La NADPH oxydase, Nox4, appartient à la famille des Nox qui génèrent les espèces radicalaires de l'oxygène, ROS, en transférant un électron à l'oxygène moléculaire. Malgré sa large distribution dans les tissus, Nox4 est encore mal comprise. Contrairement aux autres Nox, Nox4 est unique par son activité constitutive et sa capacité à former H2O2. Les ROS sont des espèces bactéricides dans les phagocytes et des outils de signalisation dans les cellules non phagocytaires en étant associés à de nombreuses pathologies inflammatoires et du vieillissement. Une étude de la structure en lien avec la fonction de Nox4 permettra de mettre l'accent sur un mécanisme de fonctionnement et sur de nouvelles cibles thérapeutiques. 5 nouveaux anticorps monoclonaux ont été générés contre une construction recombinante tronquée (AA: 206-578) de Nox4. La spécificité de 3 anticorps monoclonaux (8E9, 5F9, 6B11) a été confirmée par western blot dans les cellules HEK293 transfectées et le cortex de rein humain. L'anticorps 8E9 est le seul à permettre un marquage des cellules TRex-Nox4 sans perméabilisation par FACS. L'immunofluorescence confocale a montré que Nox4 est localisée dans la zone périnucléaire et le réticulum endoplasmique. La microscopie TIRF a confirmé sa présence dans la membrane plasmique. Un phénomène intéressant est que 5F9 ne détecte pas Nox4 à la membrane plasmique. L'épitope de 8E9 reconnaît une région sur la dernière boucle E extracellulaire de Nox4 (222H-E241), tandis que les anticorps monoclonaux, 6B11 et 5F9 marquent respectivement les régions 6B11 (389S-P416) et 5F9 (392D-F398). Par ailleurs, seuls 5F9 et 6B11 inhibent l'activité de Nox4, ce qui suggère que les deux régions marquées par ces ACm sont impliquées dans le transfert d'électrons. Une étude ciblée sur la boucle E de Nox4 a permis de montrer que le changement de 2 cystéines modifie la nature des ROS générés par Nox4 avec la production de O2- au lieu de H2O2. O2- est mis en évidence par la formation de peroxynitrite en présence de NO. Par ailleurs l'ACm 8E9 diminue la production de H2O2 dans les cellules COS7 qui expriment Nox4 à la membrane plasmique alors que celle de O2- est augmentée. Des constructions recombinantes de Nox4 (native ou tronquée) ont été générées par induction bactérienne, E.Coli, et par un système de transcription/traduction (RTS). Les protéines correspondantes, solubles, ont été produites à grande échelle et l'activité diaphorase mesurée; cette activité est constitutive. L'étude de la topologie membranaire de Nox4 et p22phox a été abordée en préparant des protéines de fusion avec l'ubiquitine marquée à la GFP. Cette méthode, TDUFA, particulièrement originale, devrait permettre d'appréhender la topologie de l'hétérodimère Nox4/p22phox, actif. / NADPH oxidase, Nox4, belongs to the Nox family which could generate reactive oxygen species by transferring an electron to molecular oxygen. Despite its wide distribution in tissues, Nox4 is still poorly understood. Unlike the other Noxes, Nox4 shows some unique characters: the constitutive activity, H2O2 formation. Nox4 involved ROS has been proposed to be implicated in several pathologies. Thus, to study the structure/function and the regulation of the activity of Nox4 will provide new ideas and new drug targets for the effective prevention and treatment of clinical diseases related with ROS. To know more about Nox4, in this study, 5 novel monoclonal antibodies were raised against a truncated recombinant protein (AA: 206-578) of Nox4. The specificity of 3 mAbs (8E9, 5F9, 6B11) was confirmed by western blot analysis in HEK293 transfected cells and human kidney cortex. In FACS studies, only mAb 8E9 could react with intact tet-induced T-RExTM Nox4 cells. Immunofluorescence confocal microscopy showed that Nox4 localized not only in the perinuclear and endoplasmic reticulum regions but also at the plasma membrane of the cells which was further confirmed by TIRF-microscopy. An interesting phenomena is that mAb 5F9 failed to detect Nox4 at the plasma membrane. Epitope determination showed that mAb 8E9 recognizes a region on the last extracellular loop of Nox4 (222H-E241), while mAb 6B11 (389S-P416) and 5F9 (392D-F398) are directed to its cytosolic tail. Cell-free oxidase assays showed a moderate but significant inhibition of constitutive Nox4 activity by mAb 5F9 and 6B11. To study the protein region which is responsible for the unique ability of Nox4 of releasing H2O2 rather than O2-, chimeric proteins and mutants were used. E-loop of Nox4 is 28 amino acid longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or change of the two cysteines in the E-loop switch Nox4 from H2O2 to O2- generation. In the presence of a NO donor, the O2--producing Nox4 mutants, but not widetype Nox4, generated peroxynitrite, excluding artifacts of the detection systems as the apparent origin of O2-. A second approach was used to confirm the responsibility of E-loop for the H2O2 formation. In Cos7 cells, which exhibit some plasma membrane expression of Nox4, addition of the mAb 8E9 decreased H2O2 production but increased O2- formation. Unlike Nox1 or Nox2, the E-loop of Nox4 contains a highly conserved histidine H222. Mutation of H222 also switched Nox4 from H2O2 to O2- formation. The structure of the E-loop might hinder O2- egress and/or provide a source for protons to accelerate dismutation to form H2O2. Two bacterial protein expression approaches (in vitro RTS and bacterial induction) were used to produce Nox4 cytosolic tail for characterizing the electronic transfer property of Nox4. The presence of rare codons (1363AGA AGA CUA1371) and high level of hydrophobicity affects the production of soluble and active recombinant Nox4Aqc and Nox4Bqc. After optimization of the conditions, soluble and active recombinant proteins were obtained by RTS or by bacteria induction. The soluble proteins were produced in large scale, purified onto affinity chromatography and were tested for the diaphorase activity (INT and cytochrome c). Results showed that electronic acceptor cytochrome c gives a higher rate than INT. Nox4Aqc produced a lower specific activity by a cell-based system compared to the protein synthesized in cell-free technology. This activity is not stimulated by the addition of cytosolic factors. A new method, topological determination by ubiquitin fusion assay (TDUFA), was used to investigate the topology of Nox4 and p22phox. ubGFP fusion proteins are used as tools to obtain details of membrane protein topology. This method was first validated by using two membrane proteins with known topology and then should get more topology information of Nox4 and p22phox further.

<p>A 95-kDa protein was purified from normal human granulocytes. The protein reacted with a monoclonal antibody against CEACAM8. MALDI-Tof and MS/MS analyses revealed the protein to be a CGM6 gene product. Thus, the protein was proved to be identical to CEACAM8. </p><p>An ELISA for CEACAM8 was developed with detection range of 1-64μg/L. Data are presented on the levels of CEACAM8 in the blood of healthy individuals and patients undergoing surgery, as well as in patients with acute infection. The highly elevated levels of CEACAM8 in the blood of these patients were significantly correlated with the surface expression of CEACAM8 on neutrophils and the number of circulating neutrophils, which suggests that CEACAM8 could serve as a biological marker for granulocyte activitiy in vivo. </p><p>The cellular content of CEACAM8 in neutrophils was estimated to be 82.4 ± 8.9 ng/10⁶ cells. Subcellular localisation and mobilisation studies showed that the majority of CEACAM8 is present in the secondary granules of human neutrophils, with a small amount on the plasma membranes. Upon stimulation, CEACAM8 translocated to the plasma membranes from the secondary granules and was also released extracellularly (5.5 ± 0.7% of the total content of CEACAM8).</p><p>In eosinophils, the cellular content of CEACAM8 was estimated to be 73.8 ± 6.0 ng/10⁶ cells. In these cells, CEACAM8 is mainly stored in secretory vesicles. Upon activation, eosinophils released 5.1 ± 1.1% of the total content of CEACAM8. </p><p>Administration of granulocyte colony-stimulating factor (G-CSF) to healthy individuals resulted in an increased content of CEACAM8 in neutrophils on day 1, and decreased on day 4. However, the content of CEACAM8 in light membrane fractions was increased on day 4. The translocation of CEACAM8 observed <i>in vivo</i> after G-CSF administration is probably not directly related to this cytokine but to other cytokines such as TNF-a. </p>

<p>Serum levels of eosinophil granule proteins namely ECP, EPO and EPX, which are stored in the matrix of specific granules, were shown to correlate with the course of disease in disorders involving eosinophils. The concentration of eosinophil proteins in serum is the result of their release <i>in vivo</i> and <i>ex vivo</i> during the sampling procedure. Generally, eosinophils release the content of their specific granules in three ways: exocytosis, piecemeal degranulation (PM) or cytolysis. Which of them is operating in circulating eosinophils has not yet been defined. The aim of this thesis was to study the mechanisms of granule protein release from blood eosinophils in respect of protein subcellular localization and cell ultrastructure.</p><p>In patients with bacterial infections, serum levels of ECP but not EPO increased, while in patients with viral infections both proteins remained within the range of healthy controls. G-CSF is a cytokine involved in the response mechanism to bacterial but not viral infections. Administration of G-CSF to healthy subjects induced an elevation of eosinophil numbers and a preferential increase of serum EPX and ECP in comparison to EPO.</p><p>The model of PM consists of the stepwise transportation of specific granule contents from the granules towards the plasma membrane. We observed that administration of G-CSF to healthy subjects and the allergen exposure of allergic subjects during the pollen season, caused changes in the ultrastructure of eosinophil specific granules such as loosening of the matrix, granule matrix lucency and ragged losses of their core. Similar alterations of morphology had been previously described for eosinophils undergoing PM.</p><p>ECP, EPX and EPO were localized not only in the specific granules but also in extra-granular compartments as shown both by immuno electron microscopy and subcelular fractionations, An extra-granular EPX compartment was present in healthy as well as in allergic and in hypereosinophilic subjects, and there were no significant differences in its size between the groups. The size of the extra-granular compartments of ECP and EPO was increased in allergics during the season, and these compartments were clearly separate from that of EPX. Results of this show the differential mobilization ofgranule proteins in blood stream eosinophils serum and indicates PM as its mechanism.</p>

