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Title: TRAP1 regulation of mitochondrial homeostasis and cellular quality control
Authors: Barbosa, Inês Biscaia de Andrade 
Orientador: Oliveira, Paulo J.
Santos, Maria Sancha
Keywords: Cancer; TRAP1
Issue Date: 14-Mar-2014
Citation: BARBOSA, Inês Biscaia de Andrade - TRAP1 regulation of mitochondrial homeostasis and cellular quality control. Coimbra : [s.n.], 2013. Tese de doutoramento. Disponível na WWW:
Abstract: Through years of evolution and in order to maintain viable protein homeostasis, cells have developed defense mechanisms against the accumulation of misfolded proteins and aggregates. This mechanism comprises a complex network of specialized proteins designated as heat shock proteins (HSPs). Tumor necrosis factor receptor (TNFR)-associated protein 1 (TRAP1) is a 90 KDa HSP family member that has received large attention over the past years. The interest in TRAP1 originated from its identification as a mitochondrial chaperone whose expression is augmented in several human neoplasias. Since its identification, TRAP1 was described to play important anti-oxidant and anti-apoptotic roles, conferring tumor cells growth advantage. Despite the increasing knowledge, the mechanisms of TRAP1 cytoprotective actions are not yet fully understood. In fact, reports in literature are sometimes contradictory or describe alterations without providing a detailed mechanism of action. The present dissertation aims to contribute with new insights on the role of TRAP1 in conferring mitochondrial protection and regulating cellular quality control systems. We hypothesize that TRAP1 contributes to tumor homeostasis allowing cell growth and survival under stressful environments by preserving mitochondrial functionality and viability as well as through the regulation of cellular quality control systems, including autophagy and apoptosis. To test the hypothesis, the A549 lung carcinoma cell line was used due to its high expression of TRAP1. TRAP1 depletion in this system was achieved through small interference RNA. In addition, a parallel study was performed using MRC-5 cells, a normal lung fibroblast cell line, with low TRAP1 content. The use of the MRC-5 cell line would allow exploring the effects of TRAP1 silencing in a non-tumor cell line, with a low basal expression of that protein. We initially verified that TRAP1 localization in A549 cells was predominantly mitochondrial, whereas TRAP1 was localized in non-mitochondrial areas in MRC-5 cells. Overall, the results presented in this thesis regarding mitochondrial function are in agreement with previous observations showing that TRAP1 contributes to the maintenance of mitochondrial membrane potential and to decrease ROS production in tumor cells, while the same was not observed in normal MRC5 cells. Although TRAP1 function in mPTP modulation has been previously described, we show for the first time the direct effect of TRAP1 silencing on basal mPTP state. Surprisingly, and contrarily to what was expected, mPTP existed in a more closed conformation in A549 TRAP1-depleted cells. Another breakthrough of the present work regards ROS modulation in the tumor cell line, which according to our results may involve p66SHC phosphorylation in Ser36 residue. Additionally, TRAP1 silencing in A549 cells resulted in mitochondrial fragmentation, possibly involving DRP-1 fission protein. Although increased lysosome content (in A549 cells) and decreased p62 levels (in both cell lines) suggest an increased autophagic flux, our data showed a decrease in the expression of several macroautophagy markers in TRAP1-depleted cells. However, these apparently contradictory results are explained by a lower ubiquitin content and increased LAMP2A levels suggesting the activation of an alternative autophagy pathway, involving chaperone-mediated autophagy (CMA). Nonetheless, this activation of CMA was only observed in A549 cells. Moreover, incubation of TRAP1-silenced cells with the autophagy inducer rapamycin resulted in increased cellular growth, mainly in A549 cells, suggesting that autophagy signaling in these cells are pro-tumorigenic. Regarding TRAP1 silencing effects on apoptotic signaling, results for both cell lines showed an increase in caspase 3/7-like activity with no alterations in the apparent activity of initiator caspases (8, 9 and 12). Additionally, TRAP1 silencing shifts the BAX/BCL-xL balance in favor of apoptosis in A549 cells suggesting that these cells have an active apoptotic signaling, whereas MRC-5 cells are not affected. In conclusion, besides TRAP1 differential expression in normal versus cancer cells, its subcellular localization may contribute to the distinct effects observed after TRAP1 silencing (or chemical inhibition). The present work also suggests that p66SHC is a good candidate to mediate TRAP1 ROS modulation in cancer. Moreover, our data suggests that TRAP1 controls mitochondrial morphology through DRP1 content and, additionally, plays an important role in the maintenance of cellular quality control systems. Our results are relevant to clarify not only the role of TRAP1 as an anti-cancer target but also as to understand off-target effects of TRAP1 silencing/inhibition in non-tumor cells.
Description: Tese de doutoramento em Biologia apresentadas à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Rights: embargoedAccess
Appears in Collections:FCTUC Ciências da Vida - Teses de Doutoramento

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