disassembly and assembly /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.

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Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2007.
Typescript. Includes bibliographical references (leaves 142-165). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

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41

Moparthi, Satish Babu. "Biophysical studies of protein folding upon interaction with molecular chaperones /." Linköping : Department of Physics, Chemistry and Biology, Linköping University, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-51604.

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42

Edkins, Adrienne Lesley. "Hsp90 co-chaperones as drug targets in cancer: current perspectives." Springer International Publishing Switzerland, 2016. http://hdl.handle.net/10962/66347.

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publisher version
Hsp90 is a molecular chaperone that regulates the function of numerous oncogenic transcription factors and signalling intermediates in the cell. Inhibition of Hsp90 is sufficient to induce the proteosomal degradation of many of these proteins, and as such, the Hsp90 chaperone has been regarded as a promising drug target. The appropriate functioning of the Hsp90 chaperone is dependent on its ATPase activity and interactions with a cohort of non-substrate accessory proteins known as co-chaperones. Co-chaperones associate with Hsp90 at all stages of the chaperone cycle and regulate a range of Hsp90 functions, including ATP hydrolysis and client protein binding and release. Given the ability of co-chaperones to organise the function of the Hsp90 molecular machine, these proteins are now regarded as potential drug targets. Herein the role of selected Hsp90 co-chaperones Hop, Cdc37, p23 and Aha1 as possible drug targets is discussed with a focus on cancer.
This work is based on the research supported by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation of South Africa (Grant No 98566), the Cancer Association of South Africa (CANSA), Medical Research Council South Africa (MRC-SA) and Rhodes University. The views expressed are those of the authors and should not be attributed to the DST, NRF, CANSA, MRC-SA or Rhodes University. We apologize if we have inadvertently missed any important contributions to the field.

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Sciacovelli, Marco. "Cell death regulation by mitochondrial chaperones in tumor cell models." Doctoral thesis, Università degli studi di Padova, 2011. http://hdl.handle.net/11577/3421645.

