Dissertations / Theses on the topic 'ER stress'

Mestrado em Bioquímica - Bioquímica Clínica
Breast cancer is the most prevalent cancer among women and also one of the oncologic pathologies that causes more deaths. In the last decades several studies have reported that solid tumors generate an immunosuppressive microenvironment. This microenvironment (acidosis, hypoxia, glucose deprivation and cytokines) is favourable to endoplasmic reticulum (ER) stress induction. ER stress is primarily a response towards the re-establishment of homeostasis; however if not resolved it usually results in cell death by apoptosis. Nevertheless, ER stress and unfolded protein response (UPR) play a paradoxical role in cancer physiopathology: the three branches of UPR, PERK, IRE1 and ATF6 actively contribute to signalling of survival and metastasis mechanisms. Recently it was reported a possible transmission of ER stress from tumor cells to immune cells, modulating the phenotype and function of recipient cells. Thus, the aim of the present work is to assess the ability and the respective mechanisms by which T-47D tumor cells transmit ER stress to THP-1 monocytes, and the consequences of this transmission. ER stress transmission was only observed when pharmacological ER stress inducers were used, such as tunicamycin, contrarily to physiological stimulation, as glucose deprivation. Additionally, it was found that tunicamycin seems to be transported within exosomes which, in turn, directly induces ER stress on monocytes. It was also observed that exosomes derived from glucose deprived T-47D cells do not transmit ER stress; however these exosomes conduct monocytes towards a particular proinflammatory profile, accompanied by the decrease of its maturation status. Overall, our results question the ER stress mechanism originally described, showing that pharmacological ER stress inducers can be transported within exosomes and directly inducing ER stress on recipient cells.
O cancro da mama é o cancro de maior incidência entre as mulheres, sendo também uma das situações oncológicas que mais mortes causa. Na última década inúmeros estudos têm demonstrado que os tumores sólidos geram um microambiente favorável à evasão/subversão do sistema imune. Esse microambiente (acidose, hipoxia, deprivação de glucose, citoquinas) é muita das vezes propicio à indução de stress do reticulo endoplasmático (RE). O stress do RE é primariamente uma resposta no sentido de restabelecer a homeostasia no entanto se não resolvido resulta normalmente na morte celular por apoptose. O stress do RE e a respetiva resposta às proteínas mal conformadas (UPR), desempenham um papel paradoxal na fisiopatologia do cancro: os três ramos da UPR, PERK, IRE1 e ATF6, contribuem ativamente para a sinalização de alguns mecanismos de sobrevivência e metastização. Recentemente, foi descrita uma possível transmissão do stress do RE das células tumorais para as células do sistema imunitário, modulando a ação destas. Desta forma, pretendeu avaliar-se com o presente trabalho a capacidade e os mecanismos pelos quais células tumorais T-47D transmitem o stress do RE para células monocíticas THP-1, e quais as consequências desta transmissão. A transmissão foi apenas observada aquando da utilização de indutores farmacológicos como a tunicamicina, não se registando para estímulos fisiológicos como a deprivação de glucose. Por outro lado, verificou-se que a tunicamicina parece ser transportada via exossomas e desta forma induzir diretamente stress do RE nos monócitos. Observou-se ainda que os exossomas provenientes das células T-47D em stress do RE por deprivação de glucose apesar de não transmitirem o referido stress conduzem os monócitos para um perfil pró-inflamatório específico diminuindo ainda a sua capacidade de maturação. Em geral, os nossos resultados questionam seriamente o mecanismo de transmissão de stress ER tal como originalmente descrito, mostrando que no uso de indutores farmacológicos o que parece ocorrer é o transporte do fármaco em vesículas e a indução direta nas células recetoras.

In Saccharomyces cerevisiae, the general stress response (GSR) protects cells from diverse stress conditions such as osmotic stress and heat stress, while the Unfolded Protein Response (UPR) is a protein folding stress signalling pathway which maintains homeostasis of the endoplasmic reticulum (ER). A mechanism of how and if at all the UPR integrates with other pathways is largely unknown. The focus of this thesis was to determine whether essential components of the UPR like the bZIP transcription factor Hac1p and the Rpd3p-Sin3p histone deacetylase integrated within osmotic stress and to identify a possible mechanism of such an integration event. Data from this thesis demonstrate that UPR components protect cells from hyperosmotic stress. Hac1p is a direct positive regulator of GSR genes. Rpd3p and Hac1p belong to the same pathway in activating GSR genes. Data also suggest that Hac1p does not contribute to the increase in nucleosomal histone acetylation levels after osmotic stress. The Gcn5 histone acetyltransferase contributes to the increase in histone acetylation observed after osmotic stress. The Rpd3p represses GSR genes in unstressed cells but also contributes to the activation of GSR genes after hyperosmotic shock. The Rpd3 large complex and not the small complex is involved regulating GSR gene expression. Subsequent investigation demonstrates that a possible mechanism by which the UPR contributes to the GSR gene activation is by the RNA polymerase II clearance at the GSR gene promoters.

2012/2013
Elevati livelli plasmatici di bilirubina non coniugata (UCB) sono responsabili dell’ittero neontale che è fisiologico nella maggior parte dei casi. L’iperbilirubinemia severa e prolungata nel tempo può causare encefalopatia da bilirubina e Kernicterus che, se non trattati, possono lasciare pesanti sequele neurologiche e nei casi più gravi condurre a morte. La neurotossicità da bilirubina è ancora una delle principali cause di malattie neurologiche nei paesi via di sviluppo ed è un problema riemergente nei paesi sviluppati a causa delle anticipate dimissioni dall’ospedale dei neonati. I meccanismi molecolari responsabili della neurotossicità da bilirubina non sono ancora completamente chiariti. Questo lavoro riporta i risultati ottenuti durante il mio progetto di dottorato volto a studiare il “molecular signalling” coinvolto nella neurotossicità da bilirubina. L’obiettivo principale è stato valutare gli effetti di concentrazioni pro-ossidanti di bilirubina sullo stato redox cellulare e sullo stress del reticolo endoplasmico (ER stress). Ci siamo focalizzati sulla pathway che coinvolge Nrf2, analizzando i geni indotti dalla bilirubina per effetto di Nrf2 e studiando il signalling a monte coinvolto nella sua attivazione. Parallelamente abbiamo anche studiato la cascata di segnali coinvolti nell’ER stress. Tutti gli esperimenti sono stati condotti nella linea cellulare di neuroblastoma umano SH-SY5Y, alcuni ripetuti anche nella linea di epatocarcinoma HepG2 e in colture primarie di astrociti dalla corteccia cerebrale di ratto. I nostri risultati mostrano che concentrazioni tossiche di bilirubina inducono un 40% di mortalità cellulare tra 1 e 4 ore di trattamento che si mantiene stabile fino alle 24 ore di trattamento. Le cellule trattate con UCB mostrano un incremento del livello dei ROS intracellulare dopo 1 ora seguito dall’accumulo nucleare dell’Nrf2 endogeno dopo 3 ore. La bilirubina aumenta l’induzione della trascrizione dell’ARE-GFP reporter gene associata ad una up-regolazione di diversi geni target di Nrf2. L’induzione dell’espressione genica può essere suddivisa in due categorie principali:la risposta precoce (4h-8h) e la risposta tardiva (16h-24h).La risposta precoce inizia con l’induzione dell’espressione di ATF3 dopo 4 ore di trattamento ed è seguita da i trasportatori di amminoacidi (xCT and Gly1) dopo 8h. Per la risposta tardiva abbiamo visto l’induzione dell’espressione genica degli enzimi coinvolti nella sintesi del glutatione. (γGCL and TNX1),nella risposta antiossidante e di detossificazione (HO-1, NQO1, FTH)e nell’omeostasi del NADPH (ME1, and G6PD). In seguito al silenziamento specifico di Nrf2, il trattamento con bilirubina diminuisce l’induzione dell’mRNA solo dell’HO-1 (75%), del NQO1 (56%) e della FTH (40%) Inoltre l’induzione dell’HO-1 è ridotta se le cellule vengono pretrattate con l’antiossidante NAC (65%) e con specifici inibitori per PKC (80%), P38α (40%) and MEK1/2 (25%). Risulta evidente che l’induzione di ATF3 è la prima risposta generata dal trattamento con UCB. Di seguito abbiamo osservato un’induzione sequenziale dei marker dell’ER stress: da quelli coinvolti nel signaling di PERK a 4h (PERK, ATF3, ATF4, CHOP), dalla diminuzione della proteina della ciclina D1 dopo 1 h e dall’induzione di IRE1 (XBP1), ATF6, e BiP dopo 8h di trattamento. Da notare però che il silenzia mento di PERK non riduce l’induzione dell’espressione dell’mRNA di ATFs/CHOP, ma induce l’espressione dell’mRNA di GCN2. Riassumendo noi abbiamo dimostrato che la bilirubina causa mortalità cellulare, produce la formazione di ROS, provoca l’accumulo di Nrf2 nel nucleo e induce la risposta antiossidante mediata dalle sequenze ARE. La bilirubina induce l’espressione di diversi geni coinvolti nella risposta antiossidante, tra tutti l’HO-1 e il NQO1 sono indotti dalla bilirubina in maniera dipendente da Nrf2. Abbiamo anche dimostrato che lo stress ossidativo (OS) e la PKC sono i principali fattori coinvolti nell’attivazione di Nrf2/HO-1. I risultati ottenuti dimostrano che l’induzione di ATFs/CHOP e di PERK sono uno dei primi eventi associati alla tossicità da bilirubina. Allo stesso tempo il silenziamento di PERK non influisce sull’induzione di ATFs/CHOP mentre induce GCN2, suggerendo un meccanismo di compensazione tra il signalling di PERK e GCN2. Concludendo i nostri dati dimostrano che lo stress ossidativo e lo stress del reticolo endoplasmico sono coinvolti nella neurotossicità indotta da UCB nella linea di neuroblastoma umano SH-SY5Y. Le cellule sviluppano una risposta adattativa alla bilirubina inducendo OS and ER stress e aumentando l’espressione dei geni coinvolti nella risposta antiossidante (in parte via Nrf2 pathway) e nello stress del reticolo endoplasmico (UPR).
Elevated levels of unconjugated bilirubin (UCB) are responsible for neonatal jaundice, and in some case, severe hyperbilirubinemia exposes babies to bilirubin encephalopathy and kernicterus with the risk of neurological sequela and death. Bilirubin neurotoxicity is still a major cause of neurological injury in the developing countries and is a re-emerged problem in the developed countries, due to the early hospital discharge of newborns after birth. The molecular mechanisms of UCB induced neurotoxicty are incompletely elucidated. Present thesis are reported the results obtained during my PhD course aimed to investigate the molecular signaling involved in UCB induced neurotoxicity .The main goal of this work was to evaluate the effects of the pro-oxidant concentration of UCB on cellular redox state and ER stress. We focused on Nrf2 pathway, analyzing the genes induced by UCB at Nrf2-dependent manner and the up-stream signaling involved in Nrf2 pathway activation. In parallel, we also studied the ER stress cascade signaling. All experiments were conducted in SH-SY5Y neuroblastoma cell line, with some performed in HepG2 cells and primary culture of cortical astrocytes. Our results showed that SH-SY5Y neuroblastoma cells incubated with toxic concentration of UCB suffer a 40% loss of cell viability between 1h to 4h, reaching a plateau until 24h after UCB treatment. Treated cells showed an increased level of intracellular ROS after 1h followed by the nuclear accumulation of endogenous Nrf2 after 3h. UCB enhanced the transcriptional activation of ARE-GFP reporter gene associated with an up-regulation of several Nrf2 target genes. Expression response could be divided into two main categories: early (4h-8h) and late response (16h-24h). As far as early genes, UCB mediates a sequential transcription starting with the ATF3 up-regulation at 4h and followed by the induction of amino acid transporters at 8h (xCT and Gly1). On the contrary, for late genes, we observed an up-regulation of the enzymes involved in GSH synthesis (γGCL and TNX1), antioxidant/detoxification (HO-1, NQO1, FTH), and NADPH homeostasis (ME1, and G6PD). Specific Nrf2 siRNA against Nrf2 decreased the induction only of HO-1 (75%), NQO1 (56%), and FTH (40%) upon UCB exposure. HO-1 induction was reduced in cells pre-treated with antioxidant NAC (65%) and with specific signaling inhibitors for PKC (80%), P38α (40%) and MEK1/2 (25%). It was evident that ATF3 up-regulation at 4h represents the earliest response to UCB exposure. We observed a sequential activation of UPR sensors starting with PERK signaling at 4h (up-regulation of PERK, ATF3, ATF4, CHOP at 4h, and loss of cyclin D1 protein at 1h), followed by IRE1 (XBP1), ATF6, and BiP at 8h after UCB treatment. Interestingly, PERK siRNA does not changed the induction of ATFs/CHOP while induced GCN2 mRNA upon UCB exposure. In summary, we demonstrated that UCB mediates loss of cell viability, ROS generation, Nrf2 nuclear accumulation and induction of ARE. Nrf2 pathway activation was associated with the induction of multiple antioxidant genes, among all, HO-1 and NQO1 are induced by UCB at Nrf2-dependent manner. We observed that OS and PKC are the major up-stream signaling involved in Nrf2/HO-1 activation. Results demonstrated ATFs/CHOP induction and ER stress (initiated by PERK signaling) as one of the earliest event associated with UCB toxicity. However, PERK siRNA does not affected ATFs/CHOP induction by UCB while induced GCN2, suggesting a compensatory mechanism between PERK and GCN2 signaling. In conclusion, our data demonstrate that OS and ER stress are involved in UCB induced neurotoxicity in SH-SY5Y cells. The cells undergo an adaptive response against UCB induced OS and ER stress, through activation of multiple antioxidant genes (in part via Nrf2 pathway), and activation of sequential UPR sensors
XXVI Ciclo
1985

