The main objective of this study was to investigate promoter sequences of putative HSR genes for the presence of unique regulatory elements and modules that might be involved in the regulation of HSR. In order to achieve this objective, an in silico promoter analysis strategy was devised, which focused on the identification of promoter sequences and regulatory elements, and modelling of promoter modules by using Genomatix software tools such as MatInspector and ModelInspector. Results showed that two modules (EGRF_SP1F_01 and SP1F_CEBP_01) were conserved in the promoter sequences of three well-known Hsp-genes (Hsp90, Hsp105β and αβ-crystallin). Screening the 60 target gene promoters for the presence of the two modules revealed that 12 genes (20 %) contained both modules. These included Moesin, Proline-4 hydroxylase, Poly(A) binding protein and Formin-binding protein. None of these genes had been previously associated with heat shock response.

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.

The main objective of this study was to investigate promoter sequences of putative HSR genes for the presence of unique regulatory elements and modules that might be involved in the regulation of HSR. In order to achieve this objective, an in silico promoter analysis strategy was devised, which focused on the identification of promoter sequences and regulatory elements, and modelling of promoter modules by using Genomatix software tools such as MatInspector and ModelInspector. Results showed that two modules (EGRF_SP1F_01 and SP1F_CEBP_01) were conserved in the promoter sequences of three well-known Hsp-genes (Hsp90, Hsp105β and αβ-crystallin). Screening the 60 target gene promoters for the presence of the two modules revealed that 12 genes (20 %) contained both modules. These included Moesin, Proline-4 hydroxylase, Poly(A) binding protein and Formin-binding protein. None of these genes had been previously associated with heat shock response.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

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

Orientadoesr: Carlos Henrique Inácio Ramos, Gonçalo Amarante Guimarães Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T06:21:14Z (GMT). No. of bitstreams: 1 Goncalves_DanieliCristina_M.pdf: 10469841 bytes, checksum: df29d5b11d3cdd27679b971b2bbcb032 (MD5) Previous issue date: 2012 / Resumo: Chaperonas moleculares (Heat Shock proteins - HSPs) são componentes chave do sistema de controle de qualidade de proteínas (PQC - Protein Quality Control), que é essencial para a vida, sendo responsável por manter a homeostase proteica e a adequada função de diversas vias. Problemas no processo de enovelamento estão relacionados a doenças degenerativas, amilóides e câncer. Em plantas, as chaperonas moleculares desempenham um papel crucial na proteção contra estresses bióticos e abióticos, pois como organismos sésseis, as plantas devem ser capazes de responder rapidamente a mudanças na temperatura, salinidade, déficit hídrico, entre outros. A chaperona molecular Hsp90 (Heat Shock protein 90 kDa) compreende uma família ubíqua, considerada um 'hub' por interagir com chaperonas, co-chaperonas e ter como clientes proteínas regulatórias essenciais como fatores de transcrição, quinases, receptores de hormônios, entre outros. A Hsp90 age em conjunto com co-chaperonas, as quais modulam e direcionam sua função. Uma destas co-chaperonas é a Hop (Hsp70-Hsp90 organizing protein), capaz de interagir simultaneamente com a Hsp90 e Hsp70, mediando a transferência de substratos. A Hop é composta por três domínios com repetições de tetratricopeptídeos (TPR) (TPR1, TPR2A e TPR2B), responsáveis pela interação com as chaperonas, porém a dinâmica desta interação não está bem entendida, uma vez que ainda não há estrutura da Hop inteira e o estado oligomérico desta co-chaperona ainda é controverso na literatura. Neste trabalho apresentamos a classificação de um gene de Hsp90 de cana-de-açúcar, e o início de sua caracterização funcional através de transgenia em Arabidopsis thaliana. Apresentamos também a caracterização biofísica de uma importante co-chaperona da Hsp90, a Hop (Hsp70-Hsp90 organizing protein) humana. Através da análise de sequências a Hsp90 de cana-de-açúcar foi classificada como Hsp90-3, uma isoforma citosólica. Plantas transgênicas de A. thaliana, produzidas a partir da inserção do gene da Hsp90-3 de cana-de-açúcar, apresentaram níveis