Made available in DSpace on 2016-06-02T20:21:34Z (GMT). No. of bitstreams: 1 6071.pdf: 2789101 bytes, checksum: 91e27d4de2c662fb669682eb6d3013b3 (MD5) Previous issue date: 2014-06-03 / Universidade Federal de Sao Carlos / Protein folding is essential for proteins proper biological function. Failures in this process can lead to the formation of poorly unfolded proteins and/or protein aggregates. In order to avoid this problem, the cells express a family of proteins known as molecular chaperones. The molecular chaperones are proteins that assist the correct folding of other proteins, and other important functions in the cells. The Hsp90 family is important for protein folding and it assists in preventing protein aggregation. Hsp90 is regulated by several co-chaperones, for example, p23. The p23 is a small acidic protein that regulates the ATPase activity of Hsp90. It has a structured N-terminal beta-sheet and an unstructured C-terminal domain. In addition to the regulatory role, as an inhibitor of ATPase activity of Hsp90, it also has chaperone activity in itself. Thus, the aim of this study was to investigate comparatively two p23 identified in the Leishmania braziliensis (Lbp23A and Lbp23B) genome. The proteins were expressed, purified and structurally and functionally characterized. Furthermore, functional assays such as intrinsic chaperone activity and inhibition of ATPase activity of Hsp90 L. braziliensis (LbHsp90) and identifying in vivo by western blotting were developed. The results indicate that these two proteins are structurally similar, however, demonstrated significant differences in chemical and thermal stability. The Lbp23 also differ in relation to chaperone activity and inhibition of ATPase activity of LbHsp90. The in vivo identification revealed the presence of both Lbp23 in extracts of L. braziliensis; besides suggesting possible post-translational modifications in Lbp23B. The results indicate that both Lbp23 are undoubtedly p23, since they show p23-like function and structural signs. / O enovelamento proteico é essencial para a correta função biológica das proteínas. Falhas nesse processo podem levar à formação de proteínas mal enoveladas e/ou agregados proteicos. Para tentar evitar esse problema, a célula expressa uma família de proteínas denominadas de chaperonas moleculares ou proteínas de choque térmico (Hsp). As chaperonas moleculares auxiliam no enovelamento correto de outras proteínas, entre outras funções importantes para as células. A família das Hsp90 são chaperonas importantes por auxiliarem no enovelamento proteico e prevenirem a agregação de proteínas. A Hsp90 é regulada por diversas co-chaperonas, como, por exemplo, a p23. A p23 é uma pequena proteína ácida que regula a atividade ATPásica da Hsp90. Ela possui um domínio N-terminal estruturado em folhas-beta e um domínio C-terminal desestruturado. Além do papel regulatório, inibindo a atividade ATPásica da Hsp90, ela possui atividade chaperona. Desta forma, o objetivo desse trabalho foi estudar comparativamente as duas p23 identificadas no genoma do protozoário Leishmania braziliensis (Lbp23A e Lbp23B). As proteínas foram expressas, purificadas e caracterizadas estrutural e funcionalmente. Além disso, foram desenvolvidos experimentos funcionais como: atividade chaperona; inibição da atividade ATPásica da Hsp90 de L. braziliensis (LbHsp90) e identificação in vivo por western blotting. Os resultados indicam que essas duas proteínas são similares estruturalmente, porém, possuem estabilidade química e térmica notavelmente diferente. Ambas Lbp23 apresentam diferenças em relação à atividade chaperona e inibição da atividade ATPásica da LbHsp90. A identificação in vivo mostrou a presença das duas Lbp23 em extratos de L. braziliensis; além de sugerir possíveis modificações pós-traducionais na Lbp23B. Os resultados indicam que ambas as Lbp23 de L. braziliensis são inequivocamente p23, pois possuem sinais estruturais e função desta co-chaperona.

