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D'Souza, Sandra Maria. "Constitutive expression of heat shock proteins hsp90, hsc70, hsp70 and hsp60 in the rat during postnatal development." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ34098.pdf.
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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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EL, YAAGOUBI ABDELHAMID. "Role des proteines de choc thermique (dont dnak/hsp70 et groel/hsp60) dans l'expression des proteines chez escherichia coli." Paris 11, 1995. http://www.theses.fr/1995PA112423.
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Cette these a ete consacree a l'etude du role des proteines de choc thermique (hsps) d'e. Coli dans l'expression de proteines chez e. Coli. D'abord, nous avons montre que le chaperon dnak/hsp70, chez e. Coli, est implique dans l'exportation de la galactose binding protein, mglb, ainsi que dans la stabilite de son arm mglb. Par ailleurs nous avons localise dnak au niveau des jonctions de bayer (sites d'adhesion des membranes interne et externe chez e. Coli). Alors que groel interagit preferentiellement avec les chaines laterales des acides amines hydrophobes et affiche une interaction moindre avec les acides amines polaires, nous avons montre que groes reduit la specificite de groel pour les acides amines hydrophobes et augmente sa specificite pour les hydrophiles. Cette modulation par groes de l'affinite de groel pour les chaines laterales des acides amines du substrat proteique pourrait avoir une implication importante dans le repliement de proteines. Nous avons montre egalement que l'activite atpase de groel est stimulee specifiquement par la forme liee au ligand de la galactose binding protein d'escherichia coli, tandis ce qu'elle est faiblement affectee par sa forme non liee. Cette interaction est refletee par la stimulation de l'activite atpase de groel par la galactose binding protein. La troisieme partie decrit la purification d'une nouvelle proteine de choc thermique membranaire de 29 kda, chez e. Coli, qui est en cours de caracterisation. La derniere partie decrit la ca racterisation du systeme de transport galactose binding protein-dependant de s. Thyphimurium dont l'expression est etudie dans la premiere partie de la these. Nous avons purifie et caracterise la proteine mgla comme etant une atpase galactose-dependante. Nous avons reconstitue ce systeme de transport de galactose dans des proteoliposomes
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Schwindel, Uwe. "Prognosefaktoren beim nichtkleinzelligen Bronchialkarzinom Inzidenz von p53, bcl-2, HER-2, HSP27, HSP60 und HSP70 in nichtkleinzelligen Bronchialkarzinomen des Stadiums IIIA /." [S.l.] : [s.n.], 2003. http://archiv.ub.uni-marburg.de/diss/z2003/0697/.
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Brinker, Achim. "Proteininteraktionsdomänen in Hsp70-Hsp90-Multichaperonkomplexen." [S.l. : s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=963295527.
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Silva, Luciana Pugliese da. "Estudo da expressão dos genes de choque térmico hsp90, hsp60 e hsp10 do fungo aquático Blastocladiella emersonii." Universidade de São Paulo, 2003. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-14052018-120842/.
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A proteína de choque térmico Hsp90 é uma chaperone molecular encontrada no citosol. O cDNA incompleto desta proteína foi isolado de uma biblioteca construída a partir de mRNA de células de esporulação de B. emersonii submetidas a choque térmico. Um clone genômico contendo a seqüência completa do gene hsp90 também foi isolado, seqüenciado e caracterizado. A região codificadora do gene hsp90 é interrompida por um único íntron de 184 nucleotídeos. A seqüência de aminoácidos deduzida indicou uma proteína de 71 O resíduos, com massa molecular calculada de 80.792 Da e um pl médio de 4,85. Experimentos de extensão de oligonucleotídeo e RACE-PCR demonstraram um sítio único de início de transcrição localizado a -65 e -70 nucleotídeos do ATG da metionina iniciadora, respectivamente. Motivos similares ao consenso do elemento de choque térmico eucariótico (HSE) e do elemento responsivo a estresse (STRE) foram encontrados na região promotora do gene a -395 e -98 nucleotídeos do ATG, respectivamente. Experimentos de \"Northern blot\" revelaram que o mRNA para a Hsp90 apresenta níveis máximos aos 90 minutos da fase de esporulação do fungo. Análise por \"western blot\" mostrou que a proteína Hsp90 está presente durante todo o ciclo de vida do fungo e os níveis máximos de acúmulo foram observados aos 90 minutos da esporulação, indicando um controle transcricional do gene. Tanto a proteína quanto o mRNA são altamente induzidos quando as células são submetidas a choque térmico e a cádmio. As proteínas Hsp60 e Hsp10 são chaperones moleculares mitocondriais (chaperoninas). Os cDNAs completos destas proteínas foram isolados e totalmente seqüenciados. A seqüência de aminoácidos deduzida da Hsp60 corresponde a uma proteína de 559 resíduos, com massa molecular calculada em 58.741 Da e um pl médio de 8, 7. Experimentos de \"Northern blot\" revelaram que o mRNA para Hsp60 tem níveis máximos de expressão aos 90 minutos da esporulação. Análise por \"western blot\" mostrou que a Hsp60 está presente durante todo o ciclo de vida do fungo, com níveis máximos da proteína 90 minutos após a indução da esporulação. Tanto a proteína quanto o mRNA são bastante induzidos quando as células são submetidas ao choque térmico. A seqüência de aminoácidos deduzida da Hsp10 corresponde um polipeptídeo de 101 resíduos com massa molecular calculada em 10.688 Da e um pl médio de 6,25. Experimentos de \"Northern blot\" revelaram que o mRNA para Hsp10 tem níveis máximos de expressão aos 120 minutos da germinação e é bastante induzido quando as células são submetidas ao choque térmico.The heat shock protein 90 (Hsp90) is a cytosolic molecular chaperone. The incomplete cDNA of this protein was isolated by immunoblot screening of a heat shock cDNA expression library. The complete genomic clone was also isolated and completely sequenced and characterized. The coding sequence is interrupted by a single intron with 184 nucleotides. The deduced amino acid sequence corresponds to a 710-residue polypeptide with a calculated molecular mass of 80,792 Da and an average pl of 4.85. Primer extension and RACE-PCR experiments demonstrated a single transcription start site localized -65 and -70 nucleotides from de ATG of the initiator methionine, respectively. Sequence motifs resembling the standard eukaryotic heat shock element (HSE) and the stress responsive element (STRE) were evident in the regulatory region -395 and -98 nucleotides from de ATG, respectively. Northern blot analysis revealed that the Hsp90 mRNA presents maximum levels by 90 minutes of the sporulation stage. Immunoblot analysis indicated that the Hsp90 is present during the entire life cycle of the fungus and maximum levels were observed 90 minutes after the induction of sporulation, indicating a transcriptional control. During heat shock both the mRNA and the Hsp90 protein are highly induced. Proteins Hsp60 and Hsp10, are mitochondrial molecular chaperones (chaperonines). The complete cDNAs encoding these proteins were and completely sequenced. The deduced amino acid sequence for Hsp60 corresponds to a 559-residue polypeptide with a calculated molecular mass of 58,741 Da and an average pl of 8.7. Immunoblot analysis showed that Hsp60 is present during the entire life cycle of the fungus and presents maximum levels by 90 minutes of the sporulation. Northern blot analysis indicated maximum levels of the Hsp60 mRNA by 90 minutes of sporulation too. Both mRNA and the protein are highly induced during heat shock. The deduced amino acid sequence for Hsp10 corresponds to a 101-residue polypeptide with a calculated molecular mass of 10,688 Da and an average pl of 6.25. Northern blot analysis indicated maximum mRNA levels by 120 minutes of germination and high levels of expression when the cells are exposed to heat shock.
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De, Simone Andrea Stefano. "Daily modulation of the Heat shock proteins (Hsps) in three different species of scleractinian corals." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amslaurea.unibo.it/8401/.
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Temperature and light intensity is the most important environmental parameters that influence circadian cycle of scleractinian corals. In this context, modulation of the biomarkers Hsp60 and Hsp70 in situ was investigated by three different healthy coral species (Acropora tenuis, Echinopora lamellosa and Porites lobata) not stress induced during time course of 24h. Significance species-specific modulation under natural conditions is displayed by all corals under study. A strong fluctuation in Hsps expression is shown by the most susceptible, branched coral A. tenuis, instead of fine and low modulation is shown by the massive coral P. lobata. From the results match between morphology difference and physiological difference response its suggest and similarity pattern between Hsps with different cellular compartments location is suggested too. Starting from this study health of coral reefs could be able to be investigated in the future with a set of biomarkers composed also by Hsps which will be set up.
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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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Njunge, James Mwangi. "Characterization of the Hsp40 partner proteins of Plasmodium falciparum Hsp70." Thesis, Rhodes University, 2014. http://hdl.handle.net/10962/d1013186.