reduzidos de Hsp90. Tal perturbação nos níveis de Hsp90 parece ter afetado a expressão de outras proteínas da rede de interações, relacionadas com processos diversos como resposta imune e fotossíntese. As plantas transgênicas também exibiram germinação mais rápida e raízes mais longas em relação ao controle. Sob estresse térmico, linhagens transgênicas apresentaram maior suscetibilidade à alta temperatura em relação ao controle. Tais resultados sugerem que a Hsp90 tem um importante papel na fisiologia celular e no desenvolvimento, e que os níveis de Hsp90 são críticos para a resposta frente a estresses. A caracterização biofísica do mutante Hop D456G, uma mutação no domínio TPR2B, mostrou que esta proteína é uma mistura de monômeros, dímeros e oligômeros maiores, porém com prevalência do estado monomérico. O resíduo D456 pode ter uma participação na dinâmica de dimerização e é possível que o estado oligomérico da Hop seja regulado entre os estados monomérico e dimérico, com a finalidade de facilitar sua atividade adaptadora / Abstract: Molecular chaperones (heat shock proteins - HSPs) are key components of protein quality-control system (PQC - Protein Quality Control), which maintains protein homeostasis and the proper function of several pathways, being essential for life. Defects in folding processes are related to degenerative diseases, amyloidosis and cancer. In plants, which as sessile organisms must be able to respond rapidly to changes in temperature, salinity, water deficit, and others, molecular chaperones play a crucial role in protecting against such biotic and abiotic stresses. Molecular chaperone Hsp90 (Heat Shock Protein 90 kDa) comprise an ubiquitous family, considered a hub as it interacts with chaperones, co-chaperones, and have as clients key regulatory proteins such as transcription factors, kinases, hormone receptors, and others. The chaperone acts together with co-chaperones, which modulate and guide Hsp90 function. The co-chaperone Hop (Hsp70-Hsp90 organizing protein), interacts simultaneously with Hsp90 and Hsp70, mediating substrate transfer. Hop has three TPR domains (TPR1, and TPR2A TPR2B) responsible for interaction with the chaperones, but this interaction dynamics remains unclear, since there is no structure of full length Hop and its oligomeric state is controversial in literature reports. This work presents the classification of an Hsp90 gene from sugarcane, and primary functional characterization studies in Arabidopsis thaliana transgenic lines. We also present the biophysical characterization of the human Hsp90 co-chaperone Hop (Hsp70-Hsp90 organizing protein). Through sequence analysis the Hsp90 from sugarcane has been classified as Hsp90-3, a cytosolic isoform. Transgenic A. thaliana, produced by Hsp90-3 insertion, exhibited reduced transcript levels of Hsp90. This disruption in Hsp90 levels seems to affect the expression of other proteins from the interaction network, which are related to various processes such as immune response and photosynthesis. Transgenics also exhibited faster germination and longer roots than the control. Under heat stress, transgenic lines showed increased susceptibility to high temperature. These results suggest that Hsp90 has an important role in cellular physiology and development; in addition the levels of Hsp90 are critical for responses to stresses. The biophysical characterization of the mutant D456G Hop, a mutation in domain TPR2B showed that this protein is a mixture of monomers, dimers and higher oligomers, but the monomeric state is majoritary. The residue D456 may be involved in dimerization dynamics, and it is possible that Hop is regulated between monomeric and dimeric species, to enable its adaptor functions / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Masters of Science / The main objective of this study was to investigate promoter sequences of putative HSR genes for the presence of unique regulatory elements and modules that might be involved in the regulation of HSR. In order to achieve this objective, an in silico promoter analysis strategy was devised, which focused on the identification of promoter sequences and regulatory elements, and modelling of promoter modules by using Genomatix software tools such as MatInspector and ModelInspector. Results showed that two modules (EGRF_SP1F_01 and SP1F_CEBP_01) were conserved in the promoter sequences of three well-known Hsp-genes (Hsp90, Hsp105β and αβ-crystallin). Screening the 60 target gene promoters for the presence of the two modules revealed that 12 genes (20 %) contained both modules. These included Moesin, Proline-4 hydroxylase, Poly(A) binding protein and Formin-binding protein. None of these genes had been previously associated with heat shock response. / South Africa