Cells invest a lot of energy in order to get their proteins to fold correctly and attain functionality. It is the functional proteome of a cell that defines the ‘life of a cell’. Cells have therefore employed dedicated machinery called chaperones to enable protein folding. One class of these chaperones is heat shock proteins named so because they were initially discovered to be heat inducible and particularly important during heat stress. However the role of heat shock proteins has now been extended from merely being important for stress tolerance. Heat shock proteins are prominently involved in maintaining the correct folding and conformation of proteins and are vital in regulating the stability between protein synthesis and degradation. One of the heat shock proteins, Hsp90, is an evolutionarily conserved molecular chaperone essential in all known eukaryotes examined so far. Unlike other chaperones, Hsp90 is unique in binding to substrate proteins, which are at a late stage of folding, poised for activation by either ligand binding or interaction with other cellular factors. The most common clients of Hsp90 are signaling proteins, the classic example being steroid hormone receptors and signaling kinases. Several other proteins including transcription factors, proteins involved in cell division and development have also been shown to rely on Hsp90 functioning for their maturation. Hsp90 has emerged as an important molecular chaperone due to the large number of proteins that depend on the activity of Hsp90 for their functionality. Hsp90 plays a central role in multiple cellular processes. Since knock-out of hsp90 is lethal to most eukaryotes, inhibitors of Hsp90 have been widely used to study its function. The most widely used inhibitor is geldanamycin (GA). GA binds to the N-terminal/ATP binding site of Hsp90 which results in the degradation of client proteins. Hsp90 clients have been shown to be proteins important for diverse cellular processes such as protein trafficking, signal transduction, cell-cycle, cellular motility and development in eukaryotes. Exploring new Hsp90 clients gives an insight into more pathways that Hsp90 regulates. Intriguingly, many proteins interact with Hsp90 in a context dependent manner, i.e., under certain environmental cue, or in a particular tissue, or only under certain diseased states. It is therefore essential to study Hsp90 functioning and examine Hsp90-client interactions in more than one model organism. Dictyostelium discoideum: a model organism to study the role of Hsp90 in development The eukaryote, Saccharomyces cerevisiae that has been explored extensively for studying the diverse clientele of Hsp90, lacks various signaling pathways important for growth and differentiation as prevalent in higher eukaryotes. It is desirable to develop a model system that would combine the advantages of a lower eukaryote, in terms of its ease of manipulation and retain the complexities of higher eukaryotes. With this motivation, the social slime mold D. discoideum was explored to examine potential roles of cytoplasmic Hsp90 in growth and development. D. discoideum is ideal for studying signaling pathways important for growth and differentiation and to understand how these pathways control cellular responses to external stimuli. Multicellular development in D. discoideum occurs in response to starvation induced stress. As in case of many other protozoans, we conjectured that Hsp90 may participate in regulating developmental transition from unicellular to multicellular stages in Dictyostelium as well. My initial study attempts, to address the role of Hsp90 (HspD), in development of D. discoideum. Towards this two approaches were taken: through genetic interference of HspD, and the other, through its pharmacological inhibition. An antisense HspD plasmid was designed which upon transfection in D. discoideum, showed a very slow growth phenotype, and the cells did not survive beyond few generations. Therefore to further study the functions of HspD, I resorted to pharmacological inhibition by using the specific, well characterized inhibitor, GA. As a first step towards this I examined whether GA was capable of binding to HspD from D. discoideum cell lysate. Towards this, GA was immobilized to NHS-sepharose beads, and bound proteins were examined. Western blot of the bound fraction, using antibody specific to HspD, identified it as a predominant protein being pulled down. This was further confirmed by mass spectrometry. To be able to compare Hsp90 from D. discoideum with Hsp90s from other model organisms, HspD was cloned, purified and biochemically characterized. Comparison of ATPase activities of HspD with Hsp90’s from other systems indicates HspD to possess a relatively low ATPase activity with a Kcat of 1.6 x 10-3 min-1. The dissociation constant of GA for HspD was found to be 0.8 µM, which was in the range similar to Hsp90s from other systems. In addition, we have now obtained structural data on HspD in collaboration with crystallography groups. The N-terminal domain of HspD has been crystallized, both in -free and ligand-bound forms. Crystal structure comparison of HspD with Hsp90 from S. cerevisiae shows overall fold similarity yet some important differences in side chain orientations of specific residues in the ATP binding domain. Interestingly, on treating D. discoideum cells with GA or another Hsp90 N-terminal inhibitor, Radicicol, it was found that, while control cells progressed to develop into fruiting bodies, GA/Radicicol treated cells resulted in delayed development, and were