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Cancer cells are endowed with the capability to evade normal apoptotic signaling, as they display a constitutive hyperactivation of kinase signaling pathways. Integration of survival and death stimuli occurs on mitochondria, where many of these signals converge in the regulation of a channel termed permeability transition pore (PTP). PTP opening commits cells to death, and it is regulated by a variety of factors, among which molecular chaperones play a pivotal role. Here I have studied how mitochondrial chaperones interact with signal transduction pathways, modulate the PTP and more in general mitochondrial bioenergetics, and how these regulatory networks control tumor cell viability. In a first part of my work, I have explored a functional connection between the Ras/ERK signaling axis, whose constitutive activation characterizes most tumors and prompts their growth and survival, and cyclophilin D (CyP-D), a mitochondrial chaperone that regulates the PTP. A fraction of active ERK was found to be located in mitochondria in RWPE-2 cells, obtained by v-Ki-Ras transformation of the epithelial prostate RWPE-1 cell line; in metastatic prostate cancer DU145 cells; and in osteosarcoma SAOS-2 cells. All these tumor cells displayed marked resistance to death caused by apoptotic stimuli like arachidonic acid and the BH3 mimetic EM20-25, which cause cell death through the mitochondrial PTP. PTP inhibition and the ensuing resistance to cell death induced by arachidonic acid or EM20-25 could be ablated by inhibiting ERK with the drug PD98059 or with a selective ERK activation inhibitor peptide. ERK inhibition enhanced GSK-3-dependent phosphorylation of CyP-D), whereas GSK-3 inhibition protected from PTP opening. Neither active ERK in mitochondria nor pore desensitization were observed in non-transformed RWPE-1 cells. Thus, in tumor cells mitochondrial ERK activation desensitizes the PTP through a signaling axis that involves GSK-3 and CyP-D. In a second part of my thesis work, I have investigated the activity of a second mitochondrial chaperone, TRAP1/HSP75, overexpressed in tumor cells and proposed to be involved in regulation of the pore. I have determined that TRAP1 interacts with CyP-D and characterized its survival function against a wide spectrum of death stimuli inducing oxidative stress, including diamide, exposure to TNFα, and glucose deprivation. Moreover, I have found that knocking-down TRAP1 expression level through RNA interference in SAOS-2 osteosarcoma cells facilitates PTP opening, thus lowering the threshold for committing cells to death. TRAP1 modulates cell metabolism and possibly the response to oxidative stress by reducing mitochondrial respiration and the activity of respiratory chain complex I, with which TRAP1 directly interacts, both in cells and in tumor samples. Notably, down-modulation of TRAP1 ablates the tumorigenic potential of SAOS-2 cells both in vitro and in-vivo. Altogether, these data indicate that mitochondrial chaperones such as CyP-D and TRAP1 play an important role in tumor progression and constitute a possible target for anti-neoplastic intervention.