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Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. This thesis details the 70 kDa heat shock protein (Hsp70) and J protein chaperone complements in malaria parasites affecting humans, primates and rodents. Heat shock proteins comprise a family of evolutionary conserved and structurally related proteins that play a crucial role in maintaining the structural integrity of proteins during normal and stress conditions. They are considered future therapeutic targets in various cellular systems including Plasmodium falciparum. J proteins (Hsp40) canonically partner with Hsp70s during protein synthesis and folding, trafficking or targeting of proteins for degradation. However, in P. falciparum, these classes of proteins have also been implicated in aiding the active transport of parasite proteins to the erythrocyte cytosol following erythrocyte entry by the parasite. This host-parasite “cross-talk” results in tremendous modifications of the infected erythrocyte, imparting properties that allow it to adhere to the endothelium, preventing splenic clearance. The genome of P. falciparum encodes six Hsp70 homologues and a large number of J proteins that localize to the various intracellular compartments or are exported to the infected erythrocyte cytosol. Understanding the Hsp70-J protein interactions and/or partnerships is an essential step for drug target validation and illumination of parasite biology. A review of these chaperone complements across the Plasmodium species shows that P. falciparum possesses an expanded Hsp70-J protein complement compared to the rodent and primate infecting species. It further highlights how unique the P. falciparum chaperone complement is compared to the other Plasmodium species included in the analysis. In silico analysis showed that the genome of P. falciparum encodes approximately 49 J proteins, 19 of which contain a PEXEL motif that has been implicated in routing proteins to the infected erythrocyte. Most of these PEXEL containing J proteins are unique with no homologues in the human system and are considered as attractive drug targets. Very few of the predicted J proteins in P. falciparum have been experimentally characterized. To this end, cell biological and biochemical approaches were employed to characterize PFB0595w and PFD0462w (Pfj1) J proteins. The uniqueness of Pfj1 and the controversy in literature regarding its localization formed the basis for the experimental work. This is the first study showing that Pfj1 localizes to the mitochondrion in the intraerythrocytic stage of development of P. falciparum and has further proposed PfHsp70-3 as a potential Hsp70 partner. Indeed, attempts to heterologously express and purify Pfj1 for its characterization are described. It is also the first study that details the successful expression and purification of PfHsp70-3. Further, research findings have described for the first time the expression and localization of PFB0595w in the intraerythrocytic stages of P. falciparum development. Based on the cytosolic localization of both PFB0595w and PfHsp70-1, a chaperone – cochaperone partnership was proposed that formed the basis for the in vitro experiments. PFB0595w was shown for the first time to stimulate the ATPase activity of PfHsp70-1 pointing to a functional interaction. Preliminary surface plasmon spectroscopy analysis has revealed a potential interaction between PFB0595w and PfHsp70-1 but highlights the need for further related experiments to support the findings. Gel filtration analysis showed that PFB0595w exists as a dimer thereby confirming in silico predictions. Based on these observations, we conclude that PFB0595w may regulate the chaperone activity of PfHsp70-1 in the cytosol while Pfj1 may play a co-chaperoning role for PfHsp70-3 in the mitochondrion. Overall, this data is expected to increase the knowledge of the Hsp70-J protein partnerships in the erythrocytic stage of P. falciparum development, thereby enhancing the understanding of parasite biology.
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Daniel, Sheril. "Molecular characterization of the Hsp70/Hsp90 organizing protein (Hop) phosphorylation, subcellular localization and interaction with Hsp90." Thesis, Rhodes University, 2008. http://hdl.handle.net/10962/d1004056.
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Hop (Hsp70-Hsp90 Organizing Protein) is a co-chaperone of two major molecular chaperones, Hsp70 and Hsp90, and acts by transferring substrates from Hsp70 to Hsp90. Although under normal conditions Hop is predominantly localized within the cytosol, Hop has been detected in the nucleus under certain conditions including cell cycle arrest. A putative nuclear localization signal (NLS) has been identified within Hop, which overlaps with the TPR2A domain (previously shown to be critical for Hop-Hsp90 interactions). Hop is phosphorylated in vitro by two cell cycle kinases, namely, casein kinase II (CKII) at S189 and cdc2-kinase at T198; both residues are found upstream of the putative NLS and TPR2A domain. Mimicking phosphorylation at either phosphorylation site appeared to affect the subcellular localization of Hop. The aim of this study was to characterize Hop with respect to its phosphorylation status in vivo, as well as its subcellular localization pattern under heat stress and determine how these properties affected its interaction with Hsp90 as a co-chaperone. Dephosphorylation of proteins under normal and heat shock conditions changed the isoform composition of Hop, providing strong evidence that Hop was phosphorylated in vivo. Surface plasmon resonance (SPR) and glutatione-S-transferase (GST) co-precipitation studies showed that a cdc2-kinase phosphorylated mimic of Hop disrupted Hop-Hsp90 binding. A full length Hop-EGFP construct, as well as substitution mutants of the predicted NLS residues within the Hop-EGFP construct, were transfected into baby hamster kidney (BHK)-21 cells in order to establish the subcellular localization of Hop under heat stress and to test whether predicted residues were critical for nuclear localization of Hop. Under normal conditions, both Hop-EGFP and the NLS mutants were predominantly cytosolic, but when the cells were subjected to heat stress, Hop and its NLS-mutants were localized to both the cytosol and the nucleus. SPR and GST co-precipitation studies showed that substitution of the residues within the major arm of the putative NLS abrogated Hop-Hsp90 interactions. The data obtained from this study, showed for the first time, that Hop was phosphorylated in vivo and suggested that phosphorylation of Hop by cdc2-kinase could inhibit Hop-Hsp90 interactions. Moreover, these results suggested that the subcellular localization of Hop was dependent on stress levels of the cell, particularly heat stress. We propose that the nuclear localization of Hop may be primarily regulated by stress and secondarily by cell cycle arrest. The major arm of the putative NLS did not affect the localization of Hop directly, but was shown to be critical for Hop-Hsp90 binding in vitro. The results of this study suggested that binding of Hop to Hsp90 sequestered Hop within the cytosol and that Hsp90 acted as a cytosolic retention factor for Hop. Both phosphorylation of Hop, and its subcellular localization, appeared to be intimately related to its interaction with Hsp90 as a co-chaperone.
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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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Schmid, Andreas B. "The adaptor protein Sti1/Hop connects the Hsp70 and Hsp90 chaperone cycle /." München : Verl. Dr. Hut, 2009. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=018860584&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Brychzy, Alexander. "Characterisation of Tpr2, a novel regulator of the Hsp70-Hsp90 multichaperone complex." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971635587.
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Smith, David. "Hsp90 and hsp70 genes of Theileria annulata : structure, regulation and molecular phylogeny." Thesis, University of York, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298437.
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Adeyemi, Samson Adebowale. "Structural bioinformatics analysis of the Hsp40 and Hsp70 molecular chaperones from humans." Thesis, Rhodes University, 2014. http://hdl.handle.net/10962/d1020962.
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HSP70 is one of the most important families of molecular chaperone that regulate the folding and transport of client proteins in an ATP dependent manner. The ATPase activity of HSP70 is stimulated through an interaction with its family of HSP40 co-chaperones. There is evidence to suggest that specific partnerships occur between the different HSP40 and HSP70 isoforms. While some of the residues involved in the interaction are known, many of the residues governing the specificity of HSP40-HSP70 partnerships are not precisely defined. It is not currently possible to predict which HSP40 and HSP70 isoforms will interact. We attempted to use bioinformatics to identify residues involved in the specificity of the interaction between the J domain from HSP40 and the ATPase domain from the HSP70 isoforms from humans. A total of 49 HSP40 and 13 HSP70 sequences from humans were retrieved and used for subsequent analyses. The HSP40 J domains and HSP70 ATPase domains were extracted using python scripts and classified according to the subcellular localization of the proteins using localization prediction programs. Motif analysis was carried out using the full length HSP40 proteins and Multiple Sequence Alignment (MSA) was performed to identify conserved residues that may contribute to the J domain – ATPase domain interactions. Phylogenetic inference of the proteins was also performed in order to study their evolutionary relationship. Homology models of the J domains and ATPase domains were generated. The corresponding models were docked using HADDOCK server in order to analyze possible putative interactions between the partner proteins using the Protein Interactions Calculator (PIC). The level of residue conservation was found to be higher in Type I and II HSP40 than in Type III J proteins. While highly conserved residues on helixes II and III could play critical roles in J domain interactions with corresponding HSP70s, conserved residues on helixes I and IV seemed to be significant in keeping the J domain in its right orientation for functional interactions with HSP70s. Our results also showed that helixes II and III formed the interaction interface for binding to HSP70 ATPase domain as well as the linker residues. Finally, data based docking procedures, such as applied in this study, could be an effective method to investigate protein-protein interactions complex of biomolecules.
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Botha, Melissa. "Characterisation of the plasmodium falciparum Hsp40 chaperones and their partnerships with Hsp70." Thesis, Rhodes University, 2009. http://hdl.handle.net/10962/d1003997.