Heat shock proteins (Hsps) are a class of molecular chaperones which were first discovered as proteins up-regulated in response to heat stress in Drosophila. Later, it was found that these set of proteins get up-regulated as a general stress response associated with destabilization of native protein structures. Over a period of time, intricate involvement of Hsps in various biological processes has been well established. Heat shock protein 90 (Hsp90) is one of the important representative of this class of proteins. Hsp90 is an essential molecular chaperone which is evolutionarily conserved. It has a selective set of proteins to chaperone called as clients, which majorly include transcription factors and protein kinases. Through its interaction with its clients it modulates cell cycle, signal transduction, differentiation, development and evolution. Previous studies from Candida, Leishmania and Plasmodium have implicated Hsp90 to be involved in stage transition and growth. It is also critically involved in regulating growth of other protozoans such as Dictyostelium, Entamoeba and Trypanosoma. Thus, selective inhibition of Hsp90 has been explored as an intervention strategy against important human diseases such as cancer, malaria and other protozoan diseases. In Plasmodium falciparum, Hsp90 plays a critical role in stage transition. The parasite inside the human RBC develops from ring to trophozoite to schizont stage and inhibition of Hsp90 using specific pharmacological inhibitor arrests the growth of parasite at ring stage. In Dictyostelium, it has been observed that Hsp90 function is required for development. Inhibition of Hsp90 causes mound arrest and stops the cells from entering to its next developmental stage, fruiting bodies. In parallel, Hsp90 in Candida has been shown to be involved in morphogenesis. In nature Candida exists as a single cell yeast form and upon entry into the human host these yeast forms undergo morphogenesis to form virulent filamentous fungi. Inhibition of Hsp90 mimics temperature mediated morphogenesis. All together, these studies suggest that Hsp90 functions in a context dependent manner and each biological system explored has given new insights into the Hsp90 biology. Giardia lamblia, a protozoan parasite of humans and animals, is an important cause of diarrheal disease causing significant morbidity and also mortality in tropical countries. In the present study we focus on the biology of Hsp90 from Giardia lamblia. Giardia has a biphasic life cycle with infective cyst stage and pathogenic trophozoite stage. These cysts are present in the environment and enter mammalian host through oral route. They undergo a process called as excystation in the intestine giving rise to trophozoites. The trophozoites so formed colonize the upper part of the small intestine which causes the symptoms of giardiasis. Some of the trophozoites escape from the nutrition rich milieu of the upper part of small intestine to the lower part. In this region, trophozoites undergo a process called as encystation, wherein each trophozoite forms a cyst which escapes through faeces back into the environment. As seen in the life cycle of Giardia there are two major biological transitions, excystation and encystation; and till date no definitive player or pathway is known to regulate these processes. With the knowledge of Hsp90 playing an important role in similar biological transitions in other organisms we were encouraged to study role of Hsp90 in Giardia lamblia. Trans-splicing based generation of a full length Hsp90 in Giardia lamblia To understand the role of Hsp90, we first carried out sequence alignment of Hsp90 predicted ORFs in Giardia genome with yeast Hsp90. On alignment we observed that Hsp90 in Giardia is discontinuous and is annotated to be encoded by two different ORFs. Hsp90 in most organisms is coded by a single ORF with none to many cis-spliced introns. In a relatively intron poor organism G. lamblia, cytosolic Hsp90 is coded by two different ORFs separated by 777 kb in the genome. On multiple