The goal of this project has been to develop therapeutic strategies for protein misfolding diseases caused by excessive degradation of misfolded proteins and loss of protein function. The focus for this work is lysosomal storage disorders (LSDs), a group of more than 50 known inherited metabolic diseases characterized by deficiency in hydrolytic enzymes and consequent buildup of lysosomal macromolecules. Gaucher’s Disease (GD) is used as a representative of the family of LSDs in this study. GD is caused by mutations in the gene encoding lysosomal glucocerebrosidase (GC) and consequent accumulation of the GC substrate, glucocerebroside. The most prevalent mutations among GD patients are single amino acid substitutions that do not directly impair GC activity, but rather destabilize its native folding. GC normally folds in the ER and trafficks through the secretory pathway to the lysosomes. GC variants containing destabilizing mutations misfold and are retrotranslocated to the cytoplasm for ER-associated degradation (ERAD). However, evidence shows that if misfolding-prone, mutated GC variants are forced to fold into their 3D native structure, they retain catalytic activity. This study describes strategies to remodel the network of cellular pathways that maintain protein homeostasis and to create a folding environment favorable to the folding of unstable, degradation-prone lysosomal enzyme variants. We demonstrated that folding and trafficking of mutated GC variants can be achieved by modulating the protein folding network in fibroblasts derived from patients with GD to i) upregulate the expression of ER luminal chaperones, ii) inhibit the ERAD pathway, and iii) enhance the pool of mutated GC in the ER amenable to folding rescue. We also demonstrated that the same cell engineering strategies that proved successful in rescuing the folding and activity of mutated GC enable rescue of mutated enzyme variants in fibroblasts derived from patients with Tay-Sachs disease, a LSD caused by deficiency of lysosomal hexosaminidase A activity. As a result, the current study provides insights for the development of therapeutic strategies for GD based on the modulation of general cellular pathways that maintain protein homeostasis that could in principle be applied to the treatment of multiple LSDs.

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.

Doctor of Philosophy / Department of Biochemistry / Michal Zolkiewski / ClpB is a bacterial heat-shock protein that disaggregates and reactivates strongly aggregated proteins in cooperation with the DnaK chaperone system. ClpB contains two ATP-binding AAA+ modules, a linker coiled-coil domain, and a highly mobile N-terminal domain. It forms ring-shaped hexamers in a nucleotide-dependent manner. The unique aggregation reversing chaperone activity of ClpB involves ATP-dependent translocation of substrates through the central channel in the ClpB ring. The initial events of aggregate recognition and the events preceding the translocation step are poorly understood. In addition to the full-length ClpB95, a truncated isoform ClpB80, that is missing the whole N-terminal domain, is also produced in vivo. Various aspects of the structure and function of ClpB were addressed in this work. The thermodynamic stability of ClpB in its monomeric and oligomeric forms, as well as the nucleotide-induced conformational changes in ClpB were investigated by fluorescence spectroscopy. Equilibrium urea-induced unfolding showed that two structural domains-the small domain of the C-terminal AAA+ module and the coiled-coil domain-were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation or interactions during formation of the ClpB rings. Several locations of Trp-fluorescence probes were also found to respond to nucleotide binding. The biological role of the two naturally-occurring ClpB isoforms was also investigated. We discovered that ClpB achieves optimum chaperone activity by synergistic cooperation of the two isoforms that form hetero-oligomers. We found that ClpB95/ClpB80 hetero-oligomers form preferentially at low protein concentration with higher affinity than homo-oligomers of ClpB95. Moreover, hetero-oligomers bind to aggregated substrates with a similar efficiency as homo-oligomers of ClpB95, do not show enhanced ATPase activity over that of the homo-oligomers, but display a strongly stimulated chaperone activity during the reactivation of aggregated proteins. We propose that extraction of single polypeptides from aggregates and their delivery to the ClpB channel for translocation is the rate-limiting step in aggregate reactivation and that step is supported by the mobility of the N-terminal domain of ClpB. We conclude that the enhancement of the chaperone activity of the hetero-oligomers is linked to an enhancement of mobility of the N-terminal domains.