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Central to this research, 40 kDa Heat shock proteins (Hsp40s) are known to partner (or cochaperone) 70 kDa Heat shock proteins (Hsp70s), facilitating the selection and transfer of protein substrate to Hsp70 and the stimulation of the protein folding ability of Hsp70. Members of the diverse Hsp70-Hsp40 protein complement of Plasmodium falciparum have been implicated in the cytoprotection of this malaria parasite, and are thought to facilitate the protein folding, assembly and translocation tasks required by the parasite to commandeer the infected human erythrocyte subsequent to invasion. In particular, the parasite has evolved an expanded and specialised 43- member suite of Hsp40 proteins, 19 of which bear an identifiable export motif for secretion into the infected erythrocyte cytoplasm where they potentially interact with human Hsp70. Although type I Hsp40 proteins are representative of typical regulators of Hsp70 activity, only two of these proteins are apparent in the parasite’s Hsp40 complement. These include a characteristic type I Hsp40 termed PfHsp40, and a larger, atypical type I Hsp40 termed Pfj1. Both Hsp40 proteins are predicted to be parasite-resident and are most likely to facilitate the co-chaperone regulation of the highly abundant and stress-inducible Hsp70 homolog, PfHsp70-I. In this work, the co-chaperone functionality of PfHsp40 and Pfj1 was elucidated using in vivo and in vitro assays. Purified recombinant PfHsp40 was shown to stimulate the ATPase activity of PfHsp70-I in in vitro single turnover and steady state ATPase assays, and co-operate with PfHsp70-I in in vitro aggregation suppression assays. In these in vitro assays, heterologous partnerships could be demonstrated between PfHsp70-I and the human Hsp40, Hsj1a, and human Hsp70 and PfHsp40, suggesting a common mode of Hsp70-Hsp40 interaction in the parasite and host organism. The functionality of the signature Hsp40 domain, the Jdomain, of Pfj1 was demonstrated by its ability to replace the equivalent domain of the A. tumefaciens Hsp40, Agt DnaJ, in interactions with the prokaryotic Hsp70, DnaK, in the thermosensitive dnaJ cbpA E. coli OD259 deletion strain. An H33Q mutation introduced into the invariant and crucial HPD tripeptide motif abrogated the functionality of the J-domain in the in vivo complementation system. These findings provide the first evidence for the conservation of the prototypical mode of J-domain based interaction of Hsp40 with Hsp70 in P. falciparum. Immunofluorescence staining revealed the localisation of PfHsp40 to the parasite cytoplasm, and Pfj1 to the parasite cytoplasm and nucleus in cultured intraerythrocytic stage P. falciparum parasites. PfHsp70-I was also shown to localise to the parasite cytoplasm and nucleus in these stages, consistent with the literature. Overall we propose that PfHsp40 and Pfj1 co-localise with and regulate the chaperone activity of PfHsp70-I in P. falciparum. This is the first study to identify and provide evidence for a functional Hsp70-Hsp40 partnership in P. falciparum, and provides a platform for future studies to elucidate the importance of these chaperone partnerships in the establishment and survival of the parasite in the intraerythrocytic-stages of development.
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Worrall, Liam. "Structural and biochemical studies of the Caenorhabditis elegans Hsp70/Hsp90 chaperone system." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/14705.
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This work presents the crystal structure of the C-terminal 10 kDa- sub-domain from C. elegans Hsp70. Despite a high degree of sequence identity, the C. elegans domain is shown to adopt a conformation distinct from the rat crystal structure, consistent with the more distantly related bacterial homologous. Comparison with the rat structure reveals an intriguing putative domain-swap dimerisation mechanism though the isolated C. elegans domain was found to exist exclusively as a monomer in solution. A previous study identified two TPR domain containing C. elegans putative proteins predicted to interact with Hsp90. These proteins were identified as the C. elegans homologues for small glutamine-rich TPR containing protein (SGT) and Hsp70/Hsp90 organising protein (HOP). These proteins have been successfully cloned, expressed and purified. SGT forms homo-dimers in solution. Its hydrodynamic dimensions in relation to its molecular weight suggest a protein with a low level of compactness and an extended conformation. SGT interacts with the C-terminal peptides from both Hsp70 and Hsp90 with equal affinities. Studies on C. elegans HOP suggested it might exist as a dimer in solution. In addition, a tight binding interaction was demonstrated with human and C. elegans Hsp90 homologues. A thorough search of the complete C. elegans proteome and genome was performed to identify the complete repertoire of TPR domain containing proteins likely to interact with Hsp70 or Hsp90. A profile HMM based search of the published C. elegans protein and DNA databases identified 12 proteins; nine of which are homologues of proteins known to interact with Hsp70 or Hsp90. The remaining three are uncharacterised putative proteins and represent targets for further study.
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Lanneau, David. "Rôle des protéines de choc thermique HSP90 et HSP70 dans la différenciation macrophagique." Phd thesis, Université de Bourgogne, 2010. http://tel.archives-ouvertes.fr/tel-00560535.
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La synthèse des protéines de choc thermique (HSPs) est un moyen de défense développé par la cellule pour faire face aux diverses agressions auxquelles elle peut être soumise. En tant que chaperons, les HSPs participent aux mouvements intracellulaires des protéines, préviennent l'agrégation des protéines altérées, éliminent les protéines anormales et contribuent à la conformation correcte des peptides nouvellement synthétisées. Mon équipe d'accueil s'intéresse aux rôles des HSPs dans des processus cellulaires tels que l'apoptose et la différenciation cellulaire. Le but de mon travail de thèse consiste à étudier le rôle des protéines de choc thermique HSP90 et HSP70 au cours de la différenciation des monocytes en macrophages. J'ai dans un premier temps étudié l'implication de HSP90 dans la différenciation macrophagique. c-IAP1 est un membre de la famille des protéines inhibitrices de l'apoptose impliqué dans la régulation de l'apoptose, dans le cycle cellulaire et dans la signalisation cellulaire. Nous avons précédemment montré que c-IAP1 migre du noyau vers le cytoplasme au cours de la différenciation cellulaire. Nous démontrons dans ce travail que c-IAP1 est une protéine cliente de la protéine de choc thermique HSP90β. Dans trois différents modèles de différenciation, ces protéines interagissent et migrent ensemble du noyau vers le cytoplasme au cours de la différenciation cellulaire. L'inhibition de HSP90 ou la déplétion spécifique de l'isoforme β par des siRNA conduisent à sa dégradation par le protéasome. La fonction de chaperon moléculaire de HSP90 envers c-IAP1 est spécifique de l'isoforme β car la déplétion de l'isoforme α n'a pas d'effets sur c-IAP1. De plus l'inhibition de HSP90 ou la déplétion de HSP90β bloquent la différenciation cellulaire tout comme la déplétion de c-IAP1 par siRNA. La deuxième partie de montre travail a consisté à étudier le rôle de HSP70 dans la différenciation macrophagique. Nous montrons que cette protéine est fortement induite après stimulation des cellules par le facteur de croissance M-CSF et que son inhibition bloque la différenciation des monocytes en macrophage. HSP70 interagit avec la protéine Spi-1/Pu.1, facteur de transcription clé de la différenciation macrophagique. L'expression de Spi-1/Pu.1 augmente également au cours de la différenciation macrophagique et ce de manière similaire à celle de HSP70. Ceci suggère l'implication des facteurs de transcription responsables de l'induction des HSPs, les Heat Shock Factor (HSF). L'étude du promoteur de Spi-1/Pu.1 a révélé la présence d'une séquence ressemblant fortement aux éléments de réponse classiques sur lesquels se fixe HSF1. HSF1 est capable de se fixer sur le promoteur de Spi-1/Pu.1 et l'inhibition de HSF1 bloque l'expression de Spi-1/Pu.1. HSF1 participe donc au contrôle de l'expression de Spi-1/Pu.1 lors de la différenciation macrophagique. HSP90 et HSP70 sont donc essentielles à la différenciation macrophagique. Comprendre les mécanismes cellulaires impliqués dans les voies de différenciation se révèle extrêmement important puisque des altérations des mécanismes de l'hématopoïèse sont retrouvées dans plusieurs types de leucémies (leucémies aiguës myéloblastiques et leucémies myélo-monocytaires chroniques). Connaître le rôle des HSPs dans la différenciation cellulaire permettrait donc de développer de nouvelles stratégies thérapeutiques pour le traitement de ces pathologies.
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Weeks, Stacey. "Characterisation of the HSP70-HSP90 organising protein gene and its link to cancer." Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/56006.
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HOP (Heat shock protein 70/ Heat shock protein 90 organising protein) is a co-chaperone essential for client protein transfer from HSP70 to HSP90 within the HSP90 chaperone machine and has been found to be up-regulated in various cancers. However, minimal in vitro information can be found on the regulation of HOP expression. The aim of this study was to analyse the HOP gene structure across known orthologues, identify and characterise the HOP promoter, and identify the regulatory mechanisms influencing the expression of HOP in cancer. We hypothesized that the expression of HOP in cancer cells is likely regulated by oncogenic signalling pathways linked to cis-elements within the HOP promoter. An initial study of the evolution of the HOP gene speciation was performed across identified orthologues using Mega5.2. The evolutionary pathway of the HOP gene was traced from the unicellular organisms to fish, to amphibian and then to land mammal. The synteny across the orthologues was identified and the co-expression profile of HOP analysed. We identified the putative promoter region for HOP in silico and in vitro. Luciferase reporter assays were utilized to demonstrate promoter activity of the upstream region in vitro. Bioinformatic analysis of the active promoter region identified a large CpG island and a range of putative cis-elements. Many of the cis-elements interact with transcription factors which are activated by oncogenic pathways. We therefore tested the regulation of HOP levels by rat sarcoma viral oncogene homologue (RAS). Cancer cell lines were transfected with mutated RAS to observe the effect of constitutively active RAS expression on the production of HOP using qRT-PCR and Western Blot analyses. Additionally, inhibitors of the RAS signalling pathway were utilised to confirm the regulatory effect of mutated RAS on HOP expression. In cancer cell lines containing mutated RAS (Hs578T), HOP was up-regulated via a mechanism involving the MAPK signalling pathway and the ETS-1 and C/EBPβ cis-elements within the HOP promoter. These findings suggest for the first time that Hop expression in cancer may be regulated by RAS activation of the HOP promoter. Additionally, this study allowed us to determine the murine system to be the most suited genetic model organism with which to study the function of human HOP.
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Höft, Maxine Allison. "Regulation of cell biology by extracellular species of the Hsp90- Hsp70 organising protein (Hop)." Thesis, Rhodes University, 2017. http://hdl.handle.net/10962/59199.
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Hageman, Jurre. "The human HSP70/HSP40 chaperone family a study on its capacity to combat proteotoxic stress /." [S.l. : [Groningen : s.n.] ; University of Groningen] [Host], 2008. http://irs.ub.rug.nl/ppn/.
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Clitheroe, Crystal-Leigh. "In-silico analysis of Plasmodium falciparum Hop protein and its interactions with Hsp70 and Hsp90." Thesis, Rhodes University, 2013. http://hdl.handle.net/10962/d1003819.
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A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.
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Rohrberg, Julia [Verfasser], Johannes [Akademischer Betreuer] Buchner, and Sevil [Akademischer Betreuer] Weinkauf. "Einfluss des Hsp70-Hsp90-Chaperonsystems auf die Reifung und Aktivierung von Hsp90-Substratproteinen / Julia Rohrberg. Gutachter: Sevil Weinkauf ; Johannes Buchner. Betreuer: Johannes Buchner." München : Universitätsbibliothek der TU München, 2011. http://d-nb.info/1033164186/34.
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O'leary, John Clarence. "The Role of Molecular Chaperones in the Etiology and Treatment of Psychiatric Diseases in the Elderly." Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4737.
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The elderly are at increased risk for developing psychiatric diseases, which include Alzheimer's disease, depression, anxiety and suicide. The probability of multiple disease comorbidity is also increased in the elderly. At the cellular level, the loss of protein homeostasis is often at the root of disease emergence, and thus the scientific community is searching for ways to help maintain this balance. A vast group of proteins that are paramount to balancing and counterbalancing protein levels is the molecular chaperone protein group, which has evolved a tremendous variety of functions in the cell. They aid in protein trafficking, folding, receptor signaling, neurotransmission, vesicle forming and fusion, protein degradation, and apoptosis, among other activities. Despite their best efforts, disease still ensues, but because of their vast number and multiple abilities, it may be possible to modulate these proteins as a way to treat and prevent disease. Chaperones are of particular interest in diseases of aging, because chaperone induction and effectiveness is reduced with age. In addition, many diseases of the elderly are brought on by aberrant protein accumulation, like Alzheimer's disease.
As a result, the hypothesis of this dissertation is whether the
modulation of molecular chaperones changes disease pathology. A molecular chaperone family that is important to protein degradation is the Hsp70 chaperone complex. Hsp70 proteins have specialized function depending on cell type and cellular compartment, but Hsp70 proteins are very important for protein synthesis and degradation. As a result, they are in a position to contribute to the regulation of proteins that become aberrant.
In recent years scientific literature has indicated that compounds that inhibit the enzymatic ATP hydrolysis of these proteins promote tau degradation, which accumulates in Alzheimer's disease. Alzheimer's disease is the sixth leading cause of death in the U.S., it is a progressive neurodegenerative disease, and is caused by the aberrant accumulation of the amyloid beta and tau proteins. Here, we show that treatment with the Hsp70 inhibitor methylene blue, reduces tau, saves neurons, and restores cognition, in a mouse model of tau accumulation (rTg4510). Cognitive rescue occurred despite a severe tangle load, equal to control treated tau transgenic mice. This study shows that reducing soluble tau can restore cognition, reducing tangles is not necessarily to ameliorate cognition, and saving neurons is not sufficient to increase cognition if they are burdened with soluble tau.
This work shows that methylene blue does not affect the the number
of tau tangles in this model, as suggested by in vitro data. It also suggests that further work into the development of Hsp70 ATPase inhibitors may find success in alleviating the soluble tau burden found in Alzheimer's disease.
The co-chaperone FKBP5 is also of extreme importance, not because it is essential, but because research has implicated this protein with a host of psychiatric diseases. Single nucleotide polymorphisms in this gene, which increase the levels of FKBP5, interact with averse traumatic events to enhance the likelihood of developing mood and anxiety disorders, including major depressive disorder, post-traumatic stress disorder, bipolar disorder, and suicide. Moreover, we have found that FKBP5 protein levels increase with age in the human brain, increasing the risk for the elderly of developing disease if exposed to traumatic stress. Here, we tested the hypothesis that FKBP5 negatively regulates resilient behavior. We found that FKBP5 levels increase with age in the wild type mouse brain, and that wild type mice display reduced resiliency with age. FKBP5-/- mice, on the other hand, show enhanced resiliency to stress at all ages tested, and are protected from aging-induced despair. At the molecular level, FKBP5 is a robust inhibitor of the glucocorticoid receptor, which is responsible for the shut-off of the hypothalamic-pituitary-adrenal axis.
In addition, excess glucocorticoid levels in the blood is a robust marker of psychiatric disease. Consequently, FKBP5 may be causing disease through enhanced levels of glucocorticoids. FKBP5-/- mice display reduced corticosterone after stress. Moreover, corticosterone production increases with age, and FKBP5-/- mice are protected from this increase. These studies are the first to show that reducing the levels of FKBP5 is a promising therapeutic option for the treatment of mood disorders in the elderly, resiliency naturally declines with age due to FKBP5, corticosterone levels after stress rise due to FKBP5, and that the ablation of this gene increases resiliency and prevents aging- induced despair.
As a whole, these data show that the modulation of chaperone proteins has the potential for developing new therapies for the treatment of psychiatric diseases of the elderly.
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Zhang, Huaqun. "Biophysical Study of the Ubiquitin Ligase CHIP and Interactions with the Molecular Chaperones Hsp70 and Hsp90." Miami University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=miami1511192922525393.
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Mokoena, Fortunate. "Characterization of the co-chaperones of Hsp70 and Hsp90 in Trypanosoma brucei and their potential partnerships." Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/54543.
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African Trypanosomiasis, which is caused by Trypanosoma brucei, is one of the crippling agents of social and economic development in Africa. T. brucei cycles between the cold-blooded insect vector, the tsetse fly (Glossina spp), and warm-blooded mammalian hosts. T. brucei, T. cruzi and L. major are mammal infecting kinetoplastid parasites that are collectively referred to as TriTryps. These parasites experience extreme environments as they move between their warm-blooded mammalian hosts and cold-blooded insect vectors which trigger extensive morphological transformations during the life-cycle of the parasite. Molecular chaperones have been implicated in parasite differentiation. TriTryps display significant expansions and diversity in the gene complements encoding molecular chaperones, especially J-proteins. Generally, J-proteins function as co-chaperones of Hsp70s, forming part of vital protein homeostasis processes. Hsp70s show a high degree of conservation, while J-proteins appear to be an extreme case of taxonomic radiation. Although several studies have focused on the molecular and cell biology of Hsp70s in some kinetoplastid parasites, knowledge is still lacking pertaining to J-proteins and their partnerships with Hsp70s. This thesis focused on the classification of kinetoplastid Jproteins into the four types by examining the domain organizations using T. brucei as a guide. The potential partnership of J-proteins and Hsp70s were postulated based on predicted subcellular localization. Kinetoplastid parasites, particularly T. brucei, have evolved an expanded and specialized J-protein machinery, likely to be a consequence of an evolutionary fitness/trait to adapt to diverse environment present in hosts and vectors. These analyses will yield insight into the process of parasite differentiation as well as provide new leads for chemotherapeutic treatments. The presence of the STI1 mediated Hsp90 hetero-complex formation has not been confirmed in T. brucei. To this end, in silico and biochemical techniques were used to characterize the role of TbSTI1, as an adaptor protein of Hsp70 and Hsp90. Through domain architecture analysis, sequence alignments, phylogenetic analysis and three-dimensional structure prediction, TbSTI1 was demonstrated to be the most conserved TPR containing co-chaperone of Hsp70 and Hsp83 in T. brucei and also shown to be highly similar to its eukaryotic homologues. Recombinant TbSTI1 was overproduced and purified in E.coli cells and subsequently shown to associate with TcHsp70 in a concentration dependent manner and associate weakly with TbHsp70.4. TbSTI1 and TbHsp83 were also demonstrated to be expressed and upregulated upon exposure to heat shock at the bloodstream stage of parasite development. In conclusion, this study is the first to report the interaction of TbSTI1 with a chaperone. Interactions between TbSTI1 and Hsp70s were demonstrated and therefore, the formation of the hetero-complex is predicted based the similarity of TbSTI1 to other STI1 proteins.
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Wingate, Ianthe. "Identification of potential novel roles for Hsp70/Hsp90 organising protein (Hop) using proteomic analysis in human cells." Thesis, Rhodes University, 2016. http://hdl.handle.net/10962/64758.
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Horáčková, Lucie. "Testování viability nádorových linií buněk po působení chemických látek a chemoterapeutik." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2018. http://www.nusl.cz/ntk/nusl-376847.
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Individual types of viability tests based on colorimetric changes of the solution are desribed in the theoretical part. Furthermore, HSP proteins are characterized, which are not connected only by heat shock, but also during other cell stresses such as exposure to UV, cold, extreme pH or heavy metals. They are important for the cell, because they help to reformulate proteins that have been damaged by cellular stress and also bind to new unpacked proteins and ensure their correct folding. Proteins that are affected by molecular chaperones are collectively called client proteins. Some HSPs also contribute to membrane transport or degradation. These proteins are co-operative with the cochaperones, which are important for heat shock proteins because they help them to pack protein, in particular by catalyzing the hydrolysis of ATP to ADP. Herein is also described cisplatin and its derivatives, including mechanism of action and adverse effects. This work was focused on detection cytotoxicity of cisplatin and its derivatives. Cells were exposed to stress condition induced by cytostatics and huge changes in heat shock proteins and cochaperon levels were observed. There was also observed colocalization of heat shock proteins and their client protein p53 by confocal microscopy in these stressing conditions.