Le cellule tumorali sono caratterizzate dalla capacità di evadere il normale signale apoptotico, così come mostrano una iper-attivazione costitutiva delle vie di signale kinasico. L’integrazione degli stimoli di sopravvivenza e morte si concentra nei mitocondri, dove molti di questi segnali convergono nella regolazione di un canale chiamato poro della transizione di permeabilità (PTP). L’apertura del PTP porta le cellule alla morte ed è regolata da una varietà di fattori e fra questi gli chaperoni giocano un ruolo fondamentale. Nel mio lavoro di tesi ho studiato come gli chaperoni mitochondriali si integrano nelle vie di trasduzione del segnale , modulando il PTP e più in generale la bioenergetica mitocondriale e come questi network regolatori controllano la vitalità cellualre. Nella prima parte del mio lavoro ho studiato una possibile connessione fra la via del segnale Ras/ERK, la cui attivazione costitutiva caratterizza molti tumori favorendo la loro crescita e sopravvivenza, e la ciclofilina D (CyP-D), uno chaperone mitocondriale che regola il PTP. Una frazione di ERK attivo è stato trovato nei mitocondri delle cellule RWPE-2, ottenute tramite trasformazione con v-ki-Ras a partire da cellule dell’epitelio prostatico RWPE-1; in cellule metastatiche di tumore prostatico DU145; e in cellule di osteosarcoma SAOS-2. Tutte queste cellule tumorali mostrano una marcata resistenza alla morte indotta da stimoli pro-apoptotici come l’acido arachidonico e il BH3 mimetico EM20-25, i quali inducono la morte cellulare attraverso il PTP mitocondriale. L’inibizione del PTP e la conseguente resistenza alla morte cellulare indotta da acido arachidonico o EM-20-25 può essere abolita dall’inibizione di ERK con il farmaco PD98059 o con un peptide selettivo inibitorio di ERK. L’inibizione di ERK aumenta la fosforilazione GSK-3 dipendente della CyP-D, mentre l’inibizione di GSK3 protegge dall’apertura del poro. Ne ERK attivo nei mitocondri, ne desensibilizzazione del poro è stata osservata in cellule non trasformate RWPE-1. In conclusione, nelle cellule tumorali l’attivazione dell’ERK mitocondriale desensibilizza il PTP attraverso un asse di segnale che coinvolge GSK3 e Cyp-D. Nella seconda parte del mio lavoro di tesi, ho studiato l’attività di un secondo chaperone mitocondriale, TRAP1/HSP75, fortemente espresso nelle cellule tumorali e che è stato proposto essere coinvolto nella regolazione del poro. Ho dimostrato che TRAP1 interagisce con la CyP-D ed ho caratterizzato la sua funzione di pro-sopravvivenza nei confronti di un vasto spettro di stimoli di morte, incluso lo stress ossidativo, la diamide, il TNFα, e la deplezione di glucosio. Inoltre ho trovato che il knocking-down dei livelli di espressione di TRAP1 attraverso la tecnica dell’RNA interference in cellule di osteosarcoma SAOS-2 facilita l’apertura del PTP, abbassando la soglia per portare le cellule alla morte. TRAP1 modula inoltre il metabolismo cellulare probabilmente la risposta allo stress ossidativo e l’attività della del complesso I della catena respiratoria, con il quale TRAP1 interagisce direttamente sia in cellule che campioni tumorali. La down- regolazione di TRAP1 abolisce il potere tumori genico delle cellule SAOS-2 sia in vitro che in vivo. Tutti insieme questi dati indicano che gli chaperoni mitocondriali come CyP-D e TRAP! Giocano un ruolo importante nella progressione tumorale e costituiscono un possibile target di nuove terapie antineoplastiche.