We recently identified a novel cyclin called Cyclin O which is able to bind and activate Cdk1 and Cdk2 in response to intrinsic apoptotic stimuli like DNA damage or ER stress. Cyclin O has been shown to be involved in the unfolded protein response (UPR) as an activator of PERK signalling. The aim of this thesis has been to study the molecular role of Cyclin O in response to ER stress. We have found that expression of Cyclin O is upregulated upon ER stress by pathways of the UPR that signal through eIF2α phosphorylation and CHOP expression. Furthermore, we have observed that Cyclin O activates the MAPK pathways independently of IRE1α signalling. This effect is most likely mediated by Cdk1 and Cdk2-dependent phosphorylation and consequent activation of MEKK4, which leads to the activation of JNK and p38. We have also found employing phosphoproteomics technology that many proteins involved in protein folding and translation depend on Cyclin O.
En nuestro laboratorio hemos identificado recientemente un nuevo miembro de la familia de las ciclinas, la Ciclina O, la cuál puede unirse y activar a Cdk1 y Cdk2 en respuesta a estímulos apoptóticos tales como el daño genético o el estrés del retículo endoplásmico. También hemos demostrado que la Ciclina O participa en la respuesta celular desencadenada por el acúmulo de proteínas mal plegadas (UPR) actuando a través de la activación de la señalización de la ruta de PERK. El objetivo de esta tesis ha sido el estudio molecular de la participación de la Ciclina O en la respuesta al estrés del retículo. Nuestros resultados indican que los niveles de expresión de la Ciclina O incrementan en respuesta al estrés del retículo a través de rutas de la UPR que señalizan a través de la fosforilación de eIF2α y de la expresión de CHOP. Además hemos observado que en respuesta al estrés reticular la Ciclina O activa la ruta de las MAPK de manera independiente de la señalización a través de IRE1α. Este efecto posiblemente tiene lugar a través de la fosforilación y consecuente activación de MEKK4 dependiente de Cdk1 y Cdk2. Esto conlleva la activación de las kinasas de stress JNK y p38. Asimismo, mediante experimentos de fosfoproteómica hemos demostrado que un gran número de proteínas involucradas en los procesos bioquímicos de traducción y plegado dependen de la expresión de la Ciclina O.

Cancer is a highly complex disease which is caused by an upset of balance between cell growth and death. During cell transformation, abnormality in the machinery of apoptosis often occurs, resulting in a chemotherapy-resistant phenotype of cancerous cells. Endoplasmic reticulum stress (ER stress) and autophagic process are now representing serious candidates as alternative pathways to induce cell death in chemotherapy-resistant tumor cells. If on the one hand they induce cytoprotective functions to reestablish normal cellular homeostasis, on the other hand they contribute to an efficient cell killing when the stress is prolonged and unresolved. Therefore, harnessing stress-induced survival response, such as ER stress and autophagy, may represent a new anticancer strategy to induce cell death in apoptosis-resistant tumors. In line with this hypothesis, it has been recently shown that some chemotherapeutic agents overcome apoptosis-resistance through induction of both autophagy and ER stress. Thus, understanding the link between ER stress/apoptosis and autophagy/apoptosis induction may offer the opportunity to find out new targets to design more effective therapeutic regimes to treat cancer malignancies. Here, we show that: i) the down-regulation of the transcription factor E2F1, a key regulator of proliferation and cell death, represents a critical event in ER stress-induced apoptosis, unveiling E2F1 inactivation as a novel therapeutic strategy to increase the response of tumor cells to ER stress based anticancer treatments; ii) oncogenic activating mutation in B-RAF confers resistance to autophagy in response to both classical inducers (serum starvation and rapamycin), and to ER stress-induced apoptosis (fenretinide and velcade), suggesting that autophagy is required for efficient melanoma cell killing; iii) Ambra1, an essential regulator of autophagy, plays a role in the regulation of apoptosis, and consequently in the interplay between autophagy and apoptosis, underlining the functional link existing between these processes.

MCDS is an autosomal dominant disorder, with a mild dwarfed phenotype and is caused by mutations in collagen X. The majority of the mutations identified so far are localized almost exclusively within the NC1 domain, which is responsible for trimerization of the collagen X protein. Little is known about the onset of MCDS, but recently, up-regulation of ER stress has been suggested as an important mechanism promoting the MCDS phenotype. Several studies have shown that the mutated collagen X protein is retained within the ER triggering the UPR, which has proved to be the key pathway responsible for the pathogenesis of the MCDS phenotype. In order to study the consequences of the expressing the MCDS-causing COL10A1p.N617K mutation at the molecular level, we selected HeLa cells as an appropriate cell line for the characterisation of the UPR response, by showing that the three branches of the UPR can be activated by ER stress inducing conditions in a similar manner to that seen in vivo in the MCDS growth plate. Importantly we have also shown that HeLa cells can be transduced with the collagen X cDNA constructs and will express, fold and secrete collagen X into the supernatant.Having established the cellular model for MCDS studies we demonstrated for the first time direct evidence for the retention of mutant collagen X within the ER. Moreover, we demonstrated that the mutant collagen X was degraded via a proteasomal pathway. Nevertheless, the level of ER stress induced by expression of mutant collagen X, based on BiP induction at the protein level, was disappointingly low. We therefore directly compared the level of ER stress induced by the COL10A1p.N617K mutation with that of the chondrodysplasias-causing MATN3p.V194D mutation. The ER stress induced by the matrillin mutation was far greater than that caused by the mutant collagen X. We showed that general protein synthesis was reduced in cells expressing either of the mutant proteins, most likely by the mechanism associated with the phosphorylation of eIF2alpha. Moreover, we showed the mutant matrilin-3 protein was also retained specifically in the ER. However, we could find no evidence for either proteasomal or autophagic/lysosomal degradation of mutant matrilin 3.We tested a broad range of ER stress-relieving compounds on cells expressing mutant collagen X and matrilin 3. Carbamazepine, which was previously shown to reduce ER stress in alpha1-antitripsin deficiency, reduced ER stress in cells expressing the mutant collagen X (but not matrilin 3) by way of enhanced proteasomal degradation of the retained protein. This drug should now be tested in vivo against the MCDS mouse to determine its capacity to reduce disease severity.The results presented within this thesis have contributed to the understanding of how cells deal with mutant collagen X and matrilin-3 proteins. We have identified a potential therapeutic compound that may be of use in the treatment of MCDS. Furthermore, the data presented support the concept that generic approaches to relieving ER stress may not be suitable for treating a broad range of diseases. Treatments may need to be tailored not only in a gene-specific manner but also may need to be tailored to address the differing consequences of different mutations in the same gene.