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Willmer, Tarryn. "The role of Hsp90/Hsp70 organising protein (Hop) in the Proliferation, Survival and Migration of Breast Cancer Cells." Thesis, Rhodes University, 2012. http://hdl.handle.net/10962/d1015720.
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Hop (the Hsp90/Hsp70 organising protein) is a co-chaperone that acts as an adapter between the major molecular chaperones Hsp90 and Hsp70 during the cellular assembly of the Hsp90 complex. The Hsp90 complex regulates the stability and conformational maturation of a range of important cellular proteins, many of which are deregulated in cancer. In this study, we hypothesised that Hop knockdown inhibits proliferation and migration of cancer cells. We characterised the expression of Hop in cell models of different cancerous status, and provided evidence that Hop was upregulated in tumour cells compared to normal cell counterparts. Using an RNA interference approach, a 60-90% knockdown of Hop was achieved for up to 144 hours in the MDA-MB-231 and Hs578T breast cancer cell lines. Hop knockdown resulted in downregulation of the Hsp90 client proteins, Akt and Stat3, as well as a change in the expression of other Hsp90 co-chaperones, p23, Cdc37 and Aha1, while no change in the levels of Hsp90 or Hsp70 was observed. Silencing of Hop impaired cell proliferation in Hs578T cells but an increase in proliferation in MDA-MB-231, suggesting that the role of Hop in cancer cell proliferation was dependent on type of cancer cell. Hop knockdown in Hs578T and MDA-MB- 231 cells did not lead to any significant changes in the half maximal inhibitory concentrations (IC50) of selected small molecule inhibitors (paclitaxel, geldanamycin and novobiocin) in these cell lines after 72 hours. Hop knockdown cells were however, more sensitive than control cells to the Hsp90 inhibitors geldanamycin and novobiocin at earlier time points and in the presence of the drug transporter inhibitor, verapamil. Hop knockdown caused a decrease in cell migration as measured by the wound healing assay in both Hs578T and MDA-MB-231 cells. Hop was present in purified pseudopodia fractions of migrating cells, and immunofluorescence analysis showed that Hop colocalised with actin at the leading edges of pseudopodia, points of adhesion and at intercellular junctions of cells that have been stimulated to migrate with the chemokine stromal derived factor-1. Hop was able to bind to actin in vitro using actin cosedimentation assays, and silencing of Hop dramatically reduced the capacity of Hs578T cells to form pseudopodia. These results establish a correlation between Hop and actin dynamics, pseudopodia formation and migration in the context of Hop silencing, and collectively suggest that Hop plays a role in cancer cell migration. This study presents experimental evidence for a promising alternative to targeting Hsp90 and Hsp70 chaperones, a novel drug target in cancer therapy.
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Mattison, Stacey. "Analysis of the human HSP70-HSP90 organising protein (HOP) gene - characterisation of the promoter and identification of a novel isoform." Thesis, Rhodes University, 2018. http://hdl.handle.net/10962/62821.
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Rosales, Villavicencio Inés-Marlene. "Characterization of the HSP70 protein homolog (HSP70h) of citrus tristeza closterovirus." [Gainesville, Fla.] : University of Florida, 2001. http://purl.fcla.edu/fcla/etd/UFE0000350.
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Thesis (Ph. D.)--University of Florida, 2001.Title from title page of source document. Document formatted into pages; contains xi, 116 p.; also contains graphics. Includes vita. Includes bibliographical references.
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Abdeen, Sanofar, Nilshad Salim, Najiba Mammadova, Corey M. Summers, Rochelle Frankson, Andrew J. Ambrose, Gregory G. Anderson, et al. "GroEL/ES inhibitors as potential antibiotics." Elsevier, 2016. http://hdl.handle.net/10150/618724.
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We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the
Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett. 2014, 24, 786]. As the GroEL/ES
chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with
small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we
report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and
Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae.
GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular
S. aureus, where lead compounds exhibited antibiotic effects from the low-lM to mid-nM range.
While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively
target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no
cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds
8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney
cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus
strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates
to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.
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Nanbu, Kanako. "Expression of heat shock proteins HSP70 and HSP90 in endometrial carcinomas : correlation with clinicopathology, sex steroid receptor status, and p53 protein expression." Kyoto University, 1999. http://hdl.handle.net/2433/181231.
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Niewidok, Natalia [Verfasser], and Michael [Akademischer Betreuer] Flentje. "Modulation of radiosensitivity of human tumor and normal cells by inhibition of heat shock proteins Hsp90 and Hsp70 / Natalia Niewidok. Betreuer: Michael Flentje." Würzburg : Universitätsbibliothek der Universität Würzburg, 2013. http://d-nb.info/1044532432/34.
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Melville, Mark Wallace. "The interferon induced serine/threonine protein kinase, PKR, is regulated by the influenza virus activated protein, P58IPK, and the molecular chaperones, Hsp40 and Hsp70 /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/11489.
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Koren, John. "The Role of Hsp70 in Cancer: A Study of the Hsp70 / Akt Relationship." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4105.
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The Hsp70 family of molecular chaperones is essential for
protein folding, re-folding misfolded client proteins, clearance
of aberrant client proteins, and can also inhibit programmed
cell death. There are two major cytosolic members of this
family: the constitutive Hsc70, and the inducible Hsp72. Under
stress conditions the Hsp70 family protects the cell from
protein related damage by the induction of Hsp72. Hsc70 and
Hsp72 are highly homologous with minor differences in
substrate binding. In cancers, Hsp72 is commonly induced and
this induction is thought to aid in cancer cell survival. In these
studies we demonstrate the differential regulation of the prosurvival
kinase Akt by Hsc70 and Hsp72. We demonstrate that
of the two cytosolic forms, Hsp72 is the primary Akt regulator.
Using a phenothiazine class inhibitor of Hsp70-family activity,
methylene blue, we demonstrate dose dependent decreases in
the levels of Akt; produced breast cancer specific cell death.
This cell death could be rescued by the use of an Hsp70 family ATPase stimulating compound, SW02. We also demonstrate a
similar phenotype with a rhodacyanine class Hsp70 family
inhibitor, YM-1, also capable of reducing Akt and causing
cancer specific cytotoxicity. The resulting Akt decreases were
sufficient to block a tamoxifen-resistance pathway, allowing
previously resistant cells to regain sensitivity to tamoxifen.
These results demonstrate the capabilities of Hsp70 family
inhibitors as potent compounds for the treatment of breast
cancer.
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Silva, Sabrina Matos de Oliveira da. "Clonagem, expressão heteróloga e caracterização da proteína de escolta da Hsp70 de Leishmania braziliensis." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/75/75132/tde-27102011-085909/.