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44

Barry, Amanda Nell. "Spectroscopic studies of the human copper chaperone for superoxide dismutase : probing the active cluster with selenocysteine variants." Full text open access at:, 2007. http://content.ohsu.edu/u?/etd,258.

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Muchowski, Paul J. "Structural and functional characterization of human alphaB-crystallin, a small heat-shock protein and molecular chaperone /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/5676.

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46

Weinhaeupl, Katharina. "Etudes de structure, interactions et dynamique dans des complexes de protéines "chaperone" à l'échelle atomique par spectroscopie RMN." Thesis, Université Grenoble Alpes (ComUE), 2018. http://www.theses.fr/2018GREAV002.

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Les chaperons moléculaires, une famille de protéines diverses en structure et taille, sont dédiés à accompagner, replier et protéger d’autres protéines afin qu’elles atteignent leur conformation finale et leur emplacement dans la cellule. Dans ce but, les chaperons moléculaires doivent être hautement spécialisés dans l’exécution de tâches spécifiques, telles que le repliement, le transport ou la désagrégation, et polyvalents dans leur motifs de reconnais- sance, afin de pouvoir interagir avec un grand nombre de protéines di érentes. Di érents chaperons moléculaires collaborent au sein de la cellule, formant ainsi un réseau complexe qui assure le contrôle de la qualité du protéome. Les interactions entre les di érents partenaires de ce réseau et entre les chap- erones et leurs substrats sont souvent dynamiques, ce qui rend leur obser- vation structurale particulièrement di cile pour les techniques de biologie structurale. Par conséquent, il y a à ce jour peu d’information sur les struc- tures et mécanismes d’interaction au sein des complexes chaperon-substrate. Dans cette thèse, je présente des études sur la structure, la dynamique et les interactions entre les substrats de deux chaperons moléculaires, en utilisant diverses méthodes biophysiques et in vivo.Dans la première partie, je montre que la chaperone TIM910, située dans l’espace inter-membranaire des mitochondries, lie ses substrats, des protéines membranaires destinées aux deux membranes mitochondriales, d’une manière très dynamique. Non seulement le complexe TIM910 est en échange constant entre les espèces monomèriques et hexameriques, mais aussi le substrat lié échange entre mulitples conformations à une échelle de millisecondes. Sur la base de la résonance magnétique nucléaire (RMN), de small-angle X-ray scat- tering (SAXS), de l’ultracentrifugation analytique (AUC) et des expériences mutationnelles in vivo et des tests fonctionnels d’import dans les mitochon- dries, je propose un modèle structurale de l’interaction entre le chaperon et la protéine membranaire. TIM910 lie ses substrats dans une poche hydrophobe à l’extérieur du chaperon. Cette interaction est modulaire et se fait avec un ou deux hexamères de TIM910, en fonction de la longueur du substrat.Dans la deuxième partie, nous avons étudié le comportement du récepteur N-terminal du unfoldase ClpC1 de M. tuberculosis en présence d’antibiotiques et de ligands di érents. Le domaine N-terminal de ClpC1 est le site de liai- son de divers antibiotiques nouveaux contre M. tuberculosis. L’antibiotique Cyclomarin A supprime complètement la dynamique induite par le ligand arginine-phosphate. Nous proposons que cette suppression de la dynamique soit le principe fondamental du mécanisme d’action de cet antibiotique.Dans les deux cas, les structures X-ray des chaperons dans leur état apo et la structure de ClpC-NTD liée à des antibiotiques étaient disponibles, mais ces structures statiques ne su sent pas pour expliquer le mécanisme d’action. La structure X-ray de TIM910 n’a pas fourni d’ indication sur l’endroit ou la façon dont les substrats sont liés. De même, les structures X-ray du domaine N-terminal de apo et de Cyclomarine A de ClpC1 ne présentent que des di érences de structure mineures. Les deux exemples montrent que les données structurelles statiques souvent ne permettent pas d’expliquer le fonctionnement d’un système moléculaire, donc la combinaison de di érentes techniques et le développement de nouvelles méthodes pour étudier les complexes chaperon-substrat sont primordiaux pour comprendre leur fonction
The diverse group of molecular chaperones is dedicated to accompany, fold and protect other proteins until they reach their final conformation and loca- tion inside the cell. To this end, molecular chaperones need to be specialized in performing specific tasks, like folding, transport or disaggregation, and versatile in their recognition pattern to engage many di erent client pro- teins. Moreover, molecular chaperones need to be able to interact with each other and with other components of the protein quality control system in a complex network. Interactions between the di erent partners in this network and between the substrate and the chaperone are often dynamic processes, which are especially di cult to study using standard structural biology tech- niques. Consequently, structural data on chaperone/substrate complexes are sparse, and the mechanisms of chaperone action are poorly understood. In this thesis I present investigations of the structure, dynamics and substrate- interactions of two molecular chaperones, using various biophysical and in vivo methods.In the first part I show that the mitochondrial membrane protein chap- erone TIM910 binds its substrates in a highly dynamic manner. Not only is the TIM910 complex in constant exchange between monomeric and hex- americ species, but also the bound substrate samples multiple conformations on a millisecond timescale. Based on nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC) and in vivo mutational experiments I propose a structural model of the chap- erone/membrane protein interaction. TIM910 binds its substrates in a hy- drophobic pocket on the exterior of the chaperone in a modular fashion, where the number of TIM910 complexes bound depends on the length of the substrate.In the second part I studied the behavior of the N-terminal receptor do- main of the ClpC1 unfoldase from M.tuberculosis in the presence of di erent antibiotics and ligands. The N-terminal domain of ClpC1 is the binding site for various new antibiotics against M.tuberculosis. The antibiotic cyclomarin completely abolishes dynamics induced by the ligand arginine-phosphate. We propose that this suppression of dynamics is the underlying principle for the mechanism of action of this antibiotic.In both cases X-ray structures of the apo or antibiotic bound form were available, but not su cient to explain the mechanism of action. The X- ray structure of TIM910 provided no evidence on where or how substrates are bound. Likewise, X-ray structures of the apo and cyclomarin-bound N-terminal domain of ClpC1 show only minor di erences in structure.Both examples show that static structural data is often not enough to explain how a molecular system works, and only the combination of di er- ent techniques, including newly developed methods enable the atomic-level understanding of chaperone/substrate complexes