sequence alignment, we noticed that these two ORFs correspond to two independent regions of the Hsp90 protein. The ORFs are designated as hspN and hspC, containing the N-terminal and the C-terminal region of the protein respectively. We began our study by sequencing whole genome of Giardia lamblia clinical strain. Our genome sequencing confirmed the split nature of hsp90 and showed high ‘synteny’ between the other sequenced isolates. Using PCR based approach we have ruled out the possibility of having a full length gene in the genome. In contradiction to the genome result, we have observed a higher molecular weight protein in the lysate on proteomic analysis which was further confirmed by western blotting. The protein was observed to have a molecular weight of 80 kDa which could be a resultant of combination of two ORFs, suggesting the presence of a full length mRNA for Hsp90. PCR amplification using primers against both the fragments resulted in amplification of 2.1 kb product from the RNA pool of Giardia. Sequencing of this product showed that hspN and hspC were stitched together to form a mature messenger for full length Hsp90. In total our results suggest a post transcriptional process, trans-splicing, to be involved in the construction of Hsp90. The transition marked by this fusion coincides with the canonical GU¬AG splice site transitions as observed in other eukaryotes. Interestingly, a 26 nt near-complementary region was observed inside and upstream of hspN and hspC ORFs respectively. Put together these results suggest that the 26 nt complementary region acts as the positioning element to bring these two precursors in spatial proximity. With efficient spliceosomal activity these two precursor forms are trans-spliced to generate a full length cytosolic Hsp90 in Giardia. There are only four genes which have cis-spliced introns in the Giardia genome and the core components of the spliceosomal machinery are also present. The presence of canonical splice site in both the transcripts suggests that these transcripts are fused together by the spliceosomal machinery by the phenomenon of trans-splicing. The formation of full length Hsp90 RNA by its fragmented gene is the first example of trans-splicing in Giardia. To understand, are there any other genes which are also similarly trans-spliced we have carried out shotgun proteomic analysis of the total cell lysate obtained from Giardia trophozoites. Using Hsp90 as template, in our proteomic datasets, we have designed an algorithm for identification of additional trans-spliced gene products at the protein level. We have identified a total of 476 proteins of which hypothetical proteins constitute the major class followed by metabolic enzymes. We have compared the theoretical molecular weights for the identified proteins with the experimentally determined mass. Any discrepancy in the molecular mass was further analyzed and we assigned a gene to be potentially trans-spliced based on three criteria: if they were encoded by two or more different ORFs (loci), absence of a single full length counterpart and presence of splice sites with branch point and positional elements. Using this algorithm we were able to identify dynein as a potential candidate of trans-splicing reaction which was confirmed by the nucleotide sequence analysis of the predicted ORFs. Interestingly, dynein gene fragments were observed to be scattered on different chromosomes with minor splice sites unlike hsp90 genes. In vivo Expression of Hsp90 sub-fragments, HspN and HspC In the mature Hsp90 mRNA formed upon trans-splicing, 33 additional codons are present right between hspN and hspC sequences and they were acquired from the upstream region of hspC ORF. The 33 codons encode for an important region of Hsp90 which harbours the conserved catalytic “Arg” residue; suggesting that the full length Giardia Hsp90 (GlHsp90) formed could be an active ATPase. To confirm the same we have carried out in vitro characterization of trans-spliced Hsp90. Towards this, we have cloned, expressed and purified His tag-GlHsp90. As a first step, highly purified protein was used to assess its efficiency in binding to it cognate ligand, ATP, and the known inhibitors. Our binding studies show that GlHsp90 binds to ATP with a dissociation constant of 628 M and to its inhibitors, GA and 17AAG with 1.5 μM and 17.5 μM respectively. The bound ATP will be subsequently cleaved by