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A Leishmaniose é uma doença infecciosa causada por protozoários flagelados do gênero Leishmania. Os parasitas como a Leishmania braziliensis, sofrem várias mudanças morfológicas durante seu ciclo de vida, incluindo a troca de organismo hospedeiro. Durante essas mudanças, proteínas de choque térmico ou chaperones moleculares, como, por exemplo, a Hsp70, são expressas em grande quantidade. A função da Hsp70 é auxiliar no processo de enovelamento protéico, no transporte de proteínas entre as membranas e em muitas outras importantes funções celulares. A Hsp70 é auxiliada por várias proteínas denominadas como co-chaperone e a Hep1 (do inglês Hsp70-escort protein 1) é uma delas. Essa co-chaperone tem seu papel descrito principalmente em mitocôndrias como estabilizadoras da Hsp70 capazes de prevenir a sua agregação. O objetivo deste trabalho foi clonar, expressar, purificar e caracterizar as proteínas Hsp70 e Hep1 de L. braziliensis (LbHsp70 e LbHep1). Os ensaios preliminares mostraram que a LbHsp70 foi expressa de forma insolúvel, sendo necessário expressar a proteína em corpos de inclusão para tentativas de reenovelamento, afim de obter a mesma na fração solúvel. Apesar da LbHsp70 se apresentar na fração solúvel após o reenovelamento, a mesma foi purificada como agregado. Ainda na tentativa de obter a LbHsp70 na forma solúvel, a mesma foi co-expressa com a LbHep1 (expressa na forma solúvel), porém a LbHsp70 continuou na fração insolúvel do lisado bacteriano. Como a LbHep1 não apresentou a atividade esperada quando co-expressa com a LbHsp70 citoplasmática, foram feitos ensaios de co-expressão da LbHep1 com a Hsp70 mitocondrial humana, que é heterologamente expressa na forma de agregados, com o intuito de confirmar a atividade estabilizadora das Hep1 sobre as Hsp70 mitocondriais. Este experimento possibilitou a obtenção de ambas proteínas na fração solúvel, de acordo com dados apresentados na literatura para este sistema em outros organismos. Uma vez mostrada à funcionalidade da LbHep1, foi feita a caracterização desta proteína por métodos biofísicos como dicroísmo circular, espectrometria de fluorescência, cromatografia de exclusão molecular analítica e ultracentrifugação analítica. Os experimentos mostraram que a LbHep1 apresenta estrutura secundária composta principalmente de folhas-β pregueadas e que o único triptofano está parcialmente exposto ao solvente. As análises hidrodinâmicas mostraram que a LbHep1 é assimétrica e em equilíbrio entre monômeros e dímeros. Por fim, dados de ultracentrifugação analítica indicam que a LbHep1 está em equilíbrio monômero-dímero.Leishmaniasis is an infectious disease caused by flagellate protozoa of the genus Leishmania. The parasites such as Leishmania braziliensis undergo various morphological changes during its life cycle, including the exchange of the host organism. During these changes, heat shock proteins or molecular chaperones like Hsp70, for example, are expressed in large amounts. The function of Hsp70 is to assist in the process of protein folding, protein transport between the membranes and many other important cellular functions. The Hsp70 is assisted by several proteins called co-chaperones and the Hsp70-escort protein (Hep1) is one of them. This co-chaperone has been described based on its role as a stabilizer of mitochondrial Hsp70 preventing their aggregation. The objective of this study was to clone, express, purify and characterize the Hsp70 and Hep1 ortologues of Leishmania braziliensis (LbHsp70 and LbHep1). The preliminary tests showed that LbHsp70 was expressed in the insoluble form, being necessary to express the protein in inclusion bodies to attempt its refolding in order to get it in the soluble fraction. Despite LbHsp70 was obtained in the soluble fraction after refolding, it was purified as aggregates. Still trying to get the LbHsp70 in the soluble form, it was co-expressed with LbHep1 (always expressed in the soluble form), but LbHsp70 remained in the insoluble fraction of the bacterial lysate. As LbHep1 showed no expected activity when co-expressed with LbHsp70, which is citoplasmatic, we tested if LbHep1 was able to act on human mitochondrial Hsp70 which is expressed as aggregates in bacterial heterologous systems. Then, we co-expressed LbHep1 with human mitochondrial Hsp70 which allowed obtaining both proteins in the soluble fraction, in according to data presented in the literature. Once the functionality of LbHep1 was showed, we characterize this protein by biophysical methods such as circular dichroism, fluorescence spectrometry, molecular exclusion chromatography and analytical ultracentrifugation analysis. The experiments showed that the secondary structure features LbHep1 composed mainly of β-sheets and that the only tryptophan is partially exposed to solvent. Hydrodynamic analysis showed that the protein is asymmetric and in equilibrium between monomers and dimers. Finally, analytical ultracentrifugation data indicate that LbHep1 is a system in equilibrium monomer-dimer.
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Fu, Peng. "Membrane Hsp70 expression in gliomas." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-149571.
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McLellan, Catherine Ann. "Interaction of HspBP1 with Hsp70." Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/289897.
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The Hsp70 family protein members are involved in many diverse processes that are essential for cell survival. The Hsp70 cochaperones control and specify Hsp70 interactions by modifying Hsp70 activity and linking Hsp70 proteins to specific cellular processes. Understanding the mechanistic details of how cochaperones co-opt Hsp70 for specific functions in a cell may provide insight into the workings of specific cellular functions as well as a broader understanding of how multifunctional proteins are regulated. HspBP1 is an Hsp70 cochaperone that binds to the ATPase domain of Hsp70 and inhibits its ATPase activity. The purpose of these studies was to gain a basic understanding of the Hsp70/HspBP1 interaction. The first goal was to define the structural domains of HspBP1 and explore the secondary structure of those domains. The next goal was to determine if HspBP1's ability to bind to Hsp70 and inhibit its activity segregated to its distinct protein domains. Finally, the effects of HspBP1 association on Hsp70 structure were investigated. It was determined from these studies that although HspBP1 is encoded by seven exons, it has only two structural domains as determined by limited proteolysis. Domain I, amino acids 1-83, is largely unstructured. Domain II, amino acids 84-359 is predicted to be 43% helical using circular dichroism. It was also shown that domain II is sufficient for binding to the Hsp70 ATPase domain and inhibiting luciferase renaturation although domain I was required for full activity. These studies also describe a novel activity for HspBP1, the ability to change the conformation of the ATPase domain. Only domain II of HspBP1 is required to bring about this conformational change. The studies presented here are the first to examine HspBP1's structure and determine how its structural domains interact with Hsp70. Prior to these studies, no structural information had been reported for an HspBPl family member. This information, along with the discovery that HspBPl can alter the conformation of the ATPase domain of Hsp70 will bring us closer to understanding this protein family's role in Hsp70 regulation.
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Martineau, Céline. "Spécificités fonctionnelles des Hsp70 cytoplasmiques chez la levure." Phd thesis, AgroParisTech, 2010. http://pastel.archives-ouvertes.fr/pastel-00617115.
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Les Hsp70 constituent une famille de chaperons moléculaires ubiquitaires qui jouent des rôles essentiels dans le repliement, le transport ou la dégradation des protéines. Le cytoplasme des cellules eucaryotes contient plusieurs paralogues de Hsp70 fortement conservés qui diffèrent essentiellement par leur expression spatio-temporelle. Plusieurs travaux suggèrent que ces paralogues ont des spécificités fonctionnelles que nous avons cherché à mettre en lumière et caractériser par des approches génétiques. Dans une première étude, nous avons comparé les activités des Hsp70 des levures Saccharomyces cerevisiae (Ssa1-4) et Yarrowia lipolytica (Ssa5-8) lorsqu'elles sont exprimées comme unique Hsp70 chez S. cerevisiae. Nous avons montré que ces Hsp70: 1) assurent la viabilité des cellules mais avec des taux de croissance très différents; 2) ont des effets variables sur la propagation et la stabilité des prions [URE3] et [PSI+]; et 3) permettent la dégradation protéasomale de CFTR avec des cinétiques comparables. Dans une seconde étude, nous avons montré que la formation de biofilms chez la levure dépend de la machinerie Hsp70 qui contrôle, via des voies distinctes, l'expression, la maturation et le recyclage d'une adhésine de surface (Flo11) requise pour ce processus. Enfin, nous avons construit et caractérisé des mutants de Y. lipolytica dans lesquels un ou plusieurs gènes codant des chaperons moléculaires ou acteurs de la protéostase (e.g. Hsp70, Hsp104, CHIP) ont été invalidés. Malgré une forte homologie et une redondance fonctionnelle, les Hsp70 possèdent des propriétés distinctes permettant aux cellules de faire face à différents types de substrats et de conditions de stress.
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Gehrmann, Mathias. "Untersuchungen zur Modulation der Hitzeschockprotein-70-(Hsp70)-Expression und Identifizierung Hsp70-assoziierter Moleküle auf der Zellmembran." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971837074.
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Manzerra, Pasquale. "Expression of constitutive hsc70 and stress-inducible hsp70 mRNA and protein in the rabbit central nervous system." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0009/NQ28007.pdf.
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Krainz, Thomas Edward. "An Analysis of Heat Shock Protein Production in Human Retinal Pigment Epithelial Cells After Different Stress-Induced States." Walsh University Honors Theses / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=walshhonors1524248740945083.
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Lavin, Claire. "The role of Hsp70 in diabetes." Thesis, University of Liverpool, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.396417.
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Javid, Babak. "Cross-presentation of antigen by HSP70." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613807.
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Petitjean, Celine. "Phylogénie et évolution des Archaea, une approche phylogénomique." Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-01070633.
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En 1977, Carl Woese sépare les procaryotes en deux grands groupes en proposant une nouvelle classification basée sur des critères phylogénétiques. Les Archaea deviennent ainsi un domaine à part entière aux cotés des Bacteria et des Eucarya. Depuis, la compréhension de ce nouveau groupe et de ses relations avec les deux autres domaines, essentielles pour comprendre l'évolution ancienne du vivant, est largement passée par l'étude de leur phylogénie. Presque 40 ans de recherche sur les archées ont permis de faire évoluer leur image : de bactéries vivant dans des milieux spécialisés, souvent extrêmes, on est passé à un domaine indépendant, très diversifié aussi bien génétiquement, métaboliquement ou encore écologiquement. Ces dernières années la barre symbolique de cent génomes complets d'archées séquencés a été franchie et, parallèlement, les projets génomiques et métagénomiques sur des groupes peu caractérisés ou de nouvelles lignées de haut rang taxonomique (e.g. Nanohaloarchaea, Thaumarchaeota, ARMAN, Aigarchaeota, groupe MGC, groupe II des Euryarchaeota, etc.) se sont multipliés. Tout ceci apporte un matériel sans précédent pour l'étude de l'histoire évolutive et de la diversité des Archaea. Les protéines ribosomiques ont été utilisées de façon courante pour inférer la position phylogénétique des nouvelles lignées d'Archaea. Néanmoins, les phylogénies résultantes ne sont pas complètement résolues, laissant des interrogations concernant d'importantes relations de parenté. La recherche de nouveaux marqueurs est donc cruciale et c'est dans ce contexte que mon projet de thèse s'inscrit. À partir de l'analyse des génomes de deux Thaumarchaeota et d'une Aigarchaeota, nous avons identifié 200 protéines conservées et bien représentées dans les différents phyla d'archées. Ces protéines sont impliquées dans de nombreux processus cellulaires, ce qui peut apporter un signal phylogénétique complémentaire à celui des marqueurs de type informationnel utilisés par le passé. En plus de confirmer la plupart des relations phylogénétiques inférées à partir de ces derniers (i.e., protéines ribosomiques et sous unités de l'ARN polymérase), l'analyse phylogénétique de ces nouveaux marqueurs apporte un signal permettant une meilleure résolution de la phylogénie des archées et la clarification de certaines relations jusqu'ici confuses. Un certain nombre de ces nouveaux marqueurs sont aussi présents chez les bactéries. Les relations entre les grands phyla d'archées restant encore non résolues, nous avons utilisé ces protéines pour essayer de placer la racine de l'arbre des Archaea en utilisant comme groupe extérieur les bactéries. Nous avons ainsi pu identifier 38 protéines, parmi les 200 sélectionnées précédemment, ayant un signal phylogénétique suffisamment fiable pour cette étude, auxquelles nous avons ajouté 32 protéines ribosomiques universelles. L'utilisation conjointe de ces données nous a permis de placer la racine entre les Euryarchaeota, d'une part, et un groupe rassemblant les Thaumarchaeota, les Aigarchaeota, les Korarchaeota et les Crenarchaeota, d'autre part. Ce nouvel éclairage sur l'évolution ancienne des archées nous a amené à proposer une révision de leur taxonomie avec, principalement, la création du nouveau phylum "Proteoarchaeota" contenant les quatre phyla actuels que nous proposons de rétrograder en classes : Thaumarchaea, Aigarchaea, Korarchaea et Crenarchaea.Finalement, l'analyse des protéines codées dans les trois génomes qui ont servi de point de départ de ma thèse nous a permis de générer une masse considérable de données qui ont révélé des traits particuliers ou encore des histoires évolutives inattendues. Un exemple est l'histoire du complexe formé par la chaperonne DnaK et de ses co-chaperonnes GrpE, DnaJ, et DnaJ-Fer chez les Thaumarchaeota, impliquant plusieurs transferts horizontaux entre les trois domaines du vivant.
