Baigiamojo darbo tikslai – nustatyti neefektyvios žmogaus virusų glikobaltymų raiškos mielėse priežastis ir sukurti mielių kamienus su integruotais žmogaus ląstelių sekrecinio kelio genais bei ištirti jų įtaką glikobaltymų sintezei ir brendimui mielėse. Darbo eigoje pirmą kartą buvo aprašytos tymų viruso hemagliutinino (TVH) sintezės galimybės mielėse Saccharomyces cerevisiae ir Pichia pastoris. Parodyta, kad mielių ko-transliacinio baltymų perkėlimo į endoplazminį tinklą (ET) ir ET baltymų sulankstymo mechanizmai nėra pritaikyti sudėtingų virusinių baltymų brendimui, todėl klasikinės mielių rūšys ir standartiniai rekombinantinių baltymų raiškos ir gryninimo protokolai nėra tinkami diagnostikai ir vakcinų kūrimui reikalingo TVH baltymo gavimui. Proteominė S. cerevisiae ląstelių, sintetinančių TVH baltymą, analizė leido nustatyti kad, TVH sintezė mielėse sukelia neseniai literatūroje aprašytą citoplazminį nesusivyniojusių baltymų atsaką (UPR-cyto). Pagrindinis šiame darbe aprašyto atsako į stresą požymis yra ypatingai didelių baltymų agregatų, kurių šerdį sudaro TVH ir mielių eEF1A baltymai, susidarymas. Žmogaus tipo ko-transliacinį baltymų pernešimą į ET mielių ląstelėse bandyta atkurti perkeliant žmogaus SRP, Sec61 kompleksų ir TRAM1 baltymus, o siekiant sukurti tinkamas TVH baltymo brendimui sąlygas, mielių ląstelių ET buvo sintetinami pagrindiniai žmogaus ląstelių ET šaperonai – BiP, kalretikulinas, kalneksinas, PDI ir ERp57. Nors šiame darbe nepavyko sukurti mielių... [toliau žr. visą tekstą] / The aims of the study were to determine the reasons for unsuccessful expression of measles virus hemaglutinin (MeH) in the yeast cells and to generate a stable yeast strains with integrated genes of protein secretory pathway of human cells and to examine influence of coded human proteins on MeH maturation. For the firs time, overexpression of MeH in yeast S. cerevisiae and P. pastoris was described. It was demonstrated that mechanisms of cotranslational translocation into the endoplasmic reticulum (ER) and protein maturation in the ER of yeast cells are not adapted to deal with for such complex virus glycoproteins. Proteomic analysis revealed, that overexpression of human virus surface protein precursors induces cytosolic unfolded protein response (UPR-cyto) in the yeast S. cerevisiae. A key feature of this response is the formation of extremely large aggregates involving macromolecular structures of eEF1A. Efficient mammalian like cotranslational translocation pathway was attempted to reconstitute in yeast cells by transferring human SRP, Sec61 complexes and TRAM1 protein. Human chaperones BiP, clanexin, calreticulin, ERp57 and PDI were transferred to the yeast cells to create suitable environment for maturation of MeH in the ER. Even though yeast strains, able to produce biologically active MeH protein, were not generated during this study, results show, that humanization of yeast secretory pathway, designed for producing active virus glycoproteins, is possible.