As chaperonas moleculares da família Hsp70 desempenham funções cruciais nas células de todos os organismos vivos, de procariotos a eucariotos. Nestes, estão presentes em todos os compartimentos celulares e nas mitocôndrias é expressa uma isoforma própria (mtHsp70), que participa dos processos enovelamento e maturação de proteínas bem como de sua importação para a matriz mitocondrial. Diante da crescente demanda de pesquisa sobre doenças tropicais negligenciadas, foi tomado como objeto de estudo neste trabalho uma Hsp70 mitocondrial de Leishmania braziliensis (LbmtHsp70) com o intuito de caracterizá-la estrutural e funcionalmente em comparação à ortóloga humana com maior identidade: a mtHsp70 também chamada de mortalina, GRP75, HspA9 ou PBP74. A LbmtHsp70 foi purificada em sua forma enovelada, em sistema monodisperso apresentando dados hidrodinâmicos condizentes com a forma monomérica, foi testada sua estabilidade quanto à influência de nucleotídeos de adenosina (ATP e ADP) à sua estrutura e, por fim, foram feitos ensaios para avaliar sua atividade ATPásica e de energia de interação com nucleotídeos. De forma geral, a LbmtHsp70 é bastante similar à mortalina, como pode ser evidenciado pelos resultados obtidos com algumas particularidades. / The molecular chaperones from the Hsp70 family perform critical cell roles in all organisms, from prokaryotes to eukaryotes. In the last ones, they are found in all cell compartments and a particular isoform is expressed in the mitochondria, where it carries out folding and maturation processes as well as the import of proteins to the mitochondrial matrix. In face of the growing demand for research about neglected tropical diseases, in this study a Hsp70 from Leishmania braziliensis\'s mitochondria was taken as object of study for further structural and functional characterization in comparison to the human orthologous which presents the highest identity to LbmtHsp70: the mtHsp70 also known as mortalin, GRP75, HspA9 or PB74. LbmtHsp70 was obtained in folded state in monodisperse system with hydrodynamic data consistent to monomeric conformer, stability and adenosine nucleotides influence to its structure were analyzed, and were performed assays for ATPase activity and nucleotide interaction energy. In a general way LbmtHsp70 is very similar to mortalin as can be shown through the results, but with some peculiarities.

Signal termination processes of GPCRs are well established, unlike processes that regulate the assembly and intracellular trafficking of these signaling complexes. Bimolecular fluorescence complementation was used to study GPCR dimer formation in two projects. Firstly, the importance of Rab GTPases on the cell surface expression and signaling of two chemokine receptors expressed on prostate cancer cells was examined. Rab GTPases necessary for CXCR4 and CCR2 cell surface expression and signaling were different from those necessary for the CXCR4/CCR2 heterodimer. Therefore, this project emphasizes the importance of studying heterodimers as unique entities from their constituent receptors. Secondly, interactions between molecular chaperones and two coreceptors necessary for HIV infection – CCR5, a chemokine GPCR, and the main HIV receptor, CD4, a glycoprotein – were investigated. Further emphasizing the unique characteristics of GPCR dimers, this project found that molecular chaperones interact differently with CCR5 homodimers, when compared to CCR5/CD4 heterodimers.

Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA.

Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in human diseases. Therefore it is important to develop new methods to allow simple and quantitative measurement of surface expression of membrane proteins. Here we describe the development and validation of a new assay for quantification of cell surface expression of GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac) to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac assay are quantitatively and qualitatively similar to well established ELISA when measuring agonist induced internalization of β2AR. We also show that measurement of GBR1 surface expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In conclusion, our results show that our newly developed βlac assay is quantitatively similar while being less expensive, more robust and higher throughput compared to an ELISA.

As chaperonas moleculares da família Hsp70 desempenham funções cruciais nas células de todos os organismos vivos, de procariotos a eucariotos. Nestes, estão presentes em todos os compartimentos celulares e nas mitocôndrias é expressa uma isoforma própria (mtHsp70), que participa dos processos enovelamento e maturação de proteínas bem como de sua importação para a matriz mitocondrial. Diante da crescente demanda de pesquisa sobre doenças tropicais negligenciadas, foi tomado como objeto de estudo neste trabalho uma Hsp70 mitocondrial de Leishmania braziliensis (LbmtHsp70) com o intuito de caracterizá-la estrutural e funcionalmente em comparação à ortóloga humana com maior identidade: a mtHsp70 também chamada de mortalina, GRP75, HspA9 ou PBP74. A LbmtHsp70 foi purificada em sua forma enovelada, em sistema monodisperso apresentando dados hidrodinâmicos condizentes com a forma monomérica, foi testada sua estabilidade quanto à influência de nucleotídeos de adenosina (ATP e ADP) à sua estrutura e, por fim, foram feitos ensaios para avaliar sua atividade ATPásica e de energia de interação com nucleotídeos. De forma geral, a LbmtHsp70 é bastante similar à mortalina, como pode ser evidenciado pelos resultados obtidos com algumas particularidades. / The molecular chaperones from the Hsp70 family perform critical cell roles in all organisms, from prokaryotes to eukaryotes. In the last ones, they are found in all cell compartments and a particular isoform is expressed in the mitochondria, where it carries out folding and maturation processes as well as the import of proteins to the mitochondrial matrix. In face of the growing demand for research about neglected tropical diseases, in this study a Hsp70 from Leishmania braziliensis\'s mitochondria was taken as object of study for further structural and functional characterization in comparison to the human orthologous which presents the highest identity to LbmtHsp70: the mtHsp70 also known as mortalin, GRP75, HspA9 or PB74. LbmtHsp70 was obtained in folded state in monodisperse system with hydrodynamic data consistent to monomeric conformer, stability and adenosine nucleotides influence to its structure were analyzed, and were performed assays for ATPase activity and nucleotide interaction energy. In a general way LbmtHsp70 is very similar to mortalin as can be shown through the results, but with some peculiarities.