Hsp90 which is an essential step in the chaperone cycle. As determined in our ATPase assay we observed that GlHsp90 hydrolyzes bound ATP with the catalytic efficiency of 4.4 × 10-5μM-1.min-1which confirms that Hsp90 generated upon trans-splicing is an active ATPase. The uniqueness of the hsp90 gene arrangement in Giardia posed a new question. Do these gene fragments also get translated? Our results suggest that HspN and HspC are poly¬adenylated. In order to determine the levels of these transcripts we performed qRT-PCR using primers specific to HspN, HspC and GlHsp90. We have observed that, in comparison with HspN transcript level, HspC and GlHsp90 transcripts are 15 and 75 folds higher respectively. To check for the presence of translation products of these transcripts, we have re-analyzed our proteomic datasets wherein we could identify peptides corresponding to HspN and HspC in their respective molecular weight region, 45 to 35 kDa. To confirm the proteomic data, western blot analysis was performed for trophozoite lysate on both 1D and 2D gels using anti-HspN antibody. Two specific bands (1D) / spots (2D) corresponding to the full length Hsp90 and HspN were identified. Gel filtration analysis revealed that HspN co¬eluted with full length Hsp90 thereby suggesting that both the proteins are in a same complex. With the background that HspN and HspC are present at the protein level, we asked if these fragments in combination can hydrolyse ATP. We reconstituted recombinant HspN and HspC in equimolar amounts and scored for the hydrolysis of ATP. However, no Pi release was observed. To determine whether HspN and HspC could modulate Hsp90 function, ATPase activity was monitored in the presence of HspN or HspC, in vitro. It was observed that ATPase activity was inhibited by both the fragments thus suggesting that HspN and HspC negatively regulate Hsp90 ATPase activity. Role of Hsp90 in Giardia encystation Giardia has a biphasic life cycle with proliferative trophozoites and latent cyst stage. In Giardia, in vitro encystation was established nearly two decades back by modulating the medium conditions. However, the mechanism and triggers underlying this transition are not well characterized. To understand whether Hsp90 has any role in this transition, in vitro conversion of trophozoites to cysts was achieved. The cysts obtained showed all the characteristic features of mature Giardia cyst with cyst wall protein 1 (CWP1) on the cyst wall and four nuclei as determined by immunofluorescence analysis. Further, the levels of Hsp90 in trophozoites were compared with mature cysts at both transcript and protein levels and it was found that cysts show more than 50% reduction in the level of Hsp90 in comparison with normal trophozoites. In accordance, exogenous inhibition of Hsp90 using 17AAG promoted the formation of cysts in vitro by 60 folds in a dose dependent manner; however, the window period of Hsp90 function compromise plays an important role in this process. Higher numbers of cysts were obtained from the cells treated with inhibitors during pre-encystation condition but inhibition of Hsp90 during encystation did not affect the formation of cysts, suggesting that Hsp90 down-regulation plays an important role during commitment towards encystation. To further show that cyst formation is a specific response to Hsp90 inhibition we have carried out encystation in the presence of metranidazole and from heat shocked cells; however, in both the conditions we did not observe any significant change in cyst formation, thus confirming that Hsp90 plays an important role during encystation in Giardia lamblia. Summary In Conclusion, Our study throws light on a unique aspect of Hsp90 biology in Giardia Lamblia, wherein the formation of the full length protein is dependent on a unique trans splicing reaction of its gene components representing different domains. We have also shown that HsP90 fragments, HspN and HspC, are also expressed in Trophozoites. Our in vitro data suggests that these fragments possibly regulate the function of Hsp90. Furthermore, the full length of Hsp90 plays an important role in stage transition in Giardia wherein inhibition of Hsp90 induces encystations. The study has opened many new avenues for research. Understanding the exact role of HspN and HspC in vivo will provide better appreciation for the evolution of such a complex biogenesis of an essential protein.