Plasmodium falciparum is responsible for the most virulent form of human malaria. The biology of the intra-erythrocytic stage of P. falciparum is the most well studied as it is this stage that marks the clinical manifestation of malaria. To establish a successful infection, P. falciparum brings about extensive remodeling of erythrocytes, its host compartment. The infected erythrocytes harbor several parasite induced membranous structures. Most importantly, pathogenesis related structures termed knobs, which impart cytoadherence, appear on the cell surface of the infected erythrocytes. For bringing about such eccentric renovations in its host compartment, the parasite exports 8% of its genome (~400 proteins) to various destinations in the host cell. Studies from our lab have shown that proteins belonging to heat shock protein40 (Hsp40) and heat shock protein70 (Hsp70) group of chaperones are also exported to the host compartment. We and others have implicated these chaperones in important processes such as protein trafficking and chaperoning assembly of parasitic proteins into the cytoadherent knobs. As detailed above, malaria parasite invests a lot of energy in exporting a large number of proteins including chaperones in the red blood cell to meet its pathogenic demands. In order to do so, it heavily relies on its secretory pathway. However, it is known that the parasite experiences a significant amount of oxidative stress on account of heme detoxification, its own metabolism and the immune system of the host. The parasite also effluxes large quantities of reduced thiols such as glutathione and homocysteine into the extracellular milieu indicative of redox perturbation. Additionally, the parasite lacks Peroxiredoxin IV, which otherwise localizes in the ER and carries out detoxification of peroxide generated as a result of oxidative protein folding. Together, these factors indicate that maintaining redox homeostasis is a challenging task for the parasite. It also implies that the ER, where the redox balance is even more critical as it requires oxidising environment for protein folding, is predisposed to stress. In light of this fact and the importance of secretory pathway in malaria pathogenesis, we decided to address the ways and mechanisms used by the parasite to tackle perturbations in its secretory pathway. Examination of a canonical unfolded protein response pathway in P. falciparum ER-stress is a condition arising whenever the load of unfolded proteins increases the folding capacity of the ER. However, eukaryotes have evolved a fairly well conserved homeostatic response pathway known as unfolded protein response (UPR) to tackle ER-stress. This signal transduction pathway is composed of three arms involving three ER-transmembrane signal transducers namely; IRE1, ATF6 and PERK. IRE1 brings about splicing of a bZIP transcription factor, XBP1/Hac1 and ATF6 becomes activated upon getting proteolytically cleaved in the Golgi. These transcription factors then migrate to the nucleus where they bind onto the ER-stress elements thereby, leading to the transcriptional up-regulation of the UPR targets such as ER chaperones and components of ER associated degradation (ERAD) pathway which rescue the function of the ER. PERK on the other hand brings about translational attenuation by phosphorylating eIF2α, thereby providing parasite the benefit of time to recover. We started our examination on UPR in Plasmodium by carrying out in silico analysis of the major components of UPR in the parasite by using Homo sapiens protein sequences as the query. We found that the parasite lacks the homologues of all the transcriptional regulators of canonical UPR. Only PERK component of the UPR was found to be present in the parasite. To rule out the existence of the canonical UPR in P. falciparum, we examined the status of UPR targets by subjecting the parasites to treatment with DTT. DTT perturbs the disulfide oxidation in the ER and thereby inhibits protein folding leading to ER-stress. Owing to the missing components of a canonical UPR, we did not find up-regulation of known UPR targets such as ER-chaperones including PfBiP, PfGrp94, PfPDI and ERAD marker Derlin1 at transcript as well as protein level. Owing to the presence of a PERK homologue, phosphorylation of eIF2α followed by attenuation of protein synthesis was observed upon subjecting the parasites to DTT mediated ER-stress. In the absence of a canonical UPR, the parasites were found to be hypersensitive to ER-stress in comparison to the mammalian counterpart. In the presence of DTT, the parasites showed perturbation in the redox homeostasis as indicated by increase in the levels of ROS. Next, we sought to examine if the parasites resorted to any alternate means of increasing the availability of chaperones in the ER. For this, we analysed the involvement of another Hsp70 family member, Hsp70-x which is homologous to BiP and which is known to traverse the ER while getting exported to the erythrocyte compartment. Interestingly, we found that upon exposure to ER-stress, the export of this protein is partially blocked and around 30% of the protein is retained in the ER. On the other hand, there was no effect on the trafficking of another exported chaperone KAHsp40. This indicates that the parasite possibly recruits this pool of retained Hsp70-x for the chaperoning of unfolded proteins in the ER. Global response to ER-stress in P. falciparum To dig deeper into the parasite specific strategies employed for dealing with ER-stress at a global level, we carried out high throughput transcriptomic and proteomic analysis upon subjecting the parasites to DTT