finally arrested at the ‘mound’ stage. This suggested potential involvement of HspD in developmental progression beyond the mound stage. In order to identify the pathways that are probably affected by HspD in D. discoideum development, cells were treated with/without GA and subjected to comparative proteomics using mass spectrometric analysis. Amongst other differences, there was an obvious absence of peptides corresponding to the protein paxillin in GA treated cells. The results were verified by Western blot analysis, using a specific antibody against paxillin, wherein a drastic decrease in paxillin levels were observed in cells treated with GA. Paxillin is a key player in focal adhesion sites that functions as an adaptor protein to recruit diverse cytoskeletal and signaling proteins into a complex, and is essential for cellular proliferation and cell-substrate adhesion. My studies suggest that one of the pathways through which HspD regulates development is through cellular motility as Hsp90 was involved in regulating proteins necessary for motility and cytoskeletal organization at focal adhesion points during development in D. discoideum. Hsp90 as a target for Trypanosoma evansi infections In addition to examining the role of Hsp90 in differentiation in D. discoideum, I have also looked at the potential of Hsp90 under diseased conditions. Towards this, I explored the protozoan parasite, T. evansi, which causes a fatal disease ‘surra’. Surra is a neglected disease that mainly affects domestic and wild animals including equines, camels, cattle and buffaloes. The parasite causes significant economic losses to livestock industry. While this infection is mainly restricted to domestic (camels, equines, cattle, buffaloes, goats, sheep, pigs, dogs etc.) and wild animals, recent reports indicate their ability to infect humans. There are no reliable sensitive and specific diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable. With a view to identifying potential diagnostic markers and drug targets I have studied the clinical proteome of T. evansi infection using mass spectrometry. I have been able to identify almost 166 proteins of T. evansi, which also included potential drug and vaccine targets. Due to absence of any genome sequence information from T. evansi, most of the peptides obtained matched to its related species, T. brucei, T. cruzi and also few from Leishmania major. Importantly, I was also able to identify peptides from Hsp90. Hsp90 from T. evansi was cloned and its sequence was also obtained. To investigate the possibility of exploring Hsp90 as a target against Surra infections, TeHsp90 protein was purified by expressing it in bacterial cells, and its drug (GA) binding ability was examined in-vitro. The dissociation constant of GA for HspD was found to be 1.4 µM, which was in the range similar to Hsp90s from other systems. The ability of 17AAG (a derivative of GA) was examined in inhibiting T. evansi infection at pre-clinical level. Towards this, swiss female mice were infected with purified parasites and then the drug was injected either immediately, in one group of mice, and in another group of mice the parasites were challenged with the drug only after the onset of infection. Interestingly, both groups of mice were found to get cured using Hsp90 inhibitor. The pre-clinical results suggested that Hsp90 was an interesting drug target and its inhibitor could indeed be used against ‘surra’ infections. Hsp90 from Giardia lamblia: An unusual case Hsp90 was also examined from another pathogenic protozoan, Giardia lamblia, one of the leading causes of diarrhea in the world. Previous studies from our lab have shown Gardial Hsp90 to be coded by two different ORFs, spliced together in trans. This is indeed the only example of trans-splicing in Hsp90 known so far. My study further characterizes this finding through analysis of transcription levels of the individual ORFs, using Northern blot analysis. Importantly, I was able to detect transcripts of all three forms of Hsp90; full-length, N terminus as well as C terminus, suggesting that these are expressed and may have biological significance. To understand the significance of these independent transcripts, I have examined relative levels of expression of all three forms by Real-time PCR analysis wherein there was almost 90 fold and 5 fold lesser transcript level of N terminus and C terminus Hsp90 observed, respectively as compared to the full-length GlHsp90 expression. Previous reports have shown Hsp90 from all known organisms, to get up regulated during heat shock. Thus it was important to examine the effect of heat stress on the expression of these independent transcripts. Interestingly, different domains were found to get independently induced during heat stress. The transcript level of HspC was seen to be almost similar to that of full-length upon heat shock. There was also a significant up regulation observed in HspN transcript upon heat shock. Taking together all these observations, these results suggest a possible role for the independent domains, HspN and HspC during heat stress in G. lamblia. Furthermore, I have cloned and purified one of the individually expressed domains, HspN and characterized it biochemically. HspN was found to be able to bind to ATP, however lacked ATPase activity. Taking together all these observations, it suggests a possible role for the independent domains, HspN and HspC which needs to be investigated further. Summary Altogether, my studies establish the importance of alternate model systems in understanding the biology of