Endochondral bone development depends on the progression of chondrocyte proliferation, hypertrophy and terminal differentiation, which requires precise transcriptional regulation and signaling coordination. Disturbance of this process would disrupt chondrocyte differentiation and lead to chondrodysplasias. In cells, a highly conserved mechanism, ER stress signaling, has been developed to sense the protein load and maintain the cellular homeostasis. In humans, mutations in COL10A1 induce ER stress and result in metaphyseal chondrodysplasia type Schmid (MCDS). Previous analysis of a MCDS mouse model (13deltg mouse) had revealed a novel mechanism of chondrocyte adaptation to ER stress. The hypertrophic chondrocytes survive ER stress by reverting to a pre-hypertrophic like state (Tsang et al., 2007). To dissect the underlying mechanisms that coordinate chondrocyte survival, reverted differentiation and adaptation to ER stress, different chondrocyte populations in the wild type and 13del growth plates were fractionated for global gene expression analyses. The genome-wide expression profiles of proliferating chondrocytes, prehypertrophic chondrocytes, hypertrophic chondrocytes and terminally differentiated chondrocytes in the wild type growth plate provide molecular bases to understand the processes underlying both physiological and pathological bone growth. Systematic analyses of these transcriptomic data revealed the gene expression patterns and correlation in the dynamics of endochondral ossification. Genes associated with sterol metabolism and cholesterol biosynthesis are enriched in the prehypertrophic chondrocytes. Selected genes (Wwp2, Zbtb20, Ppa1 and Ptgis) that may potentially contribute to endochondral ossification were identified differentially expressed in the growth plate. Bioinformatics approaches were applied to predict regulatory networks in chondrocytes at different differentiation stages, implying the essential and dominant roles of Sox9 in coordination of stage specific gene expression. We further confirmed that Sox9 directly regulates the transcription of Cyr61, Lmo4, Ppa1, Ptch1 and Trps1, suggesting that Sox9 integrates different steps of chondrocyte differentiation via regulation of its target genes and partially crosstalk with IHH signaling pathway. The information on gene expression and regulation from physiological growth plate provides important basis to understand the molecular defects of chondrodysplasia. The hypertrophic zone in 13del growth plate was fractionated into upper, middle and lower parts for microarray profiling, corresponding for the onset of ER stress, onset of reverted differentiation and adaptation phase. Comparative transcriptomics of wild type and 13del growth plates revealed genes related to glucose, amino acid and lipid metabolisms are up regulated in response to ER stress. Fgf21 was identified as a novel ER stress inducible factor regulated by ATF4. Removal of Fgf21 results in increasing cell apoptosis in 13del hypertrophic zone without affecting the reverted differentiation process. Up regulation of genes expression related to hypoxic stress (Slc2a1, Hyou1, Stc2 and Galectin3) in 13del hypertrophic chondrocytes suggested that survival and adaptation of chondrocytes to ER stress involve cross-regulation by other stress pathways. Our findings have provided a new insight into the mechanisms that facilitate chondrocyte survival under ER stress in vivo, and propose the integrative effects of hypoxic stress pathway during the stress adaptation process, which broaden the molecular horizons underlying chondrodysplasias caused by protein folding mutations.
published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy

A disruption in endoplasmic reticulum (ER) homeostasis can lead to ER stress and the accumulation of misfolded proteins, which has been implicated with the development of diabetes and many other diseases. In reaction to this the cell mounts an adaptive response termed the unfolded protein response (UPR) to improve cell survival during ER stress through the activation of three ER stress transducers PERK, ATF6 and IRE1. However, in case of unresolved ER stress, the UPR can triggers apoptosis pathway. UPR adaptive response is intended to restore ER homeostasis through decreasing ER load, increasing ER folding capacity and increasing ER associated degradation. At the centre of the UPR is transmembrane protein PERK which upon the phosphorylation of eIF2α leads to represses of global protein synthesis coextensive with preferential translation of mRNAs, such as activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP). In this study, I investigated the molecular mechanisms of translational repression in response to ER stress in MIN6 cells and how ATF protein expression is up-regulated in response to ER stress. In conclusion, I provide evidence that the eIF2α is likely responsible for the repression of protein synthesis in the presence of ER stress and that the induction of ATF4 expression in response to ER stress is dependent on its transcriptional upregulation. PERK mediated eIF2α phosphorylation is not required for increased ATF4 expression in MIN6 cells in response to ER stress. However, in MEFs, the PERK/eIF2α pathway is required for ATF4 protein expression, IRE1-XBP1 pathway is also required for ATF4 expression which might be time dependent, and protein synthesis is essential for induction of ATF4 expression in response of ER stress. Further investigations into how ATF4 expression is up-regulated in response to ER stress may extend our understanding to develop new therapies to protect ER from stress.

Mestrado em Bioquímica - Bioquímica Clínica
A capacidade das células dendríticas (CD) em iniciar e modular respostas imunes, nomeadamente a dermatite de contacto alérgica (DCA), é fortemente dependente da sua ativação / estado de maturação. Os mecanismos moleculares pelos quais os sensibilizadores de pele induzem maturação das CD não são ainda completamente conhecidos. No entanto, foi demonstrado que sinais de perigo primários como a formação de espécies reativas de oxigénio (ERO) desempenham um importante papel neste processo. Nos últimos anos, inúmeras evidências têm estabelecido uma estreita ligação entre a produção de ERO, stresse do retículo endoplasmático (RE) e a patogénese de diversas doenças inflamatórias. Deste modo, pretendeu-se no presente trabalho avaliar a capacidade do sensibilizador cutâneo DNFB desencadear stresse do RE em CD e as concomitantes consequências na imunobiologia dessas células. Os resultados obtidos revelaram que o DNFB induz uma rápida e sustentada fosforilação do fator de iniciação de tradução eucariótico 2α (eIF2α), o aumento dos níveis proteicos do fator de transcrição ATF4 e uma modificação pós translacional na principal proteína chaperone do RE, GRP78. Verificou-se ainda que estes efeitos são dose-dependentes e parcialmente revertidos pelo antioxidante N-acetilcisteína, indicando que a alteração do estado redox celular está na origem da indução do stresse do RE observado. O tratamento das células com o chaperone químico, ácido 4-fenilbutírico (4- PBA) causou um aumento na apoptose induzida por DNFB, enquanto o prétratamento com salubrinal, um inibidor seletivo da desfosforilação de eIF2α, provocou o efeito oposto. A exacerbação pelo salubrinal da fosforilação do eIF2α induzida por DNFB causou um forte aumento da transcrição de genes de destoxificação tais como o HMOX e do gene da citoquina pró-inflamatória IL-8 tendo por sua vez o 4-PBA anulado completamente estes efeitos. Globalmente, os nossos resultados indicam que a ativação pelo DNFB do eixo eIF2α/ATF4 em CD contribui fortemente para o desenvolvimento de um microambiente pró-inflamatório e para a transcrição de genes envolvidos no restabelecimento do equilíbrio redox.
The capacity of dendritic cells (DC) to initiate and modulate immune responses, namely allergic contact dermatitis (ACD), is tightly dependent on their activation/maturation state. Molecular mechanisms driving skin sensitizersinduced DC maturation are not yet completely unrevealed, however initial danger signals such as the generation of reactive oxygen species (ROS) were shown to play an important role. In recent years innumerous evidences established a close link between ROS production, endoplasmic reticulum (ER) stress and the pathogenesis of several inflammatory diseases. Therefore, we analyzed in this work the ability of the strong sensitizer DNFB to trigger ER stress in DC and the concomitant consequences to the immunobiology of these cells. Our results revealed that DNFB induces a rapid and sustained phosphorylation of the Eukaryotic translation initiation factor 2α (eIF2α), the up regulation of ATF4 and a post translational modification at the major ER chaperone GRP78. These effects were dose dependent and partially reverted by the antioxidant N-acetylcysteine, indicating that cellular redox imbalance is in the origin of evoked ER stress. Treatment of cells with the ER chemical chaperone 4 phenylbutyric acid (4-PBA) caused an increase in DNFB-induced apoptosis while pretreatment with salubrinal, an eIF2α dephosphorylation selective inhibitor, caused the opposite effect. Exacerbation of DNFB-induced eIF2α phosphorylation by salubrinal also caused a strong increase in the transcription of detoxifying genes such as HMOX and of pro-inflammatory cytokine IL-8 while 4-PBA completely abrogated these effects. Overall, our results indicate that DNFB activation of eIF2α/ATF4 stress pathways in DC strongly contributes to generation of a pro-inflammatory microenvironment and is crucial to the transcription of genes involved in remediation of cell redox imbalance.

Celiac disease (CD) is a complex inflammatory and auto-immune disorder triggered by the ingestion of gluten, a heterogeneous mixture of seed-storage proteins, such as gliadins, present in cereals as wheat, barley, rye and oats, in genetically predisposed individuals. The disease occurs in 1% worldwide population, and its onset has genetic, immunological and environmental components. Currently, the molecular mechanisms through which gliadin triggers the CD onset are not yet completely clear. The only treatment for the disease is represented by a gluten-free diet (GFD) which is not 100% effective and is difficult to adhere by patients. In this study, we demonstrated that gliadin stimulation induces ER stress in IEC, by using both in vitro and ex vivo models. Importantly, our results indicate that ER stress has a key role in the pathogenesis of CD. At molecular level we found that extracellular gliadin peptides interact with CXCR3 at plasma membrane level which, in turn, induce the release of IP3 through the stimulation of PLC. The interaction of IP3 with IP3R onto ER membranes results in calcium release by the ER compartment thus inducing ER stress. Of note, buffering the gliadin-induced ER stress by the chemical chaperone 4PBA completely abrogates the cytopathic effects of gliadin. Moreover, we also show that gliadin-induced ER stress is responsible for: i) CXCR3 gene expression upregulation, through CHOP; ii) TG2 gene expression upregulation, through both canonical and non-canonical activation of NF-kB; iii) altered intestinal permeability; and iv) induction and release of pro-inflammatory cytokines. Therefore, our results indicate that ER stress might represent a valuable target to design a new clinical therapeutic approach to treat CD patients.

Alzheimer’s Disease and other neurodegenerative diseases have been linked to dysfunction in proteostasis in the endoplasmic reticulum (ER). The ER provides an exclusive environment for protein synthesis and folding, which is vital to the cellular function. Under normal conditions, the synthesis and degradation of proteins remain in balance. During aging or during pathological states, disturbances of ER occur and consequently the failure of protein homeostasis. The cells rely on a system, the unfolded protein response (UPR), which regulates the homeostasis by three ER sensors: PERK, ATF6, and IRE-1. Perturbations of ER function result in UPR. In physiological condition, the cell may overcome the insult and regain homeostasis. However, prolonged or chronic UPR activates apoptotic pathways and may cause cell death. The sigma-1 receptor (Sig-1R) is a 25 kD polypeptide and a chaperone protein concentrated at the mitochondria-associated ER membrane domain (MAM). The Sig-1R plays significant roles governing calcium signalling, mitochondrial function, oxidative stress, protein chaperoning and ER stress. Results of this investigation demonstrate that immortalized mouse embryonic fibroblasts (MEFs) derived from Sig-1R-/—(KO) mice have higher baseline activation in all three branches of the UPR in the absence of ER stress compared to MEFs derived from Wild-type mice. Despite this increase in baseline activation, the PERK and ATF6 pathways have a significantly blunted response to acute stress. Rescue experiments by expressing the Sig-1R in KO MEFs did not recover the WT MEFs phenotype. Primary Sig-1R KO MEFs did not show baseline ER stress, but did show inhibited recovery following treatment with the acute ER stressor DTT. Overall, our data suggests that Sig-1R is important for the reestablishment of proteostasis following acute stress.

Tumor suppressor p53 is a mediator of stress-induced cell cycle arrest and apoptosis. The kinase inhibitor 2-aminopurine (2-AP) is an adenine analog shown to cause cells to bypass DNA damage-induced cellular arrest through unknown mechanisms, and may potentially target p53. Although p53 plays vital roles in adaptation to many stresses, its role in cellular response to endoplasmic reticulum (ER) stress is unclear. Here, stress-induced p53 stabilization and checkpoint control in the presence of 2-AP are examined, as well as p53 regulation upon ER stress induction. I show that 2-aminopurine suppresses p53 stabilization in response to different forms of DNA damage. Biologically, 2-AP exposure enables cells to bypass adriamycin-induced G2/M arrest in a p53-dependent manner, but rescues the clonogenic survival of cells exposed to adriamycin in a p53-independent manner. Next, I show that pharmacological and physiological inducers of ER stress can inhibit stress-induced p53 function by promoting p53 cytoplasmic retention.