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Baptista, Mauricio Zuccolotto 1975. "Padrões de expressão de GRP78 em mulheres com câncer de mama tratadas com antracíclicos." [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/309547.

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Orientadores: Gustavo Antonio de Souza, José Vassallo
Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-16T10:53:47Z (GMT). No. of bitstreams: 1 Baptista_MauricioZuccolotto_M.pdf: 8709827 bytes, checksum: 83a90faa4c0b3ee087444229cb9deee0 (MD5) Previous issue date: 2010
Resumo: Introdução: Evidências pré-clínicas implicam GRP78 como um possível marcador de resistência em quimioterapia baseada em antracíclicos em câncer de mama. Objetivos: O presente estudo avalia a relação entre a expressão de GRP78 no retículo endoplasmático (RE) e na membrana celular (MC) e a sobrevida global (SG) e a sobrevida livre de progressão (SLP) em pacientes tratadas com antracíclicos na adjuvância. Sujeitos e Métodos: Foram selecionadas 106 pacientes com estádios II e III de câncer de mama. Os dados clínicos foram obtidos de prontuários médicos. O microarranjo de tecidos (TMA) foi construído com blocos de parafina de tumores de mama. A expressão de GRP78 foi avaliada por imuno-histoquímica utilizando quatro cenários distintos: os cenários de alto e baixo limiar para o RE e os cenários de alto e baixo limiar para a MC. Resultados: O follow-up médio foi de 7.54 anos. Nos cenários de alto-limiar, 16% dos casos resultaram em GRP78-positiva para o RE e 40% em GRP78- positiva para a MC. Nos cenários de baixo-limiar, 74% dos casos resultaram em GRP78-positiva para o RE e 87% em GRP78-positiva para a MC. 10% dos casos mostraram nível forte (3+) de intensidade de coloração para GRP78 na MC. Ao término do seguimento não foi encontrada nenhuma relação entre a expressão de GRP78, a progressão de doença e o risco relativo de morte. O mesmo ocorreu com as probabilidades de sobrevida livre de progressão, exceto para mulheres acima de 50 anos de idade e pós-menopausadas, que tiveram um risco reduzido (RR=0.03; IC95% 0.01 a 0.40) de progressão de doença se positivas para GRP78. Não houve diferença estatisticamente significante entre as probabilidades de sobrevida em nenhum dos cenários examinados. Conclusões: Em nossa coorte, a superexpressão de GRP78 não foi significativamente associada à SG e à SLP das mulheres que receberam quimioterapia adjuvante baseada em antracíclicos. Este estudo fornece evidência que sustenta a forte atividade de GRP78 na membrana celular de células de câncer de mama.
Abstract: Introduction: Preclinical evidence implicates GRP78 as one possible marker of resistance to anthracycline-based adjuvant chemotherapy in breast cancer patients. Objectives: The present study assessed the relation between GRP78 expression in the endoplasmic reticulum (ER) and cell membrane (CM) of breast malignancies and overall (OS) and progression-free survival (PFS) of patients treated with anthracyclines in the adjuvant setting. Subjects and Methods: 106 stage II/III breast cancer patients were selected. Clinical data were retrieved from medical reports. Tissue Microarray was constructed from paraffin blocks of breast tumors. GRP78 expression was assessed by immunohistochemistry using four distinct scenarios: low and high GRP78 expression thresholds for ER and CM. Results: The median follow-up was 7.54 years. In the high-threshold scenarios, 16% of our cases were GRP78-positive for ER, and 40% were GRP78-positive for CM. In the low-threshold scenarios, 74% of our cases were GRP78-positive for ER, and 87% were GRP78-positive for CM. 10% of all cases showed strong (3+) CM staining of GRP78. By the end of the follow-up, no relation was found between GRP78 expression and disease progression and the relative risk of death. The same was true for the PFS probabilities, except for women above fifty years and postmenopausal, who had a reduced risk (RR=0.03; 95%CI 0.01 to 0.40) of disease progression if positive for GRP78. There was no statistically significant difference between the survival probabilities in any scenarios examined. Conclusions: In our cohort, GRP78 overexpression was not a predictor of OS or PFS of patients receiving anthracycline adjuvant chemotherapy. This study provides evidence supporting strong GRP78 activity in the CM of breast cancer cells.
Mestrado
Ciencias Biomedicas
Mestre em Tocoginecologia

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48

Seraphim, Thiago Vargas. "Estudo estrutural da co-chaperona Aha1 (Activator of Hsp90 ATPase 1) de Leishmania braziliensis e da sua ação sobre o ciclo funcional da Hsp90." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-25112015-102054/.

