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.

Žmogaus organizme širdies ir kraujagyslių sistema, kraujodaros organai bei kvėpavimo organų sistema leidžia organizmui palaikyti deguonies pusiausvyrą. Širdies ligos, vėžys, smegenų kraujagyslių ligos ir chroniška obstrukcinė plaučių liga- tai susirgimai, nuo kurių dažniausiai miršta žmonės visame pasaulyje. Visų šių ligų viena iš priežasčių yra deguonies pusiausvyros sutrikimas (Semenza, 2001). Ląstelės atsakas į stresines situacijas, tokias kaip deguonies trūkumas ląstelėje, seniai domino mokslininkus. Specifinis atsakas į deguonies koncentraciją ląstelėje buvo nustatytas bakterijose ir mielėse, bet ilgai buvo neaptinkamas aukštesniuose organizmuose. Atlikus visą eilę tyrimų, buvo aptikta sąsaja tarp deguonies kiekio ląstelėje kitimo ir transkripcijos įvykių kaskados, reguliuojančios aukštesniųjų organizmų ląstelių atsaką į hipoksiją. Vieno iš ryškiausių homeostazės atsako į hipoksiją- hematopoetinio augimo faktoriaus eritropoetino molekulinė analizė leido aptikti hipoksijos indukuojamą transkripcijos faktorių (HIF). Dabar jau nustatyta, kad ši sistema funkcionuoja visose žinduolių ląstelėse. (Schofield and Ratcliffe, 2004). HIF faktoriai- tai visa šeima baltymų, vaidinančių nemažą vaidmenį vėžinių auglių vystymesi žmogaus organizme. Tai daro šių faktorių raiškos reguliacijos tyrimus labai svarbiais, nes gauti rezultatai tiesiogiai turi ir praktinės reiksmės vėžinių auglių diagnostikoje bei gydyme. / Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric protein that consist of two proteins- HIF-1α and HIF-1β. HIF-1 activates the transcription of many genes that code for proteins that are involved in angiogenesis, glucose metabolism, cell proliferation/survival and invasion/metastasis. HIF-1α protein degradation is regulated by O2-dependent prolyl hydroxylation, whitch targets the protein for ubiquitylation by E3 ubiquitin-protein ligases. These ligases contain the von Hippel-Lindau tumour-suppressor protein (VHL), whitch binds specifically to hydroxylated HIF-1α and ubiqutinates it. Ubiquitylated HIF-1α is rapidly degraded by the proteosome. HIF-1α interacts with the molecular chaperone Hsp90. Hsp90 plays a pivotal role in mediating the proper folding and subsequent activation of its numerous “client” proteins. Hsp90 also cooperates with the proteosomal pathway to eliminate misfolded cellular proteins. The antibiotic geldanamycin (GA) binds to Hsp90 and modulates its chaperone function, thereby accelerating the degradative activity associated with Hsp90. However, whether Hsp90 modulates HIF-1α activity by stabilization of the protein or by another mechanism is not clear. We tested the hypothesis weather and how inactivation (i.e. with geldanamycin) of Hsp90 protein function influences HIF-1α protein expression levels. Our obtained results shows that Hsp90 is required to HIF-1α protein under normoxia as well as hypoxia and is... [to full text]

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Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 132-147).

Heat shock protein 90 (HSP90), a conserved molecular chaperone, has become a potential molecular target for cancer therapeutics. HSP90 inhibition (HSP90i) causes inhibition of several oncogenic pathways simultaneously and leads to anti-cancer activities in multiple cancers including in triple-negative breast cancer (TNBC). TNBC is a subtype of breast cancer with poor prognosis and lack of approved targeted therapies. Although HSP90i has shown promising initial clinical data, resistance to HSP90i can still arise in TNBC patients and its resistance mechanisms are not yet understood. In this study, using an in vitro system, we report for the first time the isolation of TNBC cells with acquired resistance to HSP90i. Proteome and whole transcriptome profiling, and a bioactive small molecule screen were performed to identify the molecular basis of resistance to HSP90i and potential therapeutic approaches to overcome acquired resistance to HSP90i in TNBC cells. Two independent HSP90i-resistant clones were acquired through prolonged exposure of a TNBC cell line (Hs578T) to HSP90i. The clones showed significant resistance to HSP90i, notably to resorcinol-based HSP90i. The HSP90i-resistant clones also shared genomic sequence variants, suggesting a pre-existing population of resistant cells within the parental cells. We demonstrate that upregulated expression of UGT1A9, possibly due to an increased intrinsic oxidative stress, is associated with acquired resistance to resorcinol-based HSP90i in TNBC cells, and sensitivity to HSP90i can be restored with a competitive inhibitor of UGT1A9. The HSP90i-resistant clones also exhibited slower growth and upregulated IL6- mediated JAK2-STAT3 survival signalling pathway, which might contribute to the crossresistance to chemotherapeutics and other targeted therapies seen in the clones. Finally, we demonstrate that inhibition of JAK2-STAT3 signalling pathway is able to increase the cytotoxic effects of HSP90i to TNBC cells. We conclude that by using in vitro assays, we are able to identify potential mechanisms of acquired resistance to HSP90i in TNBC cells. We propose that expression of UGT1A9 or STAT3 might be a potential biomarker of sensitivity to HSP90i in TNBC cells. A combined inhibition of HSP90 and JAK2 might be a potential therapeutic approach for the development of effective targeted therapies in TNBC patients.

A cell’s ability to recognise and respond to changes in its environment is crucial to its survival. The availability of nutrients is a fundamental part of the environment, and so cells must be able to identify when they are plentiful and when they are scarce, and adapt accordingly. GCN2 is a key protein kinase within the eukaryotic proteome, and it is activated by a drop in the intracellular concentration of amino acids. When activated, GCN2 phosphorylates the translation initiation factor eIF2, initiating the Integrated Stress Response. This causes the global inhibition of protein synthesis and the upregulation of stress response pathways. GCN2 has been implicated in a wide range of cellular processes in health and diseases, including the development of pulmonary veno-occlusive disease, neurological degeneration and cancer. The molecular mechanisms that control the regulation and activation of GCN2 remain unclear. Some insights have been provided through genetic experiments on yeast, but the complexities of the regulatory pathways have made it difficult to decipher precise mechanistic details. For this reason, the aim of this project was to characterise the human GCN2 kinase both functionally and structurally, and to investigate the molecular mechanisms that enable it to act as a sensor of nutritional stress. This thesis describes the development of a system to reconstitute GCN2 activation using purified components, allowing the effects of different regulators to be tested and characterised. Insights from these data alongside structural insights into the kinase allow the proposal of a model concerning how GCN2 can sense amino acid deprivation in response to various regulatory signals. While GCN2 is activated by nutritional stress, mammalian cells have evolved a panoply of responses to environmental stress. Hsp90 is a chaperone that is required for the stability and maintenance of approximately 60 % of the human kinome, yet how it recognises client kinases is still unclear. The final chapter of this thesis describes the use of biochemical methods in combination with HDX-MS to characterise the interactions between Hsp90’s co-chaperone Cdc37 and client kinases. These analyses enabled the identification of a correlation between protein stability and dependence on Hsp90/Cdc37, and revealed that Cdc37 binding causes a dramatic conformational remodelling of the N-lobe of the kinase.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Doctor Pharmaceuticae - DPharm / Natural products have been an important source of drugs and novel lead compounds in drug discovery. Their unique scaffolds have led to the synthesis of derivatives that continue to give rise to medicinally relevant agents. Thus, natural product-inspired drugs represent a significant proportion of drugs in the market and with several more in development. Cancer is among the leading public health problems and a prominent cause of death globally. Chemotherapy has been important in the management of this disease even though side effects that arise due to lack of selectivity is still an issue.

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.