mediated ER-stress. Microarray based gene expression profiling was carried out upon subjecting the parasites to DTT mediated ER-stress. We found that the parasite mounts a transcriptional response as indicated by up-regulation of 155 transcripts. In congruence with our biochemical analysis, we did not find up-regulation of ER chaperones as well as ERAD proteins. Functional grouping of the up-regulated genes revealed large number of hypothetical proteins in our list of differentially expressed genes. The genes encoding exported proteins represent yet another abundant class. In the course of examining the involvement of Plasmodium specific transcriptional regulators mediating response to DTT induced ER-stress, we identified 4 genes belonging to the family of AP2 transcription factors. AP2 (Apetela-2) are specific transcription factors which are possessed by apicomplexa and bring about regulation of developmental processes and stress response in plants. On comparing our list of up-regulated genes with the previously known targets of AP2 factors, we found that an entire cascade of AP2 factors is up-regulated upon DTT-mediated ER stress. Thus, AP2 factors appear to be the major stress response mediators as they are together responsible for the up-regulation of 60% of genes identified in this study. In addition, another striking observation made, was the up-regulation of a few sexual stage specific transcripts. 2D Gel electrophoresis and 2D-DIGE based Proteomic analysis indicated an up-regulation of secretory proteins and some components of vesicular trafficking and secretory machinery possibly to overcome the block in the functions of the secretory pathway. ER-stress triggers stage transition in P. falciparum Intrigued by the up-regulation of a few sexual stage specific genes, we were curious to examine if there was a functional significance of this observation. To this end, we decided to investigate the effect of ER-stress on induction of gametocytes, the only sexual stage found in humans. Indeed, we found a two fold induction in the numbers of gametocytes formed upon challenging the parasite with DTT mediated ER-stress. The induction of gametocytogenesis was also observed by using a clinical isolate of P. falciparum for the assay. The DTT treated cultures progressed through the gametocytogenesis pathway normally forming all the five morphologically distinct stages. Then we sought to examine if this phenomenon could be simulated in the physiological scenario as well. For this, we made use of a rodent model of malaria, P. berghei. Two different treatment regimes involving 1) direct injection of increasing concentration of DTT into P. berghei infected mice and 2) injection of DTT pretreated P. berghei infected erythrocytes into healthy mice were followed. In both cases, a significant increase in the gametocyte induction was observed. Having seen that Plasmodium undergoes gametocytogenesis upon exposure to ER-stress not only in in vitro cultures but also in in vivo scenario, we wanted to identify the players involved in the commitment to sexual stage. Recently, a transcription factor belonging to AP2 class of transcription factors, referred to as AP2-G has been implicated in committing the asexual parasites for transition to gametocyte stage. To examine the role of this factor in the phenotype observed by us, we looked at the effect of DTT on AP2-G. Interestingly, we found around 6 folds up-regulation in the expression of AP2-G levels under ER-stress. The downstream targets of AP2-G, many of which are the markers of gametocyte were also found to be up-regulated upon being exposed to DTT mediated ER-stress indicating the launch of a transcriptional program which together works in the direction of transition to gametocytes. Having seen that P. falciparum undergoes ametocytogenesis in response to DTT treatment both under in vitro and in vivo conditions, we sought to look for probable physiological analogue of DTT. Since glutathione is the major cellular redox buffer, critical for redox homeostasis, we quantitated the levels of both oxidized and reduced forms of this non protein thiol using Mass Spectrometric approach. We found that the levels of reduced forms of glutathione significantly increased upon treating the parasites with DTT. This indicates that the levels of glutathione could be one of the physiological triggers of gametocytogenesis. Conclusion In conclusion, our study analyses the ways and mechanisms employed by malaria parasite to cope with perturbations to its secretory pathway. We have established the absence of a canonical UPR in this parasite and our results suggest that Plasmodium has developed a three stage response to cope with ER stress: 1) an early adaptation to increase the local concentration of chaperones in the ER by partially blocking the export of a Hsp70 family member, 2) activation of gene expression cascade involving AP2 transcription factors and 3) a consequent switch to the transmissible sexual stage. Hence, our study throws light on a novel physiological adaptation utilised by malaria parasite to tackle stress to its secretory pathway. Gametocytogenesis, which can be transmitted to the mosquito vector, could hence serve as an effective means to escape ER-stress altogether. Importantly, while it is widely known that stress brings about switch towards sexual stages in P. falciparum, the molecular triggers involved in this process remain obscure in the field of malaria biology. Therefore, our findings also address this long standing question by providing the evidence of ER-stress being one such trigger required for switching to the transmissible sexual stages.