The fundamental function of the Endoplasmic Reticulum (ER) is to process nascent membrane and secretory proteins in a calcium-dependent manner. Disruption of ER function by the depletion of ER calcium results in ER stress, which triggers apoptosis if prolonged. ER stress has been shown to play a role in the pathogenesis of Motor Neuron Diseases (MNDs) and CAG-repeat disorders. Kennedy’s Disease (KD) is an X-linked neurodegenerative disease that is classified as both a MND and CAG-repeat disorder. In this Thesis I investigate whether ER Stress also plays a role in the pathogenesis of KD. Using a mouse model of KD, primary motoneuron cultures from both KD and wild-type (WT) embryos were established. Confocal microscopy was used to infer ER calcium levels, and markers of ER stress and ER stress-induced apoptosis were examined using western blot analysis and immunocytochemistry. KD motoneurons were found to have reduced levels of ER calcium and elevated levels of markers of ER stress and ER stress-induced apoptosis relative to WT controls. ER stress-induced apoptosis appears to contribute to the motoneuron death observed in KD mice, since inhibition of ER stress with Salubrinal increases ER Ca2+, decreases ER stress-induced apoptosis and consequentially improves KD motoneuron survival. Examination of markers of ER stress in the spinal cord of KD mice revealed higher expression levels compared to WT controls, with the most significant increase detected between E13 and 3 months of age i.e. pre-symptomatically. Mitochondrial dysfunction and impaired mitochondrial biogenesis was also observed in KD motoneurons. However, increasing mitochondrial biogenesis was not as effective as inhibition of ER stress in improving KD motoneuron viability. These results show that ER stress may play an early, causal role in the pathogenesis of KD and suggest that inhibition of ER stress may be a potential therapeutic strategy for the treatment of KD.

A mis-sense point mutation in the human VAPB gene is associated with a familial form of motor neuron disease that has been classified as Amyotrophic Lateral Sclerosis type VIII. Affected individuals suffer from a spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) or an atypical slowly progressing form of ALS. Mammals have two homologous VAP genes, vapA and vapB. VAPA and VAPB share 76% similar or identical amino acid residues; both are COOHterminally anchored membrane proteins enriched on the endoplasmic reticulum. Several functions have been ascribed to VAP proteins including membrane trafficking, cytoskeleton association and membrane docking interactions for cytoplasmic factors. It is shown here that VAPA and VAPB are expressed in tissues throughout the body but at different levels, and that they are present in overlapping but distinct regions of the endoplasmic reticulum. The disease-associated mutation in VAPB, VAPB (P56S) is within a highly conserved N-terminal region of the protein that shares extensive structural homology with the major sperm protein (MSP) from nematodes. The MSP domain of VAPA and VAPB is found to interact with the ERlocalized transcription factor ATF6. Over expression of VAPB or VAPB (P56S) attenuates the activity of ATF6-regulated transcription and the mutant protein VAPB (P56S) appears to be a more potent inhibitor of ATF6 activity. Moreover VAP proteins affect the activity of XBP1 and BiP promoter elements, two major components of the Unfolded Protein Response (UPR) of the Endoplasmic Reticulum and the different domains of VAPB have a differential effect on UPR regulation. Finally, over expression of the MSP domain of VAPB leads to cell death via apoptosis, while overexpression of other VAPB domains renders cells more susceptible to apoptotic death after ER stress. The data presented in this thesis indicate that VAP proteins interact directly with components of ER homeostatic and stress signalling systems and may therefore be parts of a previously unidentified regulatory pathway. The mis-function of such regulatory systems may contribute to the pathological mechanisms of degenerative motor neuron disease.

Master of Science
Department of Biochemistry
Gerald R. Reeck
Armet is a bifunctional protein widely distributed in animal species, vertebrate and invertebrate. It is an evidently part of the Unfolded Protein Response (UPR) and promotes survival in cells that are under endoplasmic-reticulum (ER) stress. It has also been found as a secreted protein with neurotrophic activity. The crystal and solution structures of human Armet show it is a helix-rich protein with two domains linked through a flexible linker region. In this study, immunofluorescence staining was used to verify Armet’s localization in ER and Golgi apparatus in MBA-MD-231 cells. Evidence for calcium binding by Armet was obtained by circular dichroism spectroscopy (the binding of calcium appeared to decrease helix content), by differential scanning calorimetry (binding of calcium resulted in a less structured protein) and two-dimensional (1H-15N HSQC) nuclear magnetic resonance spectroscopy. A difference HSQC spectrum of Armet, with and without calcium, showed peaks of increased intensity, of decreased intensity and of perturbed chemical shift. There were about 30 such peaks in total. Several of these affected amino acid residues appeared to form a cluster of negatively charged side chains that could possibly form a binding site for a calcium ion. Heterogeneity of three types was observed in recombinant Armet expressed in E. coli cells. Two bands of slightly different mobility were observed in SDS gels run in the absence of reducing agent. These may represent alternate arrangements of disulfide bonds, as previously reported by other investigators but not explained. Further, in the absence of reducing agent, a faint ladder was formed by human Armet, indicating formation of disulfides between Armet molecules. Oligomers with sedimentation coefficient greater than the monomeric protein, in the absence of reducing agent, disappeared in the presence of a reducing agent. Finally, minor species of mass differences of 98 and 180 with respect to the main protein component were observed by MALDI-TOF mass spectrometry. These studies provide a more thorough characterization of Armet than has been previously available and set the scene for future investigations of the binding of organic ligands to the protein.

Cardiac function is regulated by the molecular components of the sarco/endoplasmic reticulum (ER/SR). Disruptions in homeostatic balance of these proteins and calcium regulation results in activation of ER stress response. Sarcolemmal membrane-associated proteins (SLMAPs) are found in cell membrane, SR/ER, and mitochondria. Overexpression of SLMAP in the myocardium has shown to impair excitation-contraction (E-C) coupling in the transgenic (Tg) mice. ER stress response was examined in Tg mice overexpressing SLMAP in the myocardium. In Tg hearts, changes observed in the expression of proteins involved in ER stress were dependent on the age and sex. SLMAP overexpression results in maladaptive ER stress response, as the mice age. Neonatal cardiomyocytes isolated from the Tg hearts showed decreased viability, upregulation of ER stress response proteins, which were sensitized to thapsigargin-induced stress, and desensitized to palmitate-induced oxidative stress. These findings suggest that normal SLMAP levels are important for proper cardiac function, and cell viability.

Background: NUPR1 was described as a transcriptional factor involved in the regulation of various cellular stress-response genes, playing a crucial role in the condition of the endoplasmic-reticulum (ER) stress, thus emerging as a common molecular factor of different pathologies, obesity, hepatic steatosis, and cancer. In the present work we aim to explore how NUPR1 interacts with some pivotal genes that are the major modulators of the ER stress and metabolic cell functions. In particular we investigated the biochemical and molecular effects arising from the loss of NUPR1 in ER stress physiological conditions. Methods: We used prolonged high fat diet (HFD) feeding to induce ER stress physiological in Nupr1+/+ and Nupr1-/- male mice compared with their respectively normal chow diet (ND) controls. We fed mice with a HFD (60% fat, 20% protein, and 20% carbohydrate) for 10 weeks to promote chronic ER stress condition (Old-HFD group, n=5). An additional group of mice (n=5) was maintained on HFD (60% fat, 20% protein, and 20% carbohydrate) for a longer duration (15 weeks) to distinguish between age-dependent and age-independent effects. Liver were collected for histological and molecular assessments. Western blots and RT-qPCR were performed to assess the expression levels of the major ER-stress response UPR-associated proteins and metabolic genes. Results: We showed the downregulation of the majority of UPR-associated proteins: BIP (p<0.0001 for protein and mRNA), ATF4 (p<0.0001 for mRNA), XBP1 (p<0.0001 for protein and mRNA), CHOP (p<0.0001 for protein and mRNA), GADD34 (p=0.0296 for mRNA) in in-vivo NUPR1-/- compared to NUPR1+/+ 10 weeks HFD mice. Western blot for the major UPR associated proteins in NURP1-/- mice at 15 weeks HFD showed similar expression trends reported at the time-point of 10 weeks. ERDj4 mRNA resulted down-regulated in NUPR1-/- compared to NUPR1+/+ 15 weeks HFD mice (p=0.0032). Among the multiple metabolic genes, we reported a down-regulation of the majority mRNA associated to lipogenesis (SREBP, ACLY, ChREBP) and lipoprotein (APOB, PPAR-alfa, MTTP) in NUPR1-/- compared to NUPR1+/ + HFD mice 15 weeks. Both LCAD and MCAD fatty acid metabolisms mRNA were also downregulated, as consequence of PPAR-alfa deficit. Similarly betaoxidation mRNA ACOX1 and CPT1-alfa, as well as MTC4 and PGK1 were downregulated in NUPR1-/- compared to NUPR1+/ + HFD mice 15 weeks. Conclusion: The results of this work confirm that NUPR1 act downstream of the PERK branch playing a crucial role of NUPR1 in the activation of UPR response in physio-pathological ER stress condition and suggest a potential contribution of NUPR1-mediated ER stress response to the development of liver steatosis.
Background: NUPR1 was described as a transcriptional factor involved in the regulation of various cellular stress-response genes, playing a crucial role in the condition of the endoplasmic-reticulum (ER) stress, thus emerging as a common molecular factor of different pathologies, obesity, hepatic steatosis, and cancer. In the present work we aim to explore how NUPR1 interacts with some pivotal genes that are the major modulators of the ER stress and metabolic cell functions. In particular we investigated the biochemical and molecular effects arising from the loss of NUPR1 in ER stress physiological conditions. Methods: We used prolonged high fat diet (HFD) feeding to induce ER stress physiological in Nupr1+/+ and Nupr1-/- male mice compared with their respectively normal chow diet (ND) controls. We fed mice with a HFD (60% fat, 20% protein, and 20% carbohydrate) for 10 weeks to promote chronic ER stress condition (Old-HFD group, n=5). An additional group of mice (n=5) was maintained on HFD (60% fat, 20% protein, and 20% carbohydrate) for a longer duration (15 weeks) to distinguish between age-dependent and age-independent effects. Liver were collected for histological and molecular assessments. Western blots and RT-qPCR were performed to assess the expression levels of the major ER-stress response UPR-associated proteins and metabolic genes. Results: We showed the downregulation of the majority of UPR-associated proteins: BIP (p<0.0001 for protein and mRNA), ATF4 (p<0.0001 for mRNA), XBP1 (p<0.0001 for protein and mRNA), CHOP (p<0.0001 for protein and mRNA), GADD34 (p=0.0296 for mRNA) in in-vivo NUPR1-/- compared to NUPR1+/+ 10 weeks HFD mice. Western blot for the major UPR associated proteins in NURP1-/- mice at 15 weeks HFD showed similar expression trends reported at the time-point of 10 weeks. ERDj4 mRNA resulted down-regulated in NUPR1-/- compared to NUPR1+/+ 15 weeks HFD mice (p=0.0032). Among the multiple metabolic genes, we reported a down-regulation of the majority mRNA associated to lipogenesis (SREBP, ACLY, ChREBP) and lipoprotein (APOB, PPAR-alfa, MTTP) in NUPR1-/- compared to NUPR1+/ + HFD mice 15 weeks. Both LCAD and MCAD fatty acid metabolisms mRNA were also downregulated, as consequence of PPAR-alfa deficit. Similarly betaoxidation mRNA ACOX1 and CPT1-alfa, as well as MTC4 and PGK1 were downregulated in NUPR1-/- compared to NUPR1+/ + HFD mice 15 weeks. Conclusion: The results of this work confirm that NUPR1 act downstream of the PERK branch playing a crucial role of NUPR1 in the activation of UPR response in physio-pathological ER stress condition and suggest a potential contribution of NUPR1-mediated ER stress response to the development of liver steatosis.