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

A key prerequisite for cellular and organismal health is a functional proteome. A variety of human protein misfolding diseases are associated with the occurrence of amyloid protein aggregates, such as amyotrophic lateral sclerosis (ALS) or Huntington’s disease. The proteins involved in disease manifestation all contain aggregation-prone sequences of low compositional complexity. Such sequences are also known as prion-like, because of their sequence similarity to yeast prions. Yeast prion proteins are a specific subset of amyloid forming proteins with distinct physio-chemical and functional features, which give them transmissible properties. The aggregation properties of yeast prions and disease-related prion-like proteins reside in structurally independent, prion-forming domains (PrDs). These domains are highly enriched for uncharged polar amino acids, such as glutamine (Q) and asparagine (N). These compositional features can be used to predict prion-like proteins bioinformatically. To investigate the prevalence of prion-like proteins across different organisms, we analyzed a range of eukaryotic proteomes. Our analysis revealed that the slime mold D. discoideum contains the highest number of prion-like N/Q-rich proteins of all organisms. Based on this finding, we hypothesized that D. discoideum could be a valuable model system to study protein homeostasis (proteostasis) and the molecular basis of protein misfolding diseases. To explore how D. discoideum manages its highly aggregation-prone proteome, we analyzed the behavior of several well-characterized misfolding-prone marker proteins (variants of the disease-causing exon 1 of the huntingtin protein as well as wildtype and variant versions of the Q/N-rich yeast prion Sup35NM). Intriguingly, these proteins did not form cytosolic aggregates in D. discoideum, as they do in other organisms. Aggregates, however, formed as a result of heat stress, which indicates that the tested proteins have the capacity to aggregate, but are kept under tight control under normal conditions. Furthermore, when the stress level was reduced, the stress-induced aggregates dissolved, suggesting that D. discoideum has evolved mechanisms to reverse aggregation after a period of acute stress. Together, these findings reveal an unusual resilience of D. discoideum to aggregation-prone proteins, which very likely results from specific adaptations in its proteostasis network. By studying these specific adaptations, we could get important insight into the strategies that nature employs to control and maintain a highly aggregation-prone proteome. So far, our experimental investigations have revealed evidence for three specific adaptations. First, we identified the disaggregase Hsp101 as a key player in the acute stress response of D. discoideum. A functional analysis of Hsp101 in yeast and D. discoideum revealed that it supports thermotolerance. Second, we found evidence for an important role of the nucleus and nucleolus in proteostasis. We discovered that a small fraction of highly aggregation-prone proteins accumulated in the nucleus or nucleolus of D. discoideum cells. The magnitude of this nuclear accumulation could be increased by proteasome impairment, which suggests that the ubiquitin-proteasome system (UPS) is involved. This finding is consistent with previous studies in other organisms and hints at the possibility that D. discoideum disposes of aggregation-prone proteins by degrading them in the nucleus/nucleolus. Third and finally, we found that cells containing nuclear accumulations are asymmetrically distributed in the multicellular developmental stage (slug), suggesting that D. discoideum employs cell-sorting mechanisms to dispose of cells with accumulated protein damage. Although our current understanding of proteostasis in D. discoideum is preliminary, we have gained important insight into the molecular mechanisms and cellular pathways that D. discoideum uses to counteract protein aggregation. Findings from this work will inform similar comparative studies in other organisms and will impact our molecular understanding of protein misfolding diseases and aging. / Eine wesentliche Voraussetzung für die Gesundheit von Zellen und Organismen ist ein funktionales Proteom. Eine Reihe von humanen Protein- Missfaltungs-Erkrankungen, wie Chorea Huntington und Amyotrophe Lateralsklerose (ALS) werden mit dem Auftreten von amyloiden Protein- Aggregaten in Verbindung gebracht. Sämtliche Proteine, die in der Pathogenese dieser