Le rein natif et le greffon rénal peuvent être soumis à de multiples agressions conduisant à la détérioration progressive du parenchyme. Ces agressions peuvent être spécifiques (stress toxique, immunologique) et/ou non spécifiques (stress ischémique) et vont engendrer des réponses pouvant entraîner à la fois une diminution de la consommation d’énergie, une augmentation des apports afin de maintenir l’homéostasie tissulaire et la survie mais aussi une réaction inflammatoire et l’apoptose pouvant conduire à la fibrose. Parmi celles-ci, on peut nommer les voies HIF1α, mTOR, le stress du réticulum endoplasmique (RE), l’autophagie, l’activation de l’immunité innée et acquise. La réponse adaptative qui suit le stress du RE, la réponse UPR (Unfolded protein response), est une voie adaptative dont les implications sont actuellement encore peu connues dans le domaine de la pathologie rénale. Celle-ci se compose de trois effecteurs principaux : Perk, Ire1 et ATF6. A l’aide de deux modèles de stress toxique (ciclosporine) et ischémique (carence en glucose) sur deux modèles cellulaires distincts (cellulaires endothéliales artérielles et cellules tubulaires rénales), et dans des modèles in vivo, nous avons montré que le stress du RE était impliqué à la fois dans l’apparition de modifications phénotypiques endothéliales évocatrices de transition endothélio-mésenchymateuse induites par la ciclosporine et à la fois dans l’induction de réponses inflammatoire (régulation de NF-κB par Ire1) et angiogénique (régulation distincte de VEGF, bFGF et angiogénine par Perk et Ire1) induites par la carence en glucose. La réponse UPR semble modulée de façon subtile au cours de ces stress car les trois effecteurs n’engendrent pas des réponses identiques. Ces travaux apportent ainsi une meilleure compréhension des mécanismes d’adaptation au cours de stress variés, montrent que le stress du RE est impliqué dans ces réponses adaptatives et que la réponse peut être différente selon les effecteurs de la réponse UPR. Cette meilleure compréhension pourra permettre de valider des biomarqueurs précoces et des modulateurs de la réponse UPR afin de prévenir la dégradation du parenchyme rénal. / Native and grafted kidneys are stressed by multiple specific or non-specific insults leading to progressive structural deterioration. Responses to these insults are adaptive and preserve cell survival but may also promote inflammation, fibrosis and apoptosis. The most important of these adaptive pathways are HIF1α pathway, mTOR pathway, autophagy, unfolded protein response (UPR). The consequences of the UPR in kidney injuries are not well known. The objective of this study is to delineate the mechanisms and consequences of the activation of the UPR in response to toxic (cyclosporine) and ischemic (glucose starvation) stresses in two distinct cellular models (arterial endothelial cells and renal tubular cells). Here, we showed that UPR was engaged in cyclosporine-induced endothelial phenotypic changes, glucose starvation-induced inflammatory and angiogenic responses: NF-κB regulation by Ire1; distinct VEGF, bFGF and angiogenin regulation by Perk and Ire1. UPR is subtly modulated since its transducers do not induce identical processes. In conclusion these comprehensive works, we demonstrate the UPR is implicated in stress-induced adaptive pathways with different downstream responses according to the effector. Renal tissue degradation could be prevented by discovering and validating early biomarker and UPR modulators.