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As chaperonas moleculares atuam no enovelamento de proteínas, montagem de complexos, prevenção/recuperação de proteínas de agregados e encaminhamento de proteínas mal enoveladas para depuração. As Hsp90 são chaperonas moleculares que atuam estabilizando proteínas relacionadas a vias de sinalização, crescimento celular, processos transcricionais e traducionais, estabilidade do genoma, entre outras, sendo essencial para a viabilidade celular. Em protozoários do gênero Leishmania, as Hsp90 são imprescindíveis no desenvolvimento, adaptação e transformação celular. Estes fatores fazem das Hsp90 alvos potenciais para o tratamento de patologias, como a leishmaniose, uma doença tropical negligenciada. As Hsp90 são homodímeros flexíveis onde cada protômero é dividido em três domínios denominados N, M e C. As Hsp90 possuem um ciclo conformacional associado ao seu ciclo funcional e sua baixa atividade ATPásica, o qual é direcionado e regulado por proteínas auxiliares, as co-chaperonas. A co-chaperona Aha1 atua estimulando a atividade ATPásica da Hsp90, participando da maturação de proteínas quinase e receptores de hormônios. O objetivo deste trabalho foi caracterizar estruturalmente a proteína Aha1 de L. braziliensis (LbAha1) e seu mecanismo de interação com a Hsp90 desse organismo (LbHsp90). A LbAha1 é formada por dois domínios, LbAha1N e LbAha1C, conectados entre si por um linker flexível. Experimentos de identificação in vivo mostraram que a LbAha1 e LbHsp90 são proteínas cognatas. A LbAha1 e as construções de seus domínios (LbAha1N e LbAha1C) recombinantes foram obtidas puras e enoveladas. A LbAha1 é estruturada em dois domínios com diferentes estabilidades, que não interagem entre si e se enovelam independentemente, porém influenciam-se reciprocamente. Em solução, a LbAha1 se comporta como um monômero alongado e possui notável flexibilidade, com dimensão suficiente para interagir com os domínios N e M da LbHsp90. A análise da interação entre a LbAha1 e LbHsp90 revelou que a associação destas proteínas é dirigida entalpicamente, ocorrendo através de interações eletrostáticas e com estequiometria de 2 moléculas de LbAha1 por dímero de LbHsp90. O mapeamento de regiões envolvidas na interação indicou que o domínio LbAha1N e o domínio M da LbHsp90 compõem o cerne da interação e somente a LbAha1 íntegra é capaz de encaminhar a LbHsp90 para um estado fechado. Experimentos de cinética enzimática mostraram que somente a LbAha1 íntegra estimula a atividade ATPásica da LbHsp90 por meio de um mecanismo cooperativo positivo. Assim, é proposto que a conexão entre os domínios da LbAha1, via linker, é essencial para o direcionamento da LbHsp90 para um estado conformacional fechado e competente na hidrólise de ATP.
Molecular chaperones play a role in protein folding, complex assembly, prevention/recover of proteins from aggregates and targeting misfolded proteins to depuration. Hsp90 molecular chaperones work stabilizing proteins related to signaling pathways, cell growth, transcription and translation processes, genome stability, among others, and are essential to cell viability. In protozoa of the genus Leishmania, Hsp90s are indispensable for cell developing, adaptation and transformation. These factors make Hsp90s potential targets for pathologies treatment, such as leishmaniasis, a neglected tropical disease. Hsp90s are flexible homodimers and each protomer is divided into three domains named N, M and C. Hsp90s have a conformational cycle associated to its functional cycle and low ATPase activity, which is directed and regulated by auxiliary proteins, so-called cochaperones. Aha1 co-chaperone stimulates Hsp90 ATPase activity, participating on protein kinase and hormone receptors maturation. This work aimed to characterize the structure of the Aha1 from L. braziliensis (LbAha1) and its mechanism of interaction with the Hsp90 from the same organism (LbHsp90). LbAha1 is formed by two domains, LbAha1N and LbAha1C, connected to each other by a flexible linker. In vivo experiments identified LbAha1 and LbHsp90 as cognate proteins. Recombinant LbAha1 and its domains construct (LbAha1N and LbAha1C) were obtained pure and folded. LbAha1 is divided into two domains with dissimilar stabilities and they do not interact to each other. In spite of this they fold independently and influence each other reciprocally. LbAha1 behaves as an elongated monomer in solution and has a remarkable flexibility, with sufficient dimension to interact to LbHsp90 N and M domains. The analysis of the LbAha1-LbHsp90 interaction revealed that the association between these two proteins is enthalpically driven, occurring through electrostatic interactions in a stoichiometry of 2 LbAha1 molecules per LbHsp90 dimer. Domain mapping experiments indicated that LbAha1N and LbHsp90 M domains compose the core of the interaction and only full length LbAha1 is able to direct LbHsp90 toward a closed state. Enzyme kinetics experiments showed that only full length LbAha1 stimulates LbHsp90 ATPase activity through a positive cooperative mechanism. Thus, it is proposed that the connection between the LbAha1 domains, via linker, is essential to direct the LbHsp90 toward a closed and ATPase-competent conformational state.

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49

Linden, Liana de Salles van der. "Avaliação da proteína disulfeto isomerase A1 (PDIA1) como marcador para a qualidade seminal em garanhões." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2017. http://hdl.handle.net/10183/182414.

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Abstract:

O período de puberdade, em equinos, define-se pela aparição de espermatozóides maduros no ejaculado de animais jovens, bem como a maturação das funções endócrinas. Os espermatozóides precisam passar por um processo de maturação no epidídimo. Para se obter marcadores moleculares que permitam estabelecer a fertilidade de um garanhão, é necessário um melhor conhecimento das proteínas presentes em espermatozóides imaturos e maduros, uma das quais é a PDI (proteína dissulfeto-isomerase). A PDI foi descrita também como importante marcador de fertilidade no plasma seminal e espermatozóides de diversas espécies. O objetivo deste trabalho foi identificar a presença da PDI no epidídimo equino durante a puberdade, e quantificá-la no fluido e espermatozóides epididimários. Visou verificar a presença da PDI no plasma seminal e espermatozóides ejaculados, em garanhões férteis e subférteis. Foram realizados dois experimentos: Experimento 1: utilizou-se 22 equinos Crioulos saudáveis, castrados e divididos em três grupos: G1: potros até 24 meses, G2: de 25-36 meses e G3: a partir de 36 meses. Imediatamente após a castração, foi efetuada a dissecação do epidídimo para a coleta de fluido epididimário, o qual foi separado dos espermatozoides por centrifugação a 800 g por 10minutos. O sobrenadante foi removido, e criopreservado a -196º C. Os espermatozóides foram ressuspendidos em PBS e armazenados a -196º C. Para a dosagem de proteína das amostras, utilizou-se o método de Kit BCA espectrofotômetro e a eletroforese em gel de poliacrilamida a 10% SDS- Page.Para imunodetecção das proteínas, efetuou-se a incubação do anticorpo primário específico por no mínimo, 6 horas a 4º C, e incubação com anticorpo secundário conjugado com peroxidase anti-igG de camundongo ou anti-IgG de rato. Para a visualização das bandas foi utilizado o Kit de ECL em filmes de raio-X, e as bandas quantificadas pela utilização do software livre ImageJ. A PDI foi identificada nos três grupos avaliados na análise proteômica do fluido , e espermatozoides epididimários, sendo sua quantidade inferior no G1 em relação aos grupos dois (2) e três (3). Em conclusão, a expressão da PDI nos espermatozóides e fluido epididimários de potros castrados cirurgicamente, aumenta conforme o animal atinge a maturidade sexual. Experimento 2- Utilizou-se 12 garanhões adultos que já haviam sido submetidos à pelo menos duas temporadas de monta na região da Campanha do Rio Grande do Sul, efetuando-se quatro (4) coletas de cada garanhão, fora da estação de monta, respeitando-se um intervalo de 48h entre coletas. Imediatamente após a coleta, analisou-se motilidade, vigor e concentração com auxílio de microscópio óptico e retirou-se uma alíquota de sêmen para avaliação das patologias espermáticas. A partir dos dados obtidos, os garanhões foram divididos em dois grupos: Grupo 1: motilidade não inferior a 70%, tendo-se uma média de 76,04±5,89% e histórico reprodutivo de temporadas anteriores com índice de prenhez mínimo por temporada de 80%. Grupo 2: motilidade igual ou inferior a 30%, com média de 11,83±11,21%, e histórico reprodutivo de temporadas anteriores de índices de prenhez inferiores a 35%. Efetuadas as análises, as mostras foram centrifugadas a 800 g por 10 minutos para separar o plasma seminal, de mesma forma que no Exp. 1. Os pellets passaram por ressuspensão em PBS gelado e posteriormente armazenados a -196º C. As amostras foram submetidas à dosagem das proteínas, para serem submetidas à eletroforese, utilizando-se géis de poliacrilamida a 10% SDS-Page.Para imunodetecção das proteínas, efetuou-se a incubação do anticorpo primário específico por no mínimo, 6 horas a 4º C, e incubação com anticorpo secundário conjugado com peroxidase anti-igG de camundongo ou anti-IgG de rato. Na visualização das bandas foi utilizado o Kit de ECL em filmes de raio-X, e as bandas foram quantificadas pela utilização do software livre Image J. Ao analisar a presença da PDI no plasma seminal dos garanhões, verificou-se sua expressão em ambos os grupos; porém não houve diferença de expressão da PDI entre eles(p˂0,05). Não houve também relação da PDI com motilidade, nem com a concentração espermática. Considerando os dados obtidos no presente experimento, não foi possível relacionar a PDI com qualidade espermática e assim considerá-la como um potencial marcador de fertilidade em equinos. São necessários mais estudos que possam envolver outros fatores moleculares inclusive as demais proteínas da família das PDIs.
Puberty, in the equine species, may be defined by the appearance of mature spermatozoa in young animals´ ejaculates, as well as endocrine function maturation. One of the proteins found in immature and mature spermatozoa is PDI (protein dissulfide-isomerase). PDI was also described as an important fertility marker both in seminal plasma and sperm of many species. is responsible for rearranging dissulfide bonds, necessary for sperm adhesion proteins to link to the oocyte. The aim of this work was to identify PDI in equine epididymis during puberty, and quantify it in epididymal sperm and fluid of fertile and subfertile sperm. Two experiments were performed. Experiment 1-twenty-two healthy Crioulo colts were surgically castrated, and divided in three groups: G1: until 24 months; G2: from 25-36 months and G3: more than 36 months. Immediately after castration, testicles were measured, weighed, and the epididymis was dissecated for epididymal fluid collection, which was centrifuged at 800 g for 10minutes to separate epididymal fluid from sperm. Supernatant was removed, and cryopreserved at -196º C. Sperm were re-suspended in PBS and stored at -196º C. Protein dosing of samples was performed with BCA Kit and electrophoresis at 10% SDS-Page. To detect proteins, primary antibody was incubated for at least 6 hours at 4º C, and then incubation with secondary antibody conjugated with anti-mouse IgG or anti-rat IgG. To see bands, ECL Kit in X-ray films was used, and the bands quantified with softwareImageJ. In the three groups PDI was identified, in epididymal fluid and epididymal sperm, but in smaller amount in G1 when compared with Groups 2 and 3. In conclusion, expression of PDI in epididymal fluid and sperm of surgically castrated colts, increases as the animal attains sexual maturity. Experiment 2- The aim of this work was to verify the presence of PDI in equine seminal plasma and sperm, quantify it and to compare its expression on seminal plasma from fertile and subfertile stallions. Twelve adult stallions with at least two breeding season were used. For the study, four collections of each animal were performed. Immediately after collection, analysis of motility, velocity, concentration and sperm morphology were performed. Stallions were divided in two groups, according to the semen analysis and previous breeding history: Group 1: motility greater than 70% and previous history of pregnancy rates higher than 80%; Group 2: sperm motility less or equal than 30% and breeding history of less than 35% of pregnacy per season. After the analysis, samples were centrifuged at 800 g/10minutes to remove seminal plasma. Samples were prepared as described in Exp. 1. The expression of PDI in seminal plasma was seen in both groups, but with no statistical difference between them. There was no correlation of PDI with sperm motility or concentration. According to these findings, it is not possible to consider PDI as a fertility marker in stallions. More research is needed, involving other mollecular factors, including other PDIs family proteins.

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50

Ahmed, Sangita. "Functional analysis of the Salmonella flagellar export chaperone FlgN." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609708.

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Dissertations / Theses on the topic 'Chaperones'

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