Type 2 diabetes (T2D) is a growing health-care and economic burden. Obesity is a risk factor for developing T2D, but the underlying molecular mechanisms are not well understood. However, mechanisms such as lipotoxicity, endoplasmic reticulum stress and inflammation are becoming increasingly well-recognised in obesity, and may underlie the development and progression of T2D. A central player in these mechanisms is Protein Kinase R (PKR), proposed to have a role within nutrient- and pathogen-sensing pathways, and is activated by ER stress and lipotoxicity. A small molecule inhibitor Compound-16, adenoviral vectors and RNAi techniques in BRIN-BD11 rodent pancreatic β-cells, were used to demonstrate that PKR knockdown affords significant protection against palmitate-induced cell death. Furthermore, PKR knockdown potentiates palmitoleate cytoprotection during lipotoxicity, suggesting the cytotoxic and cytoprotective actions of long-chain fatty acid species may function via the PKR signalling pathway. The use of a novel 1.1B4 human pancreatic β-cell line has shown that important differences exist between human and rodent cell responses to fatty acids in vitro. In 1.1B4 cells, long-chain saturated and monounsaturated fatty acids do not provide increasing protection as their chain-length increases, in contrast to rodent cell models. Furthermore, methyl-saturated fatty acid species are well tolerated, and methyl-monounsaturated fatty acids are cytoprotective to 1.1B4 β-cells. TXNIP overexpression in an INS-TXNIP β-cell model has a proapoptotic role in conditions of glucotoxicity, but not glucolipotoxicity. Furthermore, in this cell model, succinate is cytoprotective against glucotoxicity, but not glucolipotoxicity. By contrast in 1.1B4 β-cells, succinate significantly protects against apoptosis induced by both glucotoxic and glucolipotoxic conditions. Chronic inflammation has been implicated in the development and progression of T2D. At the centre of this response is the pro-inflammatory cytokine IL-1β. The cellular origin of IL-1β is unclear, but IL-1β secretion has been linked to activation of the NLRP3 inflammasome, recently implicated in pancreatic β-cell death in T2D. Results suggest that IL-1β is secreted by INS-TXNIP and 1.1B4 pancreatic β-cells under lipotoxic conditions, thus offering a potential role for targeted IL-1β therapy in T2D.

Pt(II) based anticancer drugs—cisplatin, carboplatin, and oxaliplatin—are widely used in the treatment of a variety of cancers. Unfortunately, the clinical efficacy of these drugs is currently hindered by the development of undesirable side effects and resistance during treatment. The molecular mechanisms underlying these effects are still unclear. For decades, research has focused on DNA as the main cellular target of Pt(II) compounds. However, there is increasing interest in proteins as alternative targets of Pt(II) and contributors to cytotoxic and resistance mechanisms of cisplatin. In this work, I utilize Pt(II) compounds that have been functionalized to participate in the azide-alkyne cycloaddition ‘click’ reaction to study protein targets of platinum reagents. First, I describe the use of an azide-modified Pt(II) compound to fluorescently label and isolate Pt(II)-bound bovine serum albumin in vitro. Additionally, we discover that Pt(II) compounds form monofunctional adducts on BSA that can crosslink to DNA oligonucleotides. I then use the click-functionalized Pt(II) compound, azidoplatin, to enrich for Pt(II)-bound proteins in Saccharomyces cerevisiae using a biotin-streptavidin pull-down. I identified 152 proteins that are significantly enriched in AzPt-treated samples by LC-MS/MS analysis. A subset of these proteins are involved in proteostasis and ER stress, which I confirm is induced in both AzPt- and cisplatin-treated yeast. Of interest was the identification of the ER protein folding chaperone protein disulfide isomerase (PDI), which I observe is inhibited by Pt(II) binding in vitro. Finally, I investigate PDI activity in human cancer cell lines HeLa and MDA-MB-468 following treatment with Pt(II) compounds. Extracts from platinum-treated MDA-MB-468 cells show significant PDI inhibition at low concentrations of Pt(II), and these cells appear to have constitutive activation of the unfolded protein response. PDI activity in extracts from platinum-treated HeLa cells is inhibited only at high concentrations of Pt(II), and HeLa cells do not show significant XBP1 mRNA splicing during Pt(II) treatment. Additionally, MDA-MB-468 cells are nearly three times as sensitive to Pt(II) compounds than HeLa cells. From these data, I hypothesize that basal ER stress increases sensitivity to PDI inhibition by Pt(II) binding and that this interaction enhances Pt(II)-induced cell death.
10000-01-01

Cell cycle progression of Saccharomyces cerevisiae cells was monitored in continuous cultures limited for glucose or nitrogen. The G1 cell cycle phase, before initiation of DNA replication, did not exclusively expand when growth rate decreased. Especially during nitrogen limitation, non-G1 phases expanded almost as much as G1. In addition, cell size remained constant as a function of growth rate. These results contrast with current views that growth requirements are met before initiation of DNA replication, and suggest that distinct nutrient limitations differentially impinge on cell cycle progression. Therefore, multiple mechanisms are hypothesized to regulate the coordination of cell growth and cell division. Genetic interactions were identified between the dose-dependent cell-cycle regulator 2 (DCR2) phosphatase and genes involving in secretion/unfolded protein response pathway, including IRE1, through a genome-wide dominant negative genetic approach. Accumulation of unfolded proteins in the endoplasmic reticulum triggers the unfolded protein response (UPR). How the UPR is downregulated is not well understood. Inositol requirement 1 (IRE1) is an endoplasmic reticulum transmembrane UPR sensor in Saccharomyces cerevisiae. When the UPR is triggered, Ire1p is autophosphorylated, on Ser 840 and Ser 841, inducing the cytosolic endonuclease activity of Ire1p, thereby initiating the splicing and translational de-repression of HAC1 mRNA. Homologous to Atf/Creb1 (Hac1p) activates UPR transcription. We found that that Dcr2p phosphatase functionally and physically interacts with Ire1p. Overexpression of DCR2, but not of a catalytically inactive DCR2 allele, significantly delays HAC1 splicing and sensitizes cells to the UPR. Furthermore, Dcr2p physically interacts in vivo with Ire1p-S840E, S841E, which mimics phosphorylated Ire1p, and Dcr2p dephosphorylates Ire1p in vitro. Our results are consistent with de-phosphorylation of Ire1p being a mechanism for antagonizing UPR signaling.

There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on β-cell function and survival. The present study sought to determine the mechanism by which palmitate affects intracellular Ca²⁺ in pancreatic β-cells, and in particular the role of the endoplasmic reticulum (ER). In the MIN6 β-cell line, palmitate rapidly increased cytosolic Ca²⁺ through a combination of Ca²⁺ store release and extracellular Ca²⁺ influx. Palmitate caused a reversible lowering of ER Ca²⁺, measured directly with the fluorescent protein-based ER Ca²⁺ sensor, D1ER. Using another genetically encoded indicator, long-lasting oscillations of cytosolic Ca²⁺ in palmitate-treated cells were observed. The kinetics of pharmacological SERCA inhibition on the β-cell ER stress response were characterized, and the ER calcium sensor PERK was found to be rapidly activated in response to irreversible ER calcium depletion. ER calcium depletion in palmitate-treated cells also induced rapid phosphorylation of PERK, as well as other subsequent downstream ER stress signals. In summary, the effects of the free fatty acid palmitate on pancreatic β-cell Ca²⁺ homeostasis were characterized in this thesis. This study provides the first direct evidence that free fatty acids reduce ER Ca²⁺ and sheds light on pathways involved in β-cell ER stress, lipotoxicity and the pathogenesis of type 2 diabetes.

β-cell dysfunction is a major feature of the development of type 2 diabetes (T2D). Endoplasmic reticulum (ER) stress has been shown to play an important role in β-cell survival and death, and has been shown to be an important factior in the development of diabetes and many other diseases. The unfolded protein response (UPR) is a unique adaptive pathway which can improve cell survival during ER stress through the activation of three ER stress transducers: PERK, IRE1 and ATF6. However, if ER stress remains unresolved, UPR signalling triggers the apoptotic pathway through, for example, expression of the pro-apoptotic protein CHOP, which is a downstream target of ATF4. In addition, eukaryotic initiation factor 5 (eIF5) has been reported to play a role in the regulation of ATF4. Therefore, this thesis aimed at investigating the expression of eIF5, ATF4 (activating transcriptional factor 4) and CHOP (C/EBP homolog protein) in response to ER stress. The studies outlined in this thesis demonstrate that the URP is induced in response to thapsigargin-induced ER stress. In MIN6 cells, the up-regulation of ATF4 and CHOP in response to ER stress is independent of the PERK-eIF2α pathway. However, IRE1 activation is required for the up-regulation of ATF4 and CHOP in response to ER stress in a variety of cell lines. Surprisingly, IRE1-mediated ATF4 and CHOP expression is independent of translation and transcription. In addition, mRNA polyadenylation appears to be required for the up-regulation of ATF4 and CHOP. Preliminary evidence also suggests that inhibition of the IRE1 pathway results in inhibition of miR214 expression. This could lower the miRNA214-mediated up-regulation of ATF4.

Endoplasmic Reticulum Stress (ERS) is one hallmark of cancer cells: tumor hypoxia, glucose reduction and genome instability all promote accumulation of misfolded proteins in the endoplasmic reticulum. ER stress triggers the Unfolded Protein Response (UPR), a conserved pathway initiated by three ER-resident receptors, IRE1α, PERK, and ATF6, that activate specific and overlapping transcriptional programs aimed to overcome the stress or induce cell death. Accumulating evidence suggest a role for UPR in cancer progression, therefore uncovering functional interactions of this pathway with the oncogenic circuits that drive various tumors may be relevant for therapy. The tumor suppressor p53 is one of the most frequently mutated genes in cancer and missense mutant p53 proteins (mutp53) can acquire powerful oncogenic properties. We recently reported that mutant p53 can modulate the UPR, specifically sustaining activation of the ATF6 branch. This molecular axis may contribute to cancer aggressiveness and resistance to therapy. However, the mechanisms by which mutant p53 can modulate the UPR in cancer cells remained unexplored. In this Thesis, I describe one of the possible mechanisms exploited by mutp53 to sustain ATF6. Using breast, prostate and mammary cancer cell lines, I found that mutant p53 enhances ERS-induced activation of the stress kinase p38MAPK. I also found that inhibition of p38MAPK reduces ERS-induced proteolytic cleavage of ATF6 and its transcriptional activity. These data suggest that p38MAPK may have a pro-survival role in the context of ER stress. Indeed, pharmacologic inhibition of p38MAPK increased the sensitivity to Thapsigargin in cancer cells with mutant p53. Regarding the possible action of p38, I measured the turnover of the active ATF6 fragment, and found that inhibition of p38MAPK induced a perceptible reduction in ATF6f stability. Therefore, one mechanism by which mutp53 can reshape the UPR is by increasing the stability of the active ATF6f protein via enhanced activation of p38MAPK.

Autophagy is a major degradation mechanism, responsible for clearing damaged and dysfunctional organelles, including the endoplasmic reticulum, a structure essential for protein synthesis and myocellular hypertrophy. Alterations in autophagy throughout various tissues of the body have been linked to various negative side effects such as decreased myocellular hypertrophy and insulin resistance. High fat diets lead to changes (both increases and decreases) in autophagy in various tissues throughout the body in a tissue-specific manner. Skeletal muscle autophagy is decreased in myotubes cultured from obese women, however the mechanism by which this occurs is unknown. As the largest organ system in the human body, skeletal muscle serves an important role in overall metabolic health. Therefore, sufficient skeletal muscle autophagy is important for proper metabolic function. Moreover, a decrease in liver and pancreas autophagy has been found to lead to endoplasmic reticulum (ER) stress and the development of insulin resistance. Understanding the relationship between autophagy and ER stress in the skeletal muscle following a high fat diet may help elucidate a novel target for decreasing negative side effects. Interestingly, both acute and chronic exercise have been shown to increase skeletal muscle autophagy. This points to a potential therapeutic treatment for those suffering with decreased skeletal muscle autophagy and may help improve ER stress. The purpose of this study was to compare the in vivo and in vitro effects of high fat exposure on skeletal muscle autophagy. Additionally, the relationship of autophagy and ER stress in skeletal muscle was explored. Lastly, this project identified changes in skeletal muscle autophagy and ER stress following cyclic stretch, an in vitro model of exercise in C2C12 myotubes. Eight-week-old C57BL/6J were fed a high fat diet for 16 weeks and tibialis anterior muscle examined for changes in autophagy markers. Gene expression (mRNA content) of autophagy markers Atg3 (p=0.011, fold change 1.37), Atg12 (p=0.026, 1.38), and Atg16L (p=0.004, 1.49) were increased in skeletal muscle of obese mice. Protein content was also measured, where increases in Atg3 (p = 0.04, 1.22), Atg12 (p = 0.027, 1.21), and Atg16L1(p = 0.021, 1.59) were found. However, there was no difference in LC3 II:I ration. No changes were seen in Atg5 or LC3. Additionally, C2C12 myotubes were treated with equimolar palmitate and oleate for 24h then assessed for mRNA content of genes involved in autophagy and ER stress. Autophagy genes Atg5 (p = 0.007, fold change 1.78), Atg12 (p = 0.001, fold change 1.99), and LC3 (p = 0.01, fold change 2.02) were decreased with high fat treatment. Paradoxically, there was an increase in Atg16L (p = 0.005, fold change 1.90). There were no changes in protein content. ER stress was increased indicated by an increase of sXBP1 (p = 0.005, fold change 1.33). Furthermore, inhibition of autophagy lead to changes in ER morphology and ER stress. To identify the impact of cyclic stretch on skeletal muscle autophagy and ER stress, C2C12 myotubes were subjected to 30 minutes of equibaxial stretch and examined for changes in autophagy and ER stress. Autophagy flux, measured by tyrosine release, increased by 34% (p = 0.04) following exercise and ER stress was decreased. In conclusion, this study provides the novel finding that decreased skeletal muscle autophagy is sufficient for inducing ER stress. Additionally, cyclic stretch increases autophagy and improves ER homeostasis.

In this project I sought to analyse the effects of different free fatty acids (FFAs) on INS-1E β-cells. The saturated fatty acid palmitate is considered toxic whereas the monounsaturated fatty acid oleate is harmless. In my working hypothesis I assumed an additional protective effect of oleate when used in combination with palmitate. Furthermore I aimed to explore in detail the possible causes and signalling pathways responsible for apoptosis or sustained cell survival. I examined the Endoplasmic Reticulum (ER) stress response, called unfolded protein response (UPR), as one essential criterion deciding about cell death or life. Analysis of viability and apoptosis confirmed the deleterious effect of palmitate on INS-1E β-cells after 24h of incubation. Oleate proved not to be harmful and even reversed the toxicity of palmitate. When the main components of the UPR were assessed using Western blot analyses and quantitative PCR was performed I found positive proof that palmitate activated the UPR and ultimately led to apoptosis. By contrast, oleate completely prevented UPR signalling. I conclude that oleate rescues INS-1E β-cells by inhibiting ER stress and its signalling.

The endoplasmic reticulum (ER) is the organelle responsible for synthesis and folding of secreted and membranous protein and lipid biosynthesis. It also functions as one of the main cellular calcium stores. Pancreatic beta-cells evolved to produce and secrete insulin upon demand in order to regulate blood glucose homeostasis. In response to increases in serum glucose, insulin synthesis represents nearly 50% of the total protein biosynthesis by beta-cells. This poses an enormous burden on the ER, rendering beta-cells vulnerable to agents that perturb ER function. Alterations of ER homeostasis lead to accumulation of misfolded proteins and activation of an adaptive response named the unfolded protein response (UPR). The UPR is transduced via 3 ER transmembrane proteins, namely PERK, IRE-1 and ATF6. The signaling cascades activated downstream of these proteins: a) induce expression of ER resident chaperones and protein foldases. Increasing the protein folding capacity of the ER; b) attenuate general protein translations which avoids overloading the stressed ER with new proteins; c) upregulate ER-associated degradation (ERAD) genes, which decreases the unfolded protein load of the ER. In severe cases, failure by the UPR to solve the ER stress leads to apoptosis. The mechanisms linking ER stress to apoptosis are still poorly understood, but potential mediators include the transcription factors Chop and ATF3, pro-apoptotic members of the Bcl-2 familly, the caspase 12 and the kinase JNK.

Accumulating evidence suggest that ER stress contributes to beta-cell apoptosis in both type 1 and type 2 diabetes. Type 1 diabetes is characterized by a severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. During this autoimmune assault, beta-cells are exposed to cytokines secreted by the immune cells infiltrating the pancreatic islets. Our group has previously shown that the pro-inflamatory cytokines interleukin-1beta (IL1-beta and interferon-gamma (IFN-gamma), via nitric oxide (NO) formation, downregulate expression and function of the ER Ca2+ pump SERCA2. This depletes beta-cell ER Ca2+ stores, leading to ER stress and apoptosis. Of note, IL1-beta alone triggers ER stress but does not induce beta-cell death, while IFN-gamma neither causes ER stress nor induces beta-cell death. Together, these cytokines cause beta-cell apoptosis but the mechanisms behind this synergistic effect were unknown.

Type 2 diabetes is characterized by both peripheral resistance to insulin, usually as a result of obesity, and deficient insulin secretion secondary to beta cell failure. Obese patients have high levels of circulating free fatty acids (FFA) and several studies have shown that the FFA palmitate induces ER stress and beta-cell apoptosis.

In the present work we initially established an experimental model to specifically activate the ER stress response in pancreatic beta-cells. For this purpose, insulinoma cells (INS-1E) or primary rat beta-cells were exposed to the reversible chemical SERCA pump blocker cyclopiazonic acid (CPA). Dose-response and time course experiments determined the best conditions to induce a marked ER stress without excessive cell death (<25%).

The first goal of the work was to understand the synergistic effects of IL1-beta and IFN-gamma leading to pancreatic beta-cell apoptosis. Our group previously observed, by microarray analysis of primary beta-cells, that IFN-gamma down-regulates mRNAs encoding for some ER chaperones. Against this background, our hypothesis was that IFN-gamma aggravates beta-cell ER stress by decreasing the ability of these cells to mount an adequate UPR. To test this hypothesis, we investigated whether IFN-gamma pre-treatment augments CPA-induced ER stress and beta cell death. The results obtained indicated that IFN-gamma pre-treatment potentiates CPA-induced apoptosis in INS-1E and primary beta-cells. This effect was specific for IFN-gamma since neither IL1-beta nor a low dose CPA pre-treatment potentiated CPA-induced apoptosis in INS-1E cells. These effects of IFN-gamma were mediated via the down regulation of genes involved in beta cell defense against ER stress, including the ER chaperones BiP, Orp150 and Grp94 as well as Sec61, a component of the ERAD pathway. This had functional consequences as evidenced by a decreased basal and CPA-induced activity of a reporter construct for the unfolded protein response element (UPRE) and augmented expression of the pro-apoptotic transcription factor Chop.

We next investigated the molecular regulation of the Chop gene in INS-1E cells in response to several pro-apoptotic and ER stress inducing agents, namely cytokines (IL1-beta and IFN-gamma), palmitate, or CPA. Detailed mutagenesis studies of the Chop promoter showed differential regulation of Chop transcription by these compounds. While cytokines (via NO production)- and palmitate-induced Chop expression was mediated via a C/EBP-ATF composite and AP-1 binding sites, CPA induction required the C/EBP-ATF site and the ER stress response element (ERSE). Cytokines, palmitate and CPA induced ATF4 protein expression and further binding to the C/EBP-ATF composite site, as shown by Western blot and EMSA experiments. There was also formation of distinct AP-1 dimers and binding to the AP-1 site after exposure to cytokines or palmitate.