The endoplasmic reticulum is the primary organelle in the cell which has the responsibility of properly folding proteins belonging to the secretory pathway. Secretory proteins are essential for a variety of functions within the body like metabolism, growth and survival. Hence, proper folding of the proteins in the ER is absolutely essential to maintain cellular and body function. The environment of the ER is substantially different from that of the cytoplasm and is primed essentially to provide the optimum conditions to fold newly synthesized polypeptides following translation by the ribosomes in the cytoplasm and on the surface of the ER. In order for secretory proteins to fold properly, ER homeostasis must be maintained. ER homeostasis is defined by the dynamic balance between the ER protein load and the ER capacity to process this load. The optimum environment of the ER, or ER homeostasis, can be perturbed by pathological processes such as hypoxia, glucose deprivation, viral infections, environmental toxins, inflammatory cytokines, and mutant protein expression, as well as by physiological processes such as aging. Disruption of ER homeostasis causes accumulation of unfolded and misfolded proteins in the ER. This condition is referred to as ER stress. Cells cope with ER stress by activating the unfolded protein response (UPR). The UPR is initiated by three ER transmembrane proteins: Inositol requiring 1 (IRE1), PKR-like ER kinase, and activating transcription factor 6 (ATF6). These three master regulators sense and interpret protein folding conditions in the ER and translate this information across the ER membrane to activate downstream effectors, spliced XBP1, phosphorylated eIF2α and ATF4, and cleaved active ATF6 respectively. These effectors have two distinct outputs, homeostatic and apoptotic. Homeostatic outputs are adaptive responses that function to attenuate ER stress and restore ER homeostasis. These responses include the attenuation of protein translation to reduce ER workload and prevent further accumulation of unfolded proteins, upregulation of molecular chaperones and protein processing enzymes to enhance the ER folding activity, and the increase in ER-associated degradation (ERAD) components to promote clearance of unfolded proteins. When ER stress reaches a point where the cells cannot tolerate the load of unfolded proteins any more, apoptosis sets in. One of the major secretory proteins in mammals, vascular endothelial growth factor VEGF, is essential for either normal or pathological angiogenesis (blood vessel development). VEGFA is the primary member of this family which is expressed in all endothelial cells and is responsible for sprouting and invasion of blood vessels into the interstitium and thus helps in supplying nutrients and oxygen to growing cells. Recent studies have indicated that cells suffering from insufficient blood supply experience ER stress. The ER needs energy and oxygen for the folding process, thus nutrient deprivation (low ATP production) and hypoxia caused by insufficient blood supply leads to inefficient protein folding and ER stress in cells, especially in cancer cells that grow and spread rapidly. This condition also occurs in the development of the mammalian placenta. The placenta is an essential tissue characterized by a lot of blood vessels. It is responsible for the exchange of nutrients and growth factors between maternal and fetal blood vessels and hence is essential for survival of the embryo. Nutrient deprivation and hypoxia stimulate the production of VEGFA and other angiogenic factors, leading to protection against ischaemic injury in both cancer cells as well as the developing placenta. In this dissertation, we report that the three master regulators of the UPR, IRE1α, PERK and ATF6α, mediate transcriptional regulation of VEGFA under ER stress in cancer cells. Inactivation of any of the three master regulators leads to attenuation of VEGFA expression under ER stress. We show that IRE1α is able to regulate VEGFA through its downstream transcription factor XBP1 which activates the VEGFA promoter. IRE1α mediated VEGFA regulation is also essential for normal development of labyrinthine trophoblast cells in the placenta. ATF6α also regulates VEGFA via its promoter. PERK is able to activate VEGFA by preferential activation of its downstream effector, ATF4, which binds intron 1 of the VEGFA gene. Thus our work reveals a twopronged differential regulatory action of the UPR sensors on VEGFA gene expression. This work suggests that a fully active UPR is essential for VEGFA upregulation under ER stress. All three regulators are required in cancer cells for normal VEGFA expression. This tight regulation of VEGFA by the UPR presents a wonderful opportunity for therapeutic intervention into angiogenic growth of tumors.

Après la découverte des mutations de la calréticuline dans les néoplasmes classiques myéloproliferatifs négatifs pour le Ph1, les travaux se sont focalisés sur les deux mutations les plus fréquentes, c'est-à-dire la calréticuline del52 et l’ins5, mais il existe environ 20% de mutants rares de la calréticuline (une cinquantaine), qui ont été classés en type-1 « like » et type-2 « like », classification basée sur leur structure. Cependant il reste à déterminer si cette classification est pertinente du point de vue fonctionnel, ce qui pourrait avoir des conséquences pour la prise en charge des patients et leur traitement. Ici, nous démontrons que deux mutants rares de type-1 (del34 et del46) et un de type-2 (del19) se comportent de manière similaire aux deux mutations fondatrices de cette classification, del52 et ins5, respectivement. Ces résultats ont été validés par des expériences in vivo chez la souris. Tous les mutants de la calréticuline (del19, del34 et del46) nécessitent absolument le récepteur de la thrombopoïétine, appelé MPL, pour induire une transformation cellulaire en provoquant une activation indépendante de la thrombopoïétine de la voie MPL / JAK2-STAT, comme les mutants del52 et ins5. Dans les expériences de transplantation de moelle osseuse de souris, les mutants rares de type-1 sont associés à une progression fréquente de la maladie d’un tableau proche d’une thrombocytémie essentielle à une myélofibrose, tandis que le mutant rare de type 2 est associé à une légère thrombocytose. Du point de vue hématopoïétique, les mutants rares de type-1 provoquent une amplification au niveau des cellules souches hématopoïétiques donc à un stade précoce tandis que les mutants rares de type-2 provoquent une amplification tardive de la mégacaryopoïèse. Grâce à une modélisation protéique basée sur l'homologie des mutants de calréticuline, nous avons identifié des domaines oncogènes qui seraient potentiellement responsables de l'interaction pathologique de la calréticuline et de MPL pour conduire à une activation indépendante de la thrombopoïétine. Maintenant, ces résultats in silico doivent être absolument validés par des études structure fonction. Enfin, nous avons modélisé un nouveau mécanisme de signalisation dans la leucémie myéloïde chronique comprenant IRE-1alpha, un bras de la voie de réponse des protéines mal repliées (UPR), qui pourrait être responsable de la perte de la fonction de la p53 pendant la progression de la leucémie myéloïde chronique vers une leucémie aiguë. Un tel mécanisme pourrait être impliqué dans les autres MPN. / After the discovery of calreticulin mutations in classical Ph1- Myeloproliferative Neoplasms, extensive investigation is underway on the two most frequent mutations, i.e., del52 and ins5, but it remains that the rare calreticulin mutants, which include both type-1 like and type-2 like require a similar investigation for ascertaining whether the classification of type-1 and type-2 has a functional relevance as well as for therapeutic intervention and patient management. Here we demonstrate that type-1 like (del34 and del46) and type-2 like (del19) mutants behave similarly as del52 and ins5 mutants, respectively. Moreover, we validate our findings with in vivo experiments. All the calreticulin mutants (del19, del34 and del46) absolutely require the thrombopoietin receptor, MPL, to induce cell transformation by causing ligand independent activation of the MPL/JAK2-STAT pathway. In mouse bone marrow transplantation experiments, type-1 like mutants are associated with frequent progression from an essential thrombocythemia-like phenotype to myelofibrosis whereas type-2 like mutant is associated with mild thrombocytosis. Type-1 like mutants cause clonal amplification of early hematopoetic stem cells whereas the type-2 like mutant causes late platelet amplification. Further, by homology based protein modeling of calreticulin mutants, we have identified possible oncogenic domains responsible for pathologic interaction of CALR and MPL leading to ligand independent activation of MPL. Now they must be validated by structural-functional studies Finally, we have modelled a novel signaling mechanism in chronic myeloid leukemia comprising of IRE-1alpha, an unfolded protein response (UPR) pathway arm, which may be responsible for loss of the WT p53 function during leukemic development and progression. Such a mechanism may be involved in the other MPNs

Central driving forces in the pathogenesis of liver disease are hepatocyte death and immune cell-driven inflammation. The interplay between outcomes, stemming from these two major cell types, is present from the earliest ethanol exposure, and are both determinants in advanced stages of liver disease particularly in alcoholic liver disease (ALD). The complexities associated with advanced ALD are many and therapies are limited. Due to the liver’s role in ethanol metabolism and filtering gut-derived products, it is becoming increasingly clear that innate immunity plays a central role in triggering activation of cell death and inflammatory pathways in ALD. We identified interferon regulatory factor 3 (IRF3) activation as a mediator of hepatocyte death as the first event after ethanol exposure, and the inflammasome as a protein complex responsible for the subsequent inflammatory cascade, driven by the NLRP3 inflammasome. Our novel findings in murine samples and human patients with alcoholic hepatitis demonstrate that ethanol-induced inflammasome activity results in Caspase-1-mediated pyroptosis and extracellular ASC aggregates in the liver and circulation. Pyroptosis can be abrogated by therapeutic inhibition of inflammasome components, NLRP3 or Caspase-1. Taken together, the event leading to mtDNA release into the cytoplasm is the inception of the pathogenesis of ALD, triggering hepatocyte death, culminating in a pro-inflammatory cascade driven by the NLRP3 inflammasome and pyroptotic release of ASC.