Le cancer de la prostate est le second cancer diagnostiqué chez les hommes dans le monde. Malgré le développement de nouveaux traitements au cours de ces cinq dernières années, les chimiothérapies par taxanes, docetaxel et cabazitaxel, restent des traitements de référence dans la prise en charge des patients atteints de cancer de la prostate métastatique résistant à la castration. Cependant, des résistances primaires et acquises émergent chez environ la moitié des patients. C’est pourquoi, il est urgent de découvrir et de comprendre les mécanismes de résistance aux taxanes afin d’identifier de nouvelles cibles thérapeutiques. En effet, de nouvelles thérapies ciblées peuvent émerger de la compréhension des voies de signalisation impliquées dans le cancer de la prostate pour contourner la chimiorésistance et améliorer les traitements. Les protéines chaperonnes jouent un rôle clef dans la régulation de l’homéostasie cellulaire et dans le développement de résistance aux traitements. Elles constituent donc des cibles thérapeutiques potentielles pour contourner la chimiorésistance. En réalisant un criblage fonctionnel par siARN à partir de profils d’expression génique, nous avons identifié FKBP7, une chaperonne moléculaire encore jamais étudiée chez l’homme, impliquée dans la résistance au docetaxel et au cabazitaxel. FKBP7 est surexprimée dans les tumeurs de la prostate et son expression est corrélée avec la récurrence chez les patients ayant reçu du docetaxel en thérapie néoadjuvante. De plus, FKBP7 est surexprimée dans des lignées cancéreuses prostatiques résistantes aux taxanes et son expression est nécessaire à leur croissance in vitro et à la croissance tumorale dans un modèle murin de résistance au docetaxel. Par des approches de protéomique haut-débit, nous avons identifié la voie de signalisation régulée par FKBP7 qui est responsable de la survie des cellules chimiorésistantes. Enfin, nous proposons une stratégie thérapeutique pour contourner la chimiorésistance au docetaxel et au cabazitaxel en ciblant l’effecteur moléculaire en aval de FKBP7. / Prostate cancer is the second cancer diagnosed among men worldwide. Beside approval of new therapies in the last five years, chemotherapeutic agents, docetaxel and cabazitaxel taxanes remain key treatments for metastatic castration resistant prostate cancers. However, primary and acquired resistance to taxanes still emerged in about half of patients. There is therefore an urgent need to discover and understand the taxane resistance mechanisms in order to identify new therapeutic targets. Indeed, targeted therapies that exploit the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes. Molecular chaperones play a key role in the regulation of cellular homeostasis and the development of treatment resistance, and are promising therapeutic targets. Using high throughput siRNA functional screening based on a gene expression signature, we identified FKBP7, involved in acquired resistance to docetaxel and cabazitaxel. FKBP7 is a molecular chaperone that has not been studied in human so far. FKBP7 is overexpressed in prostate tumors and its expression is correlated with recurrence in patients who received docetaxel as neoadjuvant therapy. Moreover, FKBP7 is upregulated in taxane resistant prostate cancer cell lines and its expression sustains their growth in vitro and in a mice model of Docetaxel resistance. Using a high throughput proteomic approach, we identified the signaling pathway regulated by FKBP7 which is responsible for the survival of chemoresistant cells. Finally, we proposed a promising therapeutic strategy to overcome both docetaxel and cabazitaxel chemoresistance by targeting the downstream effector of FKBP7.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18918号 / 薬科博第32号 / 新制||薬||4(附属図書館) / 31869 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 松﨑 勝巳, 教授 加藤 博章, 教授 石濱 泰 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM