Yes / Inhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality.

No description available.

High temperatures are a significant biological stressor for mammals, which they may adapt to through behavioral changes, physiological plasticity, and via genetic adaptation. Savanna monkeys (genus Chlorocebus) have a wide climatic range in Africa south of the Sahara, making them a good model species for understanding adaptations to heat stress in primates. Savanna monkeys have been observed to behaviorally mitigate high temperatures, and genetic signs of selection in response to climate have also been found (specifically in relation to cold). In this study, I investigate whether there is genetic variation and evidence for selection related to function in a heat shock protein gene (HSPA1L) in 73 wild savanna monkeys ranging from equatorial Africa to the southern coast of South Africa. Given the important role of heat shock proteins in buffering heat stress, I hypothesized that genetic variation would be associated with maximum summer temperatures, as those are most likely to be warm enough to induce a heat shock response. I found 45 single nucleotide polymorphisms (SNPs) outside of Hardy-Weinberg Equilibrium, and 10 SNPs with significant integrated haplotype scores, only one of which was in a protein coding region (17:40210341; piHS = 2.20). Using phylogenetic least squares modeling I found that maximum temperature of the warmest month was strongly but not significantly associated with the frequency of a derived allele nested within a regulatory region for HSPA1L (17:40207386; piHS = 2.57; b = 0.044, p = 0.061) presumably experiencing selection. I discuss implications of these results for heat tolerance in primates and resilience to climate change.

Most proteins must fold into native three-dimensional structures to be functional. But, newly synthesized proteins are at high risk of misfolding and aggregating in the cell. Stress, disease or mutations can also cause protein aggregation. A cyanobacterial small heat shock protein, Hsp16.6, can act as a chaperone to prevent irreversible protein aggregation during heat stress. This thesis is focused on the properties of proteins that were associated with Hsp16.6 during heat stress, and which therefore may be “substrates” of Hsp16.6. Bioinformatics were used to determine if Hsp16.6 preferentially binds to proteins with certain properties, and biochemical studies were performed to investigate how the substrates actually behave with Hsp16.6 during heat stress. It was found that Hsp16.6 preferentially binds to proteins with higher molecular weight, higher acidity, higher percentage of charged residues (especially negatively charged residues), and a lower percentage of hydrophobic residues compared to all proteins encoded by the Synechocystis genome. Proteins bound to Hsp16.6 were also slightly enriched in VQL motifs. The potential substrate fructose bisphosphate aldolase class II (FBA) was expressed in E.coli and purified. FBA could be protected by Hsp16.6 from aggregation through forming a complex with Hsp16.6 during heat stress in vitro, consistent with it being a substrate of Hsp16.6. Another potential substrate, elongation factor G1 (EF-G1) was also expressed in E.coli and purified. EF-G1 did not form insoluble aggregates even at 47°C, but circular dichroism spectroscopy revealed the secondary structure has melted at this temperature, and the protein eluted earlier than unheated protein on size exclusion chromatography. Thus, EF-G1 appears heat sensitive, and may also be an in vivo substrate of Hsp16.6. Lastly, in vivo study studies were performed to determine the amount of FBA and EF-G1 in Synechocystis cells. Both proteins are abundant, with FBA levels (around 2% of total cell protein) being about twice that of EF-G1. Further in vivo experiments will be needed to confirm that FBA and EF-G1 are substrates of Hsp16.6.

No description available.

Amyotrophic Lateral Sclerosis (ALS) is an always lethal motor neuron disease with unknown pathogenesis. Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) have limited neuroprotection in some models of motor neuron degeneration. However the direct effect of Hsp90 inhibition on motor neurons is unknown. Here we show that Hsp90 inhibition induced motor neuron death through activation of the P2X7 receptor. Motor neuron death required phosphatase and tensein homolog (PTEN)-mediated inhibition of the PI3K/AKT pathway leading to Fas receptor activation and caspase dependent death. The relevance of Hsp90 for motor neuron survival was investigated in mutant Cu/Zn superoxide dismutase (SOD) transgenic animal models for ALS. Nitrated Hsp90, a posttranslational modification known to induce cell death (Franco, Ye et al. 2013), was present in motor neurons after intracellular release of zinc deficient (Zn, D83S) and the SOD in which copper binding site was genetically ablated (Q) but not after copper deficient (Cu) wild type SOD. Zn deficient and Q mutant SOD induced motor neuron death in a peroxynitrite mediated and copper dependent mechanism. Nitrated Hsp90 was not detected in the spinal cord of transgenic animals for ALS-mutant SOD animal models until disease onset. Increased nitrated Hsp90 concentrations correlated with disease progression. Addition of Zn or Q SOD to nontransgenic brain homogenate treated with peroxynitrite led to an increase level of nitrotyrosine in comparison to wild type controls. However, in the same samples there was a 2 to 10 time increase in Hsp90 nitration as compared to nitrotyrosine. The selective increase is likely due to the binding of Hsp90 to Zn deficient and Q SOD as oppose to wild type SOD. These results suggest that Hsp90 nitration facilitated by mutant SOD may cause motor neuron degeneration in ALS. Targeted inhibition of nitrated Hsp90 may be a novel therapeutic approach for ALS. An alternative therapeutic strategy is to target the production of survival factors by glial cells. Riluzole is the only FDA approved drug for the treatment of ALS and it shows a small but significant increase in patient lifespan. Our results show that acute riluzole treatment stimulated trophic factor production by astrocytes and Schwann cells. However long-term exposure reversed and even inhibited the production of trophic factors, an observation that may explain the modest increase in patient survival in clinical trials. Discontinuous riluzole treatment can maintain elevated trophic factor levels and prevent trophic factor reduction in spinal cords of nontransgenic animals. These results suggest that discontinuous riluzole administration may improve ALS patient survival. In summary, we demonstrated that Hsp90 has an essential function in the regulation of motor neuron survival. We have also shown that Hsp90 was nitrated in the presence of mutant SOD and was present during symptom onset and increases as disease progresses, which may explain the toxic gain of function of mutant SOD. Finally we demonstrate a biphasic effect of riluzole on trophic factor production and propose changes in administration to improve effects in ALS patients.

Ultrasound contrast agents (CAs) are typically used in clinic for perfusion studies (blood flow through a specific region) and border delineating (differentiate borders between tissue structures) during cardiac imaging. The CAs used during ultrasound imaging usually consist of gas filled microbubbles (MBs) (diameter 1-5 μm) that are injected intravenously into the circulatory system. This thesis partially involves a novel polymer-shelled ultrasound CA that consists of air filled MBs stabilized by a polyvinyl alcohol (PVA) shell. These MBs could be coupled with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as a combined CA for ultrasound and magnetic resonance imaging. The first three papers (Paper A-C) in this thesis investigate the structural characteristic and the elimination process of the CA. In Paper A, two types (PVA Type A and PVA Type B) of the novel CA were analyzed using transmission electron microscopy (TEM) images of thin sectioned MBs. The images demonstrated that the SPIONs were either attached to the PVA shell surface (PVA Type A) or embedded in the shell (PVA Type B). The average shell thickness of the MBs was determined in Paper B by introducing a model that calculated the shell thickness from TEM images of cross-sectioned MBs. The shell thickness of PVA Type A was determined to 651 nm, whereas the shell thickness of PVA Type B was calculated to 637 nm. In Paper C, a prolonged blood elimination time was obtained for PVA-shelled MBs compared to the lipid-shelled CA SonoVue used in clinic. In addition, TEM analyzed tissue sections showed that the PVA-shelled MBs were recognized by the macrophage system. However, structurally intact MBs were still found in the circulation 24 h post injection. These studies illustrate that the PVA-shelled MBs are stable and offer large chemical variability, which make them suitable as CA for multimodal imaging. This thesis also involves studies (Paper D-E) of the molecular chaperones (Hsp21 and DNAJB6). The small heat shock protein Hsp21 effectively protects other proteins from unfolding and aggregation during stress. This chaperone ability requires oligomerization of the protein. In Paper D, cryo-electron microscopy together with complementary structural methods, obtained a structure model which showed that the Hsp21 dodecamer (12-mer) is kept together by paired C-terminal interactions.The human protein DNAJB6 functions as a very efficient suppressor of polyglutamine (polyQ) and amyloid-β42 (Aβ42) aggregation. Aggregation of these peptides are associated with development of Huntington’s (polyQ) and Alzheimer’s (Aβ42) disease. In Paper E, a reconstructed map of this highly dynamic protein is presented, showing an oligomer with two-fold symmetry, indicating that the oligomers are assembled by two subunits. / <p>QC 20160527</p>

Orientador: Ilda de Godoy / Resumo: O tabagismo é responsável pelo maior número de mortes evitáveis ​​no mundo e está relacionado ao desenvolvimento de várias doenças, principalmente a doença pulmonar obstrutiva crônica (DPOC). Assim, a busca por biomarcadores precoces torna-se relevante para sua identificação e para o sucesso terapêutico. Os objetivos do nosso estudo foram avaliar a concentração da proteína de choque térmico 70 (HSP70), expressão do gene HSP70, anticorpos anti-HSP70 auto, marcador inflamatório sistêmico através da citocina interleucina-8 (IL-8) e proteína C reativa (PCR), alterações imunológicas e danos no DNA no sangue periférico de fumantes crônicos assintomáticos e não fumantes. Nossos resultados mostraram concentrações séricas aumentadas de HSP70, anti-HSP70, IL-8, PCR e neutrófilos, e danos no DNA de células sanguíneas de fumantes em comparação ao não-fumantes. Portanto, o tabagismo foi responsável por levar a alteração nos parâmetros fisiológicos e moleculares associados ao risco de desenvolver DPOC e outras doenças pulmonares. Com base nos dados, sugerimos que a HSP70 pode ser responsável pelo aumento dos níveis de citocinas inflamatórias e, consequentemente, o aumento do influxo de neutrófilos para os pulmões e aumento dos danos ao DNA e auto-anticorpos anti-HSP70. / Abstract: Smoking is responsible for the largest number of preventable deaths in the world, and is related to the development of several diseases, mainly chronic obstructive pulmonary disease (COPD). Thus, the search for early biomarkers of such diseases becomes relevant for their identification and for successful therapy. The objectives of our study were to evaluate the concentration of heat shock protein 70 (HSP70), expression of the HSP70 gene, anti-HSP70 auto antibodies, the systemic inflammatory marker through cytokine interleukin-8 (IL-8) and CPR, immunological changes and DNA damage in peripheral blood of chronic asymptomatic smokers and non-smokers. Our results showed increased serum concentrations of HSP70, anti-HSP70, IL-8, CPR and neutrophils, and DNA damage in blood cells of smokers than in non-smokers. Therefore, cigarette smoking was confirmed as a noxious agent on physiological and molecular parameters associated with the risk for developing COPD and other lung diseases. Based on the data we suggest that HSP70 can be responsible for the increased levels of inflammatory cytokines, and consequently, the increased influx of neutrophils into the lungs and increased DNA damages e anti-HSP70 auto antibodies. / Doutor

Tyrosine kinase inhibitors (TKIs) are currently the first therapy option for chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients. However, many patients affected by CML and AML may develop resistance to TKIs or may not recover under this treatment regime. New potential and more effective treatments are recently emerging. Heat shock protein inhibitors (HSPIs) and the proteasome inhibitor Bortezomib are drugs which have been yet to be successfully tested on leukemic patients, despite being successful on other malignancies such as multiple myeloma (MM). The combination between HSPIs and Bortezomib could potentially be successful in killing leukemic cells, by enhancing their respective molecular mechanisms. Indeed, HSPIs would bind to HSP72 avoiding the protein to exert its ligase function to the proteasome, whilst Bortezomib could stop the ubiquitinated proteins to enter the proteasome and ultimately inducing apoptosis. To test the effects of such combination, cell viability was measured via MTS assay, apoptosis levels were tested through Annexin V\PI assays. Involvement of HSP72 and pro-survival protein Bcl-2 were measured via flow-cytometry. The cells were administered with HSPIs and Bortezomib first as single agents for 24 hours, to establish working minimal concentration. Also, the drugs were tested for a shorter time, to understand when the drugs start to be effective. It emerged that one hour is sufficient for the drugs to give an initial effect in terms of cell viability and apoptosis. Following, combination experiments of HSPIs and Bortezomib were performed; the first drug was administered for one hour, the second following one hour and the cells were incubated for 24 hours. This was repeated alternatively for both type of drugs on the different cell lines. MTS and Annexin V\PI showed that there is not a synergistic effect between drugs, but instead there is antagonism. No necrosis was found at any level of the study. The cells were then probed for HSP72 and Bcl-2, to investigate their involvement in apoptosis mechanisms. Following 6 hours of combined and single agent treatment, both type of drugs inhibit HSP72 but failed to reduce the expression of Bcl-2, particularly on AML cells. It is thus proposed that CML and AML cells may die by apoptosis following a short time of treatment with HSPIs and Bortezomib by an extrinsic pathway of apoptosis, independent from Bcl-2 involvement and from mitochondrial pathway of apoptosis. This study may be the first to indicate a potential use of HSPIs and Bortezomib on CML and AML patients for a short time of treatment, although not in combination. Future studies are needed to further investigate the mechanisms of action of these drugs, aiming to potentially give CML and AML patients another successful therapy option to overcome resistance to canonic chemotherapy.

La myélofibrose (MF) est la plus agressive des néoplasies myéloprolifératives (NMP). Elle porte à elle seule le plus mauvais pronostic pour les patients puisqu’elle s’accompagne d’une fibrose de la moelle osseuse évoluant vers une insuffisance médullaire. Les inhibiteurs de la kinase JAK2 ont apporté de nouveaux espoirs pour le traitement des NPM mais leurs effets ont été essentiellement bénéfiques sur les symptômes et non sur la fibrose elle-même ni sur le cours de la maladie. Plus récemment, la protéine de choc thermique 90 (HSP90) - connue pour stabiliser JAK2 - est apparue comme une cible thérapeutique prometteuse pour les NMP. Cependant, les inhibiteurs de la HSP90 ont montré une toxicité importante accompagnée d’une expression compensatoire des HSPs inductibles (i.e HSP70, HSP27), connues pour favoriser l’émergence de phénomène de résistance. Par ailleurs, des études ont montré que HSP27 était fortement exprimée chez les patients présentant une fibrose pulmonaire idiopathique ou rénale montrant l’importance de HSP27 dans les processus fibrotiques. Sur la base de l’ensemble de ces données, nous avons évalué d’une part l'efficacité chez l’animal d'un oligonucléotide inhibiteur spécifique de HSP27 appelé OGX-427 (en essai clinique dans plusieurs cancers). D’autre part, nous avons déterminé le niveau d’expression intra- et extracellulaire de HSP27 chez des patients atteints de MF. L'effet de l'OGX-427 a été évalué dans deux modèles murins de myélofibrose, laquelle est induite soit par la sécrétion excessive de thrombopoïétine (TPOhigh) soit par la mutation JAKV617F. Nous avons mis en évidence dans les souris traitées par l’OGX-427, une réduction de la taille de la rate, de la prolifération mégacaryocytaire et de l’hématopoïèse extramédullaire par rapport aux souris contrôles, révélant ainsi un effet bénéfique de l’inhibition de HSP27 sur la progression de la maladie. De toutes récentes observations complémentaires à ce travail ont également montré une diminution de la fibrose réticulinique dans la moelle osseuse de souris JAKV617F. Au niveau moléculaire, nous démontrons que l'effet prolifératif induit par la voie de signalisation exacerbée - JAK2/STAT5 - est régulé par HSP27 via des interactions directes. Pour finir, nous avons détecté une augmentation de l'expression de HSP27 aussi bien dans les progéniteurs circulants CD34+ que dans le sérum des patients atteints de NMP avec MF. Ce travail révèle pour la première fois le rôle intra et extracellulaire de HSP27 dans la physiopathologie de la MF et le bénéfice thérapeutique potentiel de l’utilisation des inhibiteurs de HSP27 dans cette maladie. / Myelofibrosis (MF) is the most aggressive myeloproliferative neoplasms (MPN) with the highest degree of morbidity and mortality, including progressive bone marrow fibrosis resulting into bone marrow failure. JAK2 kinase inhibitors have been successfully used for a few years in MPN and more particularly for MF treatment. Nevertheless, their beneficial effects are mainly restricted on symptoms and not on the course of the disease. Recently, heat shock protein 90 (HSP90) - known to stabilize JAK2 - has been reported as a promising therapeutic target in MPN. However HSP90 inhibitors show toxicity and induce the expression of stress-inducible proteins such as HSP70 and, most likely HSP27 as previously shown in other cancers. In addition, we and others have shown that HSP27, was strongly expressed in patients with idiopathic pulmonary or kidney tubulointerstitial fibrosis, underlying a relevant role of HSP27 in fibrotic processes. Taking into account both the beneficial effects of HSP inhibitors in leukemia and in MPN, and the possible implication of HSP27 in fibrosis, we have evaluated here the status of HSP27 in MF patient’s samples and assess the effectiveness of an HSP27 oligonucleotide inhibitor called OGX-427 in murine models. We report here the effect of OGX-427 in two murine models of thrombopoietin- and JAKV617F-induced myelofibrosis. OGX-427 limited the progression of the disease associated with a reduction of spleen weight and of megakaryocytic expansion. And more recently, our additional results show a decrease of reticulin fibrosis in JAK2V617F’s bone marrow. We show that HSP27 regulates JAK2/STAT5 proliferative effect through direct interactions, and we report an increase expression of HSP27 both in CD34+ circulating progenitors and in the serum of patients with NMP with fibrosis. Taking altogether, this work supports that extra and intracellular HSP27 plays a key role of in the pathophysiology in MF and highlight the potential therapeutic benefit of HSP27 inhibitors in this disorder.