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Gobbo, Jessica. "Inhibition de HSP70 : une nouvelle piste thérapeutique contre le cancer." Thesis, Dijon, 2013. http://www.theses.fr/2013DIJOS088/document.
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Les HSP ou protéines de stress ont été découvertes chez la drosophile par Ritossa en 1962. Hautement conservées entre les espèces, elles sont essentielles à l’homéostasie cellulaire et plus encore à la survie lors de stress d’origine diverse (chimique, physique, métabolique etc…). Actuellement, chez les mammifères, il existe cinq principales familles d'HSPs en fonction de leur poids moléculaires : HSP110, HSP90, HSP70 et HSP60 et les petites HSPs à laquelle appartient HSP27 (Kampinga et al., 2009). Parmi les HSPs, HSP70 est la plus fortement induite que ce soit par des agressions telles que le stress oxydatif, les agents anticancéreux ou les radiations ionisantes. HSP70 est, contrairement aux cellules « normales », fortement exprimée dans les cellules cancéreuses et confère à ces cellules une résistance aux drogues anticancéreuses (Goloudina, Demidov & Garrido, 2012) favorisant ainsi le développement tumoral. Les propriétés cyto-protectrices de HSP70 ont été jusqu’à présent attribuées par ces fonctions intracellulaires, principalement en inhibant le processus apoptotique à différentes étapes clés de la signalisation cellulaire (Ravagnan et al., 2001). Cependant une forme membranaire de HSP70 a été détectée à la surface des exosomes dérivant des cellules tumorales (Kuppner et al., 2001). HSP70 membranaire participerait au processus de tumorigenèse en inhibant l’activation des cellules myéloïdes suppressives (MDSCs) et en conséquence, la réponse immune anti-tumorale (Pfister et al., 2007; Schmitt, Gehrmann, Brunet, Multhoff, & Garrido, 2007). Par cette double facette, HSP70 représente donc une cible thérapeutique de choix pour la thérapie anticancéreuse. Compte tenu du rôle clé de HSP70, à la fois par ses fonctions intracellulaires et extracellulaires dans le développement tumoral, l’un des projets phare de notre groupe a été de développer des inhibiteurs spécifiques de HSP70, notamment des aptamères peptidiques et des peptides. Dans un premier travail, nous avons démontré in vitro que deux aptamères A8 et A17 (et son dérivé P17), interagissent avec HSP70 sur des domaines différents et sensibilisent les cellules cancéreuses à la mort induite par des agents chimiothérapeutiques. Des études in vivo réalisées chez la souris et le rat confirment ces résultats et mettent en évidence une réduction significative de la croissance tumorale par ces aptamères peptidiques. Dans un deuxième travail, nous avons généré un dérivé de l’aptamère A8, le peptide P8.1. Nous avons démontré que ce peptide est capable de neutraliser la forme membranaire de HSP70, présente à la surface des exosomes, bloquant ainsi l’activation des MDSCs et restaurant la réponse immunitaire anti-tumorale. A plus long terme, ce travail vise à mettre au point et à valider en clinique une thérapie anticancéreuse plus efficace, en associant aux traitements anticancéreux actuels, les inhibiteurs de HSP70Heat shock proteins (HSPs) were first discovered in Drosophila by Ritossa in 1962. As stress proteins, HSPs are induced in response to a wide variety of physiological and environmental insults. HSPs have a cyto-protective function and act as molecular chaperones by assisting the folding of nascent or misfolded proteins and by preventing their aggregation. Mammalian HSPs have been classified into 5 families according to their molecular weight: HSP110, HSP90, HSP70, HSP60 and the family of small HSPs such as HSP27 (Kampinga et al., 2009). The most well-known inducible stress chaperone HSP70 is hardly detectable at basal level in normal “non-stressed” cells, but in cancer cells HSP70 is constitutively highly expressed. In that respect, this HSP play a key role in oncogenesis and in resistance to chemotherapeutic drugs (Goloudina et al., 2012).Until now, the cytoprotective properties of HSP70 were attributed to its intracellular functions mainly via its ability to block the apoptotic process at key points of the signal (Ravagnan et al., 2001). More recently, a membrane bound form of HSP70 was detected but also at the surface of exosomes derived from tumor cells but not non-cancerous cells (Kuppner et al., 2001). Moreover, growing evidence support the critical role of this membrane-bound HSP70 in the process of tumorigenesis (Pfister et al., 2007; Schmitt et al., 2007) via the activation of myeloid suppressor cells (MDSCs), which inhibit the anti-tumor immune response (Chalmin et al., 2010). Thereby, HSP70 by this dual action represents an attractive target for new anti-cancer therapy.In that aim, we developed specific inhibitors of HSP70, including peptide aptamers and peptides. In this work, we demonstrated that two aptamers A8, A17 (and the peptide P17), interact with different domains of HSP70 and, significantly sensitized cancer cells to apoptosis induced by chemotherapeutic drugs. Accordingly, in vivo studies in mice and rats showed a significant reduction of tumor growth by these inhibitors. Finally, we generate an A8 derived peptide called P8.1 that specifically neutralized the extracellular region of HSP70 at the surface of exosomes. Our results demonstrated that this peptide P8.1 inhibits MDSC activation and restored the antitumor immune response in vitro and in vivo, respectively.Overall, our work will help to develop and validate more effective cancer therapy based on the association of conventional chemotherapy with HSP70 inhibitors
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Ferreira, Anna Paula Baptista Ribeiro. "Bovinos submetidos a estresse vacinal com imunógenos sintéticos e desafiados com Rhipicephalus (Boophilus) microplus e Babesia bovis: expressão imunoistoquímica de proteínas de choque térmico (HSP70 e HSP90) e bioquímica sangüínea." Universidade Federal de Viçosa, 2007. http://locus.ufv.br/handle/123456789/4970.
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Previous issue date: 2007-03-20The expression of HSP70 and HSP90 was evaluated through immunoistochemistry studies in bovine inoculated with synthetic peptides SBbo23290 and SBm7462 applied simultaneously, in association or in individual presentation. The animals received three applications with 30 days of interval among them and after challenged with the Babesia bovis strain UFV1 - 9th passage. The histopathological studies showed reactivity of the lymphoid organ seven days after the first immunization with germinative centers increased. After the second immunization, it was evident the hyperplasia of the paracortical and medular areas, with subsequent decrease of the reactivity of the germinative centers after this period, suggesting formation of a memory response to the immunogens. The heat shock proteins presented basal expression. HSP70 expressed preferentially in the paracortical area, while HSP90 showed more visible immunomarker in the paracortical and medular areas. Immunoistochemical studies showed a coincidence in the expression of HSP90 and of the peptides SBbo23290 and SBm7462, when were considered the same periods of immunization, suggesting the formation of a complex HSP- peptide. The glucose, lactate, haptoglobin and C-reactive protein levels stayed inside of the values considered physiologic for the bovine species. The basal cellular immunoreaction of HSP70 and HSP90, as well as the maintenance of the normal levels of the biochemical parameters during the whole period of the experiment, suggest that the stress provoked by the synthetic peptides was insufficient to cause damage to the organism. In other hand, the maintainance of the health condition of the animals influenced the immunological response, and the basal levels of the heat shock proteins might have been enough to promote cytoprotection after the challenge.
A expressão de HSP70 e HSP90 foi avaliada por meio de estudos imunoistoquímicos, em bovinos inoculados com peptídeos sintéticos SBbo23290 e SBm7462, aplicados simultaneamente, em associação ou em apresentação individual. Os animais receberam 3 aplicações em intervalos de 30 dias entre si e posteriormente desafiados com cepa de Babesia bovis (UFV1- 9ª passagem). Os estudos histopatológicos mostraram reatividade do órgão linfóide sete dias após a primeira imunização, com centros germinativos aumentados. Após a segunda imunização, ficou evidente a hiperplasia da região paracortical e medular, com subseqüente diminuição da reatividade dos centros germinativos após este período, sugerindo formação de uma resposta de memória aos imunógenos. As proteínas de choque térmico apresentaram expressão basal. A HSP70 expressou, preferencialmente, na região paracortical, enquanto que a HSP90 mostrou imunomarcação mais visível nas regiões paracortical e medular. Estudos imunoistoquímicos mostraram uma coincidência na expressão de HSP90 com os peptídeos SBbo23290 e SBm7462, quando se consideraram os mesmos períodos de imunização, sugerindo a formação de um complexo HSP-peptídeo. Os níveis de glicose, lactato, haptoglobina e proteína C-reativa mantiveram-se dentro dos valores considerados fisiológicos para a espécie bovina. A imunomarcação celular basal de HSP70 e HSP90, bem como a manutenção dos níveis normais das variáveis bioquímicas durante todo o período do experimento, sugerem que o estresse provocado pelos peptídeos sintéticos foi insuficiente para causar dano ao organismo. Em contrapartida, a manutenção do estado de saúde dos animais influenciou a reposta imunológica, e os níveis basais das proteínas de choque térmico podem ter sido suficientes para promover citoproteção após o desafio.
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Gastou, Marc François Philippe. "Rôle de la protéine HSP70 au cours de l'anémie de Blackfan-Diamond." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC231/document.
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L’anémie de Blackfan-Diamond (ABD) est une érythroblastopénie congénitale rare, secondaire à un blocage de la maturation érythroïde entre les stades BFU-e et CFU-e. L’ABD est le plus souvent la conséquence d’une mutation germinale affectant un gène codant pour une protéine ribosomique (RP) de la petite ou de la grande sous-unité du ribosome. Quatorze gènes distincts ont été identifiés. Les gènes les plus fréquemment mutés sont les gènes RPL5, RPL11 et RPS19 (37% des patients). Plus rarement, l’ABD est la conséquence de mutations dans le gène TSR2 ou dans le gène GATA-1. Ce dernier code pour un facteur de transcription majeur de l’érythropoïèse. Chez les patients ABD, les mutations de GATA-1 induisent une perte quasi-totale de la forme longue de GATA-1 qui est nécessaire à la différenciation de la cellule érythroïde. Notre groupe a identifié deux phénotypes de l’ABD in vitro en fonction du gène muté. En cas d’haploinsuffisance RPS19, la prolifération érythroïde est moins réduite qu’en cas d’haploinsuffisance RPL5 ou de RPL11. Une haploinsuffisance RPS19 n’altère pas la différenciation érythroïde et n’induit pas d’apoptose contrairement à l’haploinsuffisance RPL5 ou RPL11 où il existe un retard de différenciation érythroïde et un excès net d’apoptose responsable au moins en partie de la diminution drastique de la prolifération érythroïde dans ces phénotypes.HSP70 est impliquée dans la survie cellulaire et la différenciation érythroïde en protégeant GATA-1 du clivage par la caspase-3, une protéase activée lors de la différenciation érythroïde terminale. Comme la différence entre les deux phénotypes d’ABD in vitro concernait la différenciation érythroïde et la survie cellulaire, nous avons émis l’hypothèse selon laquelle la mutation de certains gènes RP provoque un défaut d’expression d’HSP70 conduisant au blocage de la différenciation érythroïde et à l’excès d’apoptose retrouvés dans les phénotypes sévères d’ABD.Nous avons étudié différents patients atteints d’ABD, porteurs de mutations dans les gènes RPS19, RPL5 ou RPL11 et généré un modèle in vitro d’ABD en exprimant, dans des cellules CD34+ humaines issues de sang de cordon, des ARN interférents ciblant RPL5, RPL11 ou RPS19. Chez les patients comme dans le modèle reproduisant l’ABD, l’haploinsuffisance RPL5 ou RPL11 diminue drastiquement l’expression protéique de HSP70 et de GATA-1 (Western blot, microscopie confocale et en cytométrie couplée à des techniques d’imagerie, (technologie ImageStream) à la différence de 1’haploinsuffisance RPS19. Dans tous les cas, HSP70 est normalement transcrite et traduite. Les inhibiteurs du protéasome (MG132, lactacystine, bortezomib) restaurent l’expression10de HSP70. La diminution d’expression de HSP70 est donc liée à une dégradation protéasomale. L’invalidation de RPL11 induit une polyubiquitinylation importante de HSP70. La transduction lentivirale de l’ADN complémentaire d’HSP70 dans les cellules primitives invalidées pour RPL11 permet de restaurer l’expression de HSP70 et de GATA-1 à un niveau similaire aux contrôles et de rétablir la prolifération cellulaire et la différenciation érythroïde, confirmant le rôle clé de HSP70 dans le phénotype sévère RPL5+/Mut ou RPL11+/Mut. Les formes les plus sévères de l’ABD sont associées à la dégradation de HSP70 par le protéasome. La perte de la protéine chaperone de GATA-1 induit la perte de GATA-1, facteur de transcription majeur de la différenciation érythroïde. Une augmentation de l’expression de HSP70 pourrait ainsi constituer une nouvelle approche thérapeutique dans l’ABDDiamond-Blackfan anemia (DBA) is the first ribosomopathy identified and is characterized by a moderate to severe, usually macrocytic aregenerative anemia associated with congenital malformations in 50% of the DBA cases. This congenital rare erythroblastopenia is due to a blockade in erythroid differentiation between the BFU-e and CFU-e stages. The link between a haploinsufficiency in a ribosomal protein (RP) gene that now encompass 15 different RP genes and the erythroid defect is still to be fully defined. Recently, mutations in TSR2 and GATA-1 genes have been identified in a few DBA families. The GATA-1 gene encodes for the major transcription factor critical for erythropoiesis and mutation in this gene that lead to loss of expression of the long form of the protein, necessary for the erythroid differentiation accounts for erythroblastopenia of DBA phenotype. Our group and others (Dutt et al., Blood 2011) have shown previously that p53 plays an important role in the DBA erythroblastopenia, inducing cell cycle arrest in G0/G1 and depending on the nature of RP gene mutation, a delayed erythroid differentiation and an increased apoptosis. Indeed, we identified two distinct DBA phenotypes (H. Moniz, M. Gastou, Cell Death Dis, 2012): a haploinsufficiency in RPL5 or RPL11 reduced dramatically the erythroid proliferation, delayed the erythroid differentiation, and markedly increased apoptosis, while RPS19 haploinsufficiency while reduced the extent of erythroid proliferation without inducing significant apoptosis. While p53 pathway has been found to be activated in RP haploinsufficient erythroid cells in DBA patients or shRNA-RPS19, -RPL5, or -RPL11 infected CD34+ erythroid cells, the intensity of the p53 activation pathway (p21, BAX, NOXA) is different depending on the mutated RP gene. Since the differences between the two phenotypes involved the eytrhoid differentiation and the degree of apoptosis we hypothesized that HSP70, a chaperone protein of GATA-1 may play a key role in the erythroid defect of DBA. Indeed, HSP70 protects GATA-1 from the cleavage by the caspase 3, a protease activated during erythroid differentiation. As such reduced levels of HSP70 related to a RP haploinsufficiency could account for increased apoptosis and delayed erythroid differentiation of erythroid cells in DBA. Indeed, a defect in RPL5 or RPL11 decreased dramatically the expression level of HSP70 and GATA-1 in primary human erythroid cells from DBA patients and following in vitro knockdown of the proteins in CD34+ cells by RPL5 or RPL11 shRNA. Importantly, RPS19 haploinsufficiency did not exhibit this effect in conjunction with normal levels of HSP70 expression. Furthermore, we found that the decreased expression level of12HSP70 was independent on the p53 activation. Strikingly, HSP70 was noted to be degraded by the proteasome since the bortezomib, the MG132, or the lactacystin were able to restore both the HSP70 expression level and intracellular localization in the cell. The lentiviral infection of depleted RPL11 cord blood CD34+ cells with a wild type HSP70 cDNA restored both the erythroid proliferation and differentiation, and reduced apoptosis, confirming a critical role for HSP70 in the erythroid defect in the RPL11+/Mut DBA phenotypes. The loss of HSP70 may explain the loss of GATA-1 in DBA and also the erythroid tropism of the DBA disease. Restoration of the HSP70 expression level may be a viable and novel therapeutic option for management of this debilitating and difficult to manage erythroid disorder
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Siegal, Mark, and Joanna Masel. "Hsp90 depletion goes wild." BioMed Central, 2012. http://hdl.handle.net/10150/610309.
Full textAbstract:
Hsp90 reveals phenotypic variation in the laboratory, but is Hsp90 depletion important in the wild? Recent work from Chen and Wagner in BMC Evolutionary Biology has discovered a naturally occurring Drosophila allele that downregulates Hsp90, creating sensitivity to cryptic genetic variation. Laboratory studies suggest that the exact magnitude of Hsp90 downregulation is important. Extreme Hsp90 depletion might reactivate transposable elements and/or induce aneuploidy, in addition to revealing cryptic genetic variation.See research article http://wwww.biomedcentral.com/1471-2148/12/25 webcite