Krankheiten eine Rolle spielen, enthalten aggregations-anfällige Sequenzen mit geringer Sequenzkomplexität. Solche Sequenzen werden als Prion-ähnlich bezeichnet, da sie in ihrer Zusammensetzung den Prionen aus der Hefe S. cerevisiae gleichen. Die Prion-Proteine der Hefe gehören zu einer Unterart von amyloid-aggregierenden Proteinen, die durch bestimmte physikochemische und funktionelle Eigenschaften einen infektiösen Charakter erhalten. Die Aggregations-Eigenschaften von Hefeprionen und aggregationsanfällige Proteinen, die mit Erkrankungen in Verbindung gebracht werden, basieren auf strukturell unabhängigen, Prion-bildenden Domänen (prion domain, PrD). Diese Domänen sind angereichert mit polaren Aminosäuren wie Glutamin und Asparagin. Diese Zusammensetzung kann dazu verwendet werden prion-ähnliche Proteine bioinformatisch vorherzusagen. Um die Verbreitung von Prion-ähnlichen Proteinen in verschiedenen Organismen zu untersuchen, analysierten wir eine Reihe von eukaryotischen Proteomen. Unsere Analyse zeigte, dass der Schleimpilz D. discoideum die höchste Anzahl von Prion-ähnlichen N/Q-reichen Proteinen aufzeigt. Aufgrund dieser Erkenntnisse erstellten wir die Hypothese, dass D. discoideum ein nützlicher Modellorganismus sein könnte, um Protein Homöostase (Proteostase) sowie die molekulare Basis von Proteins-Missfaltungs-Erkrankungen zu ergründen. Um zu analysieren, wie D. discoideum mit seinem höchst aggregations-anfälligen Proteom umgehen kann, untersuchten wir das Verhalten mehrerer bereits charakterisierter aggregations-anfälliger Marker-Proteine in D. discoideum. Hierbei verwendeten wir Varianten des krankheits-erzeugenden Exon 1 des humanen Huntingtin Protein sowie den wild-typ und Varianten des N/Q-reichen Hefe Prions Sup35. Interessanterweise bildeten diese Proteine, anders als in anderen Organismen, keine zytosolischen Aggregate in D. discoideum aus. Aggregate wurden jedoch unter Hitzestress-Bedingungen gebildet. Dies deutet darauf hin, dass die getesteten Proteine durchaus das Vermögen zu aggregieren besitzen, jedoch unter normalen Wachstumsbedingungen streng kontrolliert werden. Wenn, darüberhinaus das Stress- Level gesenkt wurde, kam es zur Auflösung der stress-induzierten Aggregate. Dies deutet darauf hin, dass D. discoideum Mechanismen entwickelt hat, um Aggregate nach Perioden von akutem Stress wieder aufzulösen. Zusammengenommen enthüllen diese Erkenntnisse eine ungewöhnliche Widerstandsfähigkeit gegenüber aggregations-anfälligen Proteinen. Diese beruht höchstwahrscheinlich auf spezifischen Modifikationen im Proteostase Netzwerk. Durch die Analyse dieser spezifischen Anpassungen könnten wichtige Einblicke in die Strategien gewährt werden, welche die Natur benutzt, um ein höchst aggregations-anfälliges Proteom zu erhalten und zu kontrollieren. Bisher erbrachten unsere Experimente Anhaltspunkte für drei spezifische Anpassungen. Erstens zeigten wir, dass die Disaggregase Hsp101 eine Schlüsselrolle in der akuten Stressantwort in D. discoideum einnimmt. Eine funktionale Analyse von Hsp101 in D. discoideum und Hefe zeigte, dass die Disaggregase Thermotoleranz fördert. Zweitens haben wir Anhaltspunkte, dass der Nukleus und der Nukleolus eine wichtige Rolle in der Proteostase einnehmen. Eine geringe Fraktion der überaus aggregations-anfälligen Proteine akkumuliert im Nukleus oder Nukleolus von D. discoideum. Das Ausmaß der nuklearen Akkumulation konnte erhöht werden, wenn das Proteasom beeinträchtigt wird. Dies deutet darauf hin, dass das Ubiquitin-Proteasom-System involviert sein könnte. Diese Beobachtung ist im Einklang mit jüngsten Berichten aus anderen Organismen und daraus folgt, dass D. discoideum möglicherweise aggregations-anfällige Proteine durch Abbau im Nukleus entsorgt. Drittens konnten wir feststellen, dass Zellen, die nukleare Akkumulationen enthalten, asymmetrisch in der multizellulären Entwicklungs-Struktur des Pseudoplasmodiums verteilt sind. Dies deutet darauf hin, dass D. discoideum möglicherweise den Zellsortierungsmechanismus während der Entwicklung nutzen kann, um Zellen mit angereicherten Protein-Schäden zu beseitigen. Auch wenn das gegenwärtige Verständnis der Proteostase in D. discoideum nur vorläufig ist, haben wir wichtige Einblicke in die molekularen Mechanismen und zellulären Prozesse erhalten, die D. discoideum verwendet, um Protein-Aggregation zu verhindern. Die Ergebnisse dieser Arbeit werden ähnliche vergleichende Studien in anderen Organismen beeinflussen und Auswirkungen auf unser molekulares Verständnis über Protein-Missfaltungs-Erkrankungen und das Altern haben.