Hsp90. The importance of Hsp90 was first established in growth and development of a nonpathogenic protozoan D. discoideum. My results provide significant insights into the additional pathways that Hsp90 regulates during D. discoideum development. One such important pathway was delineated to be cellular locomotion and motility. Further, I have also studied the importance of Hsp90 in neglected infectious diseases. In addition to providing a glimpse into the pathways operational during disease manifestation in T. evansi, we have shown Hsp90 to be effective in pre-clinical trials against T. evansi infections. Hsp90 from another pathogenic protozoan, G. lamblia, has also been studied. This is by far the only organism, in which there is an independent expression of the N-and C-terminal domain of Hsp90. The rare gene organization, coupled with independent expression of domains of Hsp90, makes this organism important to examine novel functions of this chaperone.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22412号 / 工博第4673号 / 新制||工||1729(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 森 泰生, 教授 生越 友樹 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Microtubules (MTs) are one of the essential cell structure that participate in a number of key events in the plant cells and their properties and functions are influenced and modified by many other proteins. These proteins belong to a group of microtubule- associated proteins (MAPs, microtubule-associated proteins). One of the MAPs, the molecular chaperone Hsp90, examines and fulfills a large number of different functions in the cell. Its colocalization with MTs has been demonstrated previously by Freudenreich and Nick (1998) and Petrášek et al. (1998). However, direct interaction with MTs was described only recently using cosedimentation assay. The specific cytosolic isoform of tobacco Hsp90 bound to MTs was called Hsp90_MT due to its ability to bind MTs. It has been also found that the binding to MTs is independent on the activity of ATP (Krtková et al., 2012). The authors also described a positive effect of Hsp90_MT on MT recovery after their exposure to cold stress. Although MT cytoskeleton dynamics is influenced by a large number of MAPs, it is surprising that the molecular mechanism of MAPs interaction with MTs and their MT-binding domains have not been described yet. Therefore, we decided to determine the tobacco Hsp90_MT MT-binding domain by production of a set of recombinant proteins...

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L’allogreffe de cellules hématopoïétiques est une stratégie thérapeutique importante dans les hémopathies malignes. La maladie du greffon contre l’hôte (GvH) en est une complication majeure menaçant le pronostic vital. Elle est due à la reconnaissance des antigènes du receveur par les lymphocytes T du donneur et à l’activation de ceux-ci, à l’origine de dommages tissulaires. L’altération de la barrière intestinale joue un rôle critique dans la GvH. La famille des protéines de choc thermique (HSP)90 comporte cinq membres dont trois cytosoliques dénommés HSP90, et un localisé dans le réticulum endoplasmique (RE), Gp96, qui peut être sécrété en cas de stress. Nous montrons dans nos travaux de thèse que la 17AAG, un inhibiteur des HSP90, réduit la mortalité liée à la GvH dans un modèle murin. Cet effet est associé à une augmentation de la réponse au stress du RE dans les cellules épithéliales intestinales comme en atteste l’augmentation de l’épissage du facteur de transcription XBP-1, corrélée à une diminution du dommage tissulaire intestinal. Ces résultats permettent d’envisager une place pour la 17AAG ou d’autres inhibiteurs de HSP90 dans la prévention de la GvH chez l’homme. D’autre part, nous montrons que Gp96 est sécrétée dans le sérum de patients développant une GvH aiguë sévère avec atteinte intestinale. Nous suggérons de valider la pertinence de Gp96 comme biomarqueur de GvH intestinale dans une étude de plus grande ampleur. Enfin, nous trouvons que Gp96 s’associe avec le composant 3 du complément, une protéine impliquée dans l’immunité innée et adaptative, et inhibe certaines de ses fonctions. Les conséquences fonctionnelles de cette association sont discutées. / Allogeneic hematopoietic cell transplantation is a treatment for certain disorders including hematologic malignancies. Graft-versus-host-disease (GvHD) is a major, life-threatening complication. It is due to the recognition of recipient antigens by donor T cells, which activate and damage tissues. Intestinal barrier alteration plays a critical role in GvHD. Heat shock proteins (HSP)90 include five members, three cytosolic members named HSP90, and one member localized in endoplasmic reticulum (ER) called Gp96 and able to gain extracellular level in case of stress. We show in our thesis that 17AAG, a HSP90 inhibitor, reduces GvHD mortality in a mouse model. This effect is associated with an increase in ER stress pathway in intestinal epithelial cells as figured by transcription factor XBP-1 splicing, correlated to a decrease in intestinal tissue damage. These results suggest that 17AAG could be considered in GvHD prevention in human. Moreover, we show that Gp96 is secreted in serum of patients developing an acute GvHD with intestinal involvement. We propose to validate the relevance of Gp96 as an intestinal GvHD biomarker is a larger study. Finally, we find that Gp96 associate with complement component 3, a protein involved in innate and adaptive immunity, and inhibit some of its functions. Functional consequences of this association are discussed.