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Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Endoplasmic Reticulum (ER) stress signal is a cellular response to various insults including abnormal protein folding load, activating the unfolded protein response. Under severe ER stress, apoptosis will occur in most cell types. Interestingly, this does not happen in a disease model for Metaphyseal chondrodysplasia type Schmid (MCDS), where ER stress was activated in the hypertrophic zone of the growth plate where mutant collagen X proteins that cannot be folded correctly is expressed. Instead of normal progression from proliferating chondrocytes (PCs) to hypertrophic chondrocytes (HCs) and conversion to bone, HCs in MCDS mice undergo re-differentiation to PCs as a survival strategy due to an activation of ER stress. Transcription factors are known to be important in regulating differentiation. p53 family members, as transcription factors, are known to play important roles in developmental processes including cellular reprogramming, thus, we hypothesize that the ectopic expression of key transcription factors, p53 and TAp63, which are activated by ER stress is involved in HC re-differentiation. p53 is normally expressed in late PCs, Pre-HCs, and upper HCs, while TAp63 is expressed in PCs and Pre-HCs suggesting they may have roles in chondrocyte differentiation. p53 activated under ER stress in HCs are nuclear localized in MCDS mice, but did not invoke the apoptotic programme. In this project, using quantitative analyse to study the expression level of p53 and p63 isoforms, it was confirmed that p53 and TAp63γ are in part transcriptionally activated upon ER stress. From functional study by inactivating p53 in MCDS mice, it was shown that p53 alone was not sufficient to mediate re-differentiation. Given that TAp63γ isoforms is also highly upregulated upon ER stress, and the negative regulator, ΔNp63, is downregulated, this combination of change in gene expression also need to be considered. Furthermore, known regulators of p53 and p63 activity such as ASPP1 and iASPP are also differentially expressed in HCs, and are altered upon activation of ER stress favouring cell survival. Thus, it would be important to evaluate the combination of TAp63 in the re-differentiation process from conditional inactivation of p63 or in combination with p53 to gain a clearer understanding of the contribution and relationship of these transcription factors in the survival strategy of stressed HCs.
published_or_final_version
Biochemistry
Master
Master of Philosophy

The objective of an effective therapy against cancer is to destroy every single cancerous cell, including cancer stem cells, in order to allow the survival of the patient. Some anti-cancer therapies tend to “educate” the host’s immune system to recognize the remaining tumour cells, in order to minimize the risk of relapse. Chemotherapy and immunotherapy are often hardly compatible. In fact, most of the chemotherapeutic agents induce DNA damage, in order to trigger apoptosis of the cancer cells. These treatments often result into the induction of massive immune system effector’s depletion. Etoposide and mitomycin C, two widely used drugs, induce non-immunogenic cell death while, other compounds, such as anthracyclines, are able to induce immunogenic cell death. In addition, there are reports suggesting that anthracyclines are able to induce immunogenic apoptosis, have a direct effect on cancer and also they are able to mediate the immune response. It has been demonstrated that those compounds are able to stimulate the exposure of Calreticulin (CRT) on the plasma membrane of tumour cells in a pre-apoptotic step. One of the peculiar biochemical aspects of the immunogenic cell death is this pre-apoptotic translocation of CRT from the Endoplasmic reticulum (endo-CRT) to the cell surface (ecto-CRT). Calreticulin is a Ca2+-binding molecular chaperone expressed in the endoplasmic reticulum, where it takes part in calcium homeostasis. It can be found also in other cellular compartments, such as the nucleus and the plasma membrane, and it is a multi functional protein able to interact with the α-subunit of integrins and with proteins involved in the ER-stress response, such as PDI and ERp57. In addition, it has been shown that CRT expression on the surface of damaged cells might function as an “eat-me” signal, which elicits cellular recognition and removal by macrophages or dendritic cell. Type 2 Transglutaminase (TG2) belongs to a family of Ca2+-dependent enzymes capable of covalently modifying proteins by cross-linking them via the formation of ε(γ-glutamyl)lysine bonds. TG2 is a peculiar member of the family, which could be externalized on the cell surface and thus it mediates the interaction of integrins with fibronectin and cross-links proteins of the extra-cellular matrix. On the basis of its sub-cellular localisation TG2 may also act as a protein disulphide isomerase (PDI) at mitochondrial level or as a G-protein at plasma membrane level. In fact, it has been shown that α-subunit of the etherotrimeric G-proteins is TG2. Interestingly, it has been have shown that the β-subunit of these G-proteins is CRT. CRT interacts with TG2, when TG2 bounds GDP. We hypothesised that TG2-CRT interaction might modulate not only their intra-cellular activities but also their relationships with the plasma-membrane and, possibly, CRT exposition on cell surface. In order to address our hypothesis we assessed the presence of CRT on the plasma membrane of the human neuroblastoma cell line Sk-n-BE(2), which does not express detectable levels of TG2, in respect to the TGA cell line, transfected to achieve high TG2 expression levels. We used different approaches, such as flow cytometry, surface protein purification, to analyse cell surface exposition of CRT in these two cell lines. Our results indicate that, at steady state, the SK-n-BE(2) cell line express about 2 fold more CRT on cell surface, as compared to TGA cells, thus suggesting a role for TG2 in CRT exposure. In addition, we showed that pre-treatment of the cells with cystamnine, a pan inhibitor of TG2 transamidasic activity, lowered the amount of cell surface exposed CRT. On the basis of these results we hypothesised that TG2, when bound to GDP and acting as a G-protein, might bind CRT and prevent its translocation on cell surface. TG2-dependent modulation of CRT translocation is detectable even upon anthracyclines treatment. In fact, when we analysed the presence of CRT on cell surface after doxorubicin treatment, we spotted the same differences detected at steady state. This aspect might be of relevance, as doxorubicin not only induces high level of CRT exposure but also has been show to induce apoptosis and that TG2 might be involved in the modulation of this process (Fesus and Szondy, 2005). It is know that anthracyclines induce ER-stress. On the basis of this evidence we would like to assess whether the various TG2’s biological activities could be involved in cell death regulation cancer. During the last years, many groups demonstrated that TG2 can not only carry out different activities in relation to its sub-cellular localisation (Jones et al., 1997; Malorni et al., 2009; Rodolfo et al., 2004; Siegel et al., 2008) but also that different localisation of the enzyme may result in both pro-survival or pro-death action (Fesus and Szondy, 2005). We previously showed that TG2 may localise on mitochondria, where it’s involved in the maintenance of the organelle’s homeostasis and in the regulation of the mitochondrial pathway of apoptosis. Mitochondria have also a prominent role in the decoying and integration of cellular stress signals, such as DNA damage and ER-stress, and anthracycline, like other chemotherapeutic drugs, are not only DNA damaging agents but also powerful induces of ER-stress. This side of the picture might be very relevant to our project as CRT is an ER resident protein, involved in the activation of the ER-stress response. In addition, during metastasis formation, tumour’s cells are subjected to high levels of stress that involves both ER and mitochondria. We then decided to investigate the possible involvement of TG2 in the overall regulation of ER-stress induced cell death, in our neuroblastoma cell model, by treating cells with two different compounds: tunicamycin and thapsigargin. The choice of these drugs has been dictated by their different modes of action. In fact thapsigargin, is a specific inhibitor of SERCA ATPase (Sarco/endoplasmic reticulum Ca2+ ATPase) and causes the depletion of Ca2+ stores from the endoplasmic reticulum and the increase of the cytosolic Ca2+ concentration. Tunicamycin is an inhibitor of one of major post-translational modifications that take place in the endoplasmic reticulum, the protein’s glycosylation process. We started our investigation by performing time and dose response curve, for the two drugs, and analysing cell death induction by means of flow cytometry. After treatment with 4 and 8 µg/ml of tunicamycin for 24 and 48h, the TGA cell line seems to be less sensitive to cell death induction as compared to the SK-n-BE(2) cell line, thus suggesting TG2 over-expression as protective against tunicamycin induced cell death. Treatments were also carried out with thapsigargin at 2 and 4 µg/ml for the same times. After 24h of treatment there are no differences in cell death levels between the two cell lines for both the doses used while, after 48 h of treatment, there is a clear protection of TGA cells at the highest dose we used. These data suggest TG2 over-expression as protective against thapsigargin induced cell death, and highlighted a dose-dependent effect. The results obtained with thapsigargin were somehow un-expected because thapsigargin causes an increase in the cytosolic calcium concentration, that we hypothesised to be high enough to activate the TG2 transamidasic activity and then to induce apoptosis. We then decided to investigate whether the two cell lines displayed a different response to the ER-stress. To this aim we performed western blot analysis of the expression of ER-stress induced factors, like GRP78 protein, and of cell death markers, like PARP. The results obtained demonstrated that there were no differences between the two cell lines despite the inducer used. As we do not see any difference in the upstream regulation of cellular response to ER-stress, we decided to investigate whether there was any difference in the induction of cell death. It is well known that the ER-stress dependent induction of apoptosis relies on the activation of the pro-apoptotic members of the Bcl-2 family Bax and Bak, and on their action on mitochondrial and ER membranes. In addition, the Ca2+ release induced by Thapsigargin might have an effect on both TG2 activation and mitochondrial release of cytochrome c. In keeping with these considerations, we decided to analyse what happens in the two cell lines after treatment with 4 µg/ml of thapsigargin for 48 hours, by means of an immuno-fluorescence approach. The results obtained showed that cytochrome c release and caspase-3 activation seem to happen at the same extent in the two cell lines. The only difference that we might spot is on the number of fragmented nuclei we observed, again confirming a protection of the TGA cells. Besides these observation it remains still unclear how TG2 over-expression might protect cells from thapsigargin induced cell death. The data obtained by this kind of approach were purely descriptive and not quantitative, so we decided to investigate these events by means of western blot analysis. To this aim we treated the cells in the same way as before and we performed sub-cellular fractionation in order to obtain cytosolic and mitochondrial fraction. The western blot analysis of PARP cleavage, a marker of cell death, revealed a more extensive processing of this protein in the SK-n-BE(2) cell line, in respect to the TGA. This result indicates a more proneness of these cells to cell death induction after thapsigargin treatment, in keeping with what we previously observed by FACS analysis. The analysis of Bax and Bak activation and translocation as well as of the cytochrome c release, does not revealed, in the two cell lines, such striking differences to justify the less sensitivity of the TGA cell line to cell death. On the contrary, the TGA cell lines showed an even marked translocation of Bax and Bak to the mitochondria, suggesting a massive induction of cell death. These results indicates that, upon thapsigargin treatment, the onset of the ER-stress response and the induction of cell death takes places in a similar way in the two cell lines and they does not justify the minor sensitivity displayed by the TGA cell lines. Recently, in vitro experiments have demonstrated that caspase-3 might be a protein substrate for the cross-linking activity of TG2. Following thapsigargin treatment, the increase of the cytosolic Ca2+ concentration activates TG2, which might act on caspase-3 leading to the formation of polymers with a molecular weight of about 64 kDa. This polymerisation inactivates the caspase and leads to the inhibition of cell death. In order to verify whether this hypothesis was true in our model system, we performed a western blot analysis of caspase-3 activation, after thapsigargin treatment, by means of an antibody able to recognize the processed forms of caspase-3. After 48 hours of treatment with 4 µg/ml of Thapsigargin, we observed the decrease of the 32 kDa signal, corresponding to the pro-caspase, in both cell line at the same extent. When we checked for the appearance of the two active forms of caspase-3 at 17 and 10 kDa, we could detect their appearance only in the SK-n-BE(2) cell line. On the other hand the TGA cell line show the appearance of a faint signal corresponding to the 10 kDa active form of caspase-3, but we detected the appearance of a strong signal at a molecular mass of about 34 kDa, as well as the increase of the signal at about 27 kDa. The molecular mass of these anti-caspase 3 positive bands suggests that they are polymers of the processed caspase-3 forms and that they are dependent on the thapsigargin induced activation of the TG2 cross-linking activity, even if slightly visible also in the SK-n-BE(2) cell line. These data are also supported by the measurement of the TG2 activity we performed upon thapsigargin treatment. In fact, upon thapsigargin treatment, we detected TG2 cross-linking activation in both cell lines even if more pronounced in the TGA cell line. The more evident formation of the caspase-3 oligomers, as well as the quite complete disappearence of the signal corresponding to the 17 kDa form, in the TGA cell line could explain the reduced sensitivity to cell death induction displayed by these cells. As a final verification we checked out if also the tunicamycin treatment might induce this polymerisation of the caspase-3 and then explain the slight protection observed in the TGA cell line. Even in this case we detected the polymerised form of caspase-3 in TGA cells only but at a minor extent in respect to the thapsigargin treatment. The data we obtained support the hypotesis that during ER-stress, the increase in cytosolic Ca2+ concentration is able to activate TG2as a cross-linking enzyme, which in turn acts on casp-3. The polymerisation of casp-3 inhibits its proteolytic activity thus protecting TG2 over-expressing cells from apoptosis.

Pulmonary arterial hypertension (PAH), a common complication of limited cutaneous systemic sclerosis (lcSSc), is associated with alterations of markers of inflammation and vascular damage. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) have been implicated in various diseases. The presence of the HLA-B35 allele, Human antigen class I, has emerged as an important risk factor for the development of PAH in patients with lcSSc, however the mechanisms underlying this association have not been fully elucidated. We have recently reported that the presence of HLA-B35 contributes to human dermal microvascular endothelial cell (HDMEC) dysfunction by significantly increasing production of endothelin-1 (ET-1) and significantly decreasing endothelial NO synthase (eNOS). Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the ER stress and UPR in ECs and this mechanism can mediate the induction of ET-1 in patients with PAH. The goal of this study was to better understand the role of HLA-B35-induced ER stress/UPR in the development/progression of PAH disease in lcSSc patients. First we focused on the molecular mechanisms of ET-1 induction by HLA-B35. ER stress inducer, Thapsigargin (TG) and HLA-B35 induced ET-1 expression with similar potency in HDMECs. HLA-B35 or ER stress activated the PERK/eIF2α/ATF4 branch of the UPR and modestly increased the spliced variant of X-box binding protein (XBP1), but did not affect the Activating Transcription Factor -6 (ATF6) pathways. Depletion of ATF4 decreased basal expression levels of ET-1 mRNA and protein, and completely prevented upregulation of ET-1 by HLA-B35/ER stress. Additional experiments have demonstrated that the JNK and NF-B pathways are also required for ET-1 upregulation by HLA-B35/ER stress. Formation of the ATF4/c-JUN complex, but not the ATF4/NF-B complex was also increased. The functional role of c-JUN in responses to HLA-B35/ER stress was further confirmed in ET-1 promoter assays. This study identified ATF4 as a novel activator of the ET-1 gene. Then we focus on whether markers of ER stress/UPR were present in PBMCs from lcSSc-PAH patients and if the presence of HLA-B35 contributes to activation of the immune cells. Several ER stress/UPR genes, including Immunoglobulin-heavy-chain binding protein (BiP), ATF4 and ATF6 and a spliced form of XBP1 were upregulated in lcSSc PBMCs, with the highest levels in patients with PAH. Also selected HSP genes, particularly DNAJB1, and IFN-related genes were found at significantly elevated levels in PBMCs from lcSSc patients, while IRF4 was significantly decreased. There was a positive correlation between DNAJB1 and severity of PAH disease (PAP) (r = 0.56, p<0.05) and between ER stress markers and IL-6 levels (r = 0.53, p< 0.0001) in lcSSc PBMCs. When we stratified all PBMC samples based on the presence of the HLA-B35 allele, we could observe that HLA-B35 positive individuals showed higher levels of selected ER stress markers when compared to HLA-B35 negative individuals. Furthermore, patients carrying HLA-B35 antigen expressed higher levels of IL-6, a key inflammatory cytokine associated with development of PAH. This study demonstrates association between select ER stress/UPR markers and lcSSc-PAH suggesting that ER stress/UPR may contribute to the altered function of circulating immune cells in lcSSc. All these associations were enhanced by the presence of HLA-B35. In conclusion, we hypothesize that HLA-B35 may play a role in EC dysfunction inducing ET1 via ER stress/UPR. Also activation of ER stress/UPR, in combination with presence of HLA-B35, might drive the inflammatory process in lcSSc-PAH.

MCDS is an autosomal dominant skeletal dysplasia disorder caused by mutations in collagen X. In most cases, mutations in collagen X result in a misfolded protein which is retained within the ER of hypertrophic chondrocytes, causing increased ER stress. It has previously been demonstrated that increased ER stress causes hypertrophic chondrocytes to de-differentiate in an attempt to avoid the stress. The altered differentiation results in reduced cell hypertrophy and impaired vascular invasion accounting for reduced bone growth. The presence of increased ER stress in hypertrophic chondrocytes is sufficient to cause the MCDS pathology; therefore reducing ER stress may be beneficial in terms of improving the associated pathology. The autophagy enhancing drug carbamazepine (CBZ) has been shown to be capable of reducing ER stress in cells expressing the MCDS-causing p.N617K collagen X mutation. I show in this thesis that CBZ treatment reduced ER stress in HeLa cells transiently expressing a further 3 MCDS-causing collagen X mutations. I have also demonstrated that CBZ treatment induced the degradation of mutant collagen X proteins either through autophagy or proteasomal degradation depending on the nature of the mutation. The drug was tested in vivo using the p.N617K collagen X mouse model of MCDS. In MCDS mice, CBZ reduced the severity of the disease pathology based on histological analyses, restored hypertrophic chondrocyte differentiation toward normal, increased long bone growth rates and decreased the severity of the hip dysplasia. Gene expression analyses on RNA isolated from microdissected hypertrophic chondrocytes revealed that CBZ shifted the pattern of hypertrophic differentiation markers in MCDS mice toward the wild-type pattern, most likely through its stimulation of gene expression associated with intracellular proteolytic pathways. The results presented in this thesis have contributed to the identification of a potential treatment strategy for MCDS- the stimulation of intracellular proteolysis of mutant collagen X. CBZ is FDA approved for the use of epilepsy and bipolar disorder and has a strong safety record in humans. Therefore CBZ could be a potential treatment strategy for MCDS.

Carnitine palmitoyltransferase 1C (CPT1C) is the brain-specific isoform of the CPT1 family which is located at the endoplasmic reticulum (ER) of neurons and exhibits low catalytic activity, but still maintains the capacity to bind the metabolic intermediary malonyl-CoA (which levels highly fluctuate depending on the energetic status). CPT1C controls spine maturation and spatial learning, mainly through regulating synthesis and trafficking of the major AMPA receptor (AMPAR) subunit: GluA1. AMPARs mediate fast excitatory neurotransmission in the brain, and play a key role in synaptic plasticity. Some authors proposed CPT1C as a malonyl-CoA sensor, though whether this sensing is involved in AMPAR trafficking remains unknown. In the current PhD project, GluA1 surface expression was examined in cortical neurons under different metabolic stresses known to affect intracellular malonyl-CoA levels, such as glucose starvation. Moreover, CPT1C is known to interact with the phosphatidyl-inositol-4-phosphate (PI(4)P) phosphatase SAC1, which regulates vesicular transport, including GluA1 transport, by modulating the PI(4)P pool at the trans Golgi network (TGN). For that, the putative role of CPT1C in regulating SAC1 functionality under energetic stress was also evaluated. The results obtained in this thesis demonstrate that CPT1C is able to sense malonyl-CoA and consequently modulate GluA1 trafficking through SAC1. Under basal conditions, CPT1C downregulated SAC1 activity, which was necessary for proper GluA1 trafficking. Under low malonyl-CoA levels, CPT1C favored SAC1 translocation to the ER-TGN contact sites and released its inhibition on SAC1, which decreased the Golgi PI(4)P pool and caused the retention of GluA1 at TGN. This PhD study reveals that GluA1 trafficking is regulated by CPT1C sensing of malonyl-CoA and describes the first inhibitor of SAC1 activity, which shed light on how nutrients and energy metabolism can affect synaptic function and cognition.

Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia. The pathogenesis of these diseases involves β-cell dysfunction and death. The primary function of β-cells is to tightly regulate the secretion, production, and storage of insulin in response to blood glucose levels. In order to manage insulin biosynthesis, β-cells have an elaborate endoplasmic reticulum (ER). The ER is an essential organelle for the proper processing and folding of proteins such as proinsulin. Proteins fold properly when the ER protein load balances with the ER folding capacity that handles this load. Disruption of this ER homeostasis by genetic and environmental stimuli leads to an accumulation of misfolded and unfolded proteins, a condition known as ER stress. Upon ER stress, the unfolded protein response (UPR) is activated. The UPR is a signaling network that aims to alleviate ER stress and restore ER homeostasis promoting cell survival. Hence, the UPR allows β-cells to handle the physiological fluctuations of insulin demand. However upon severe unresolvable ER stress conditions such as during diabetes progression, the UPR switches to pathological outputs leading to β-cell dysfunction and apoptosis. Severe ER stress may also trigger inflammation and accumulating evidence suggests that inflammation also contributes to β-cell failure, but the mechanisms remain elusive. In this dissertation, we demonstrate that thioredoxin interacting protein (TXNIP) mediates ER stress induced β-cell inflammation and apoptosis. During a DNA microarray analysis to identify novel survival and death components of the UPR, we identified TXNIP as an interesting proapoptotic candidate as it has been linked to glucotoxicity in β-cells. During our detailed investigation, we discovered that TXNIP is selectively expressed in β-cells of the pancreas and is strongly induced by ER stress through the IRE1α and PERK-eIF2α arms of the UPR and specifically its transcription is regulated by activating transcription factor 5 (ATF5) and carbohydrate response element binding protein (ChREBP) transcription factors. As TXNIP has been shown to activate the Nod-like receptor protein 3 (NLRP3) inflammasome leading to the production of the inflammatory cytokine interleukin-1β (IL- 1β), we hypothesized that perhaps TXNIP has a role in IL-1β production under ER stress. We show that ER stress can induce IL-1β production and that IL-1β is capable of binding to IL-1 type 1 receptor (IL-1R1) on the surface of β-cells stimulating its own expression. More importantly, we demonstrate that TXNIP does indeed play a role in ER stress mediated IL-1β production through the NLRP3 inflammasome. Furthermore, we also confirmed that TXNIP is a mediator of β-cell apoptosis under ER stress partially through IL-1β signaling. Collectively, we provide significant novel findings that TXNIP is a component of the UPR, mediates IL-1β production and autostimulation, and induces cell death under ER stress in β-cells. It is becoming clear that TXNIP has a role in the pathogenesis of diabetes and is a link between ER stress, oxidative stress and inflammation. Understanding the molecular mechanisms involved in TXNIP expression, activity, and function as we do here will shed light on potential therapeutic strategies to tackle diabetes.