Serum levels of eosinophil granule proteins namely ECP, EPO and EPX, which are stored in the matrix of specific granules, were shown to correlate with the course of disease in disorders involving eosinophils. The concentration of eosinophil proteins in serum is the result of their release in vivo and ex vivo during the sampling procedure. Generally, eosinophils release the content of their specific granules in three ways: exocytosis, piecemeal degranulation (PM) or cytolysis. Which of them is operating in circulating eosinophils has not yet been defined. The aim of this thesis was to study the mechanisms of granule protein release from blood eosinophils in respect of protein subcellular localization and cell ultrastructure. In patients with bacterial infections, serum levels of ECP but not EPO increased, while in patients with viral infections both proteins remained within the range of healthy controls. G-CSF is a cytokine involved in the response mechanism to bacterial but not viral infections. Administration of G-CSF to healthy subjects induced an elevation of eosinophil numbers and a preferential increase of serum EPX and ECP in comparison to EPO. The model of PM consists of the stepwise transportation of specific granule contents from the granules towards the plasma membrane. We observed that administration of G-CSF to healthy subjects and the allergen exposure of allergic subjects during the pollen season, caused changes in the ultrastructure of eosinophil specific granules such as loosening of the matrix, granule matrix lucency and ragged losses of their core. Similar alterations of morphology had been previously described for eosinophils undergoing PM. ECP, EPX and EPO were localized not only in the specific granules but also in extra-granular compartments as shown both by immuno electron microscopy and subcelular fractionations, An extra-granular EPX compartment was present in healthy as well as in allergic and in hypereosinophilic subjects, and there were no significant differences in its size between the groups. The size of the extra-granular compartments of ECP and EPO was increased in allergics during the season, and these compartments were clearly separate from that of EPX. Results of this show the differential mobilization ofgranule proteins in blood stream eosinophils serum and indicates PM as its mechanism.

A 95-kDa protein was purified from normal human granulocytes. The protein reacted with a monoclonal antibody against CEACAM8. MALDI-Tof and MS/MS analyses revealed the protein to be a CGM6 gene product. Thus, the protein was proved to be identical to CEACAM8. An ELISA for CEACAM8 was developed with detection range of 1-64μg/L. Data are presented on the levels of CEACAM8 in the blood of healthy individuals and patients undergoing surgery, as well as in patients with acute infection. The highly elevated levels of CEACAM8 in the blood of these patients were significantly correlated with the surface expression of CEACAM8 on neutrophils and the number of circulating neutrophils, which suggests that CEACAM8 could serve as a biological marker for granulocyte activitiy in vivo. The cellular content of CEACAM8 in neutrophils was estimated to be 82.4 ± 8.9 ng/106 cells. Subcellular localisation and mobilisation studies showed that the majority of CEACAM8 is present in the secondary granules of human neutrophils, with a small amount on the plasma membranes. Upon stimulation, CEACAM8 translocated to the plasma membranes from the secondary granules and was also released extracellularly (5.5 ± 0.7% of the total content of CEACAM8). In eosinophils, the cellular content of CEACAM8 was estimated to be 73.8 ± 6.0 ng/106 cells. In these cells, CEACAM8 is mainly stored in secretory vesicles. Upon activation, eosinophils released 5.1 ± 1.1% of the total content of CEACAM8. Administration of granulocyte colony-stimulating factor (G-CSF) to healthy individuals resulted in an increased content of CEACAM8 in neutrophils on day 1, and decreased on day 4. However, the content of CEACAM8 in light membrane fractions was increased on day 4. The translocation of CEACAM8 observed in vivo after G-CSF administration is probably not directly related to this cytokine but to other cytokines such as TNF-a.

Since a proper balance between anabolic and catabolic reactions is essential for all eukaryotes, the basic mechanisms for regulation of the energy and carbon metabolism have been conserved throughout evolution. The moss Physcomitrella patens, which belongs to one of the basal clades among land plants, has many unique properties that make it an excellent plant model system. We have used a yeast two-hybrid system to identify novel possible regulators or targets of the moss Snf1-related kinases, previously shown to regulate energy homeostasis. The function of the identified interactors PpSki1 and PpSki2 was analyzed in order to better understand the biological role of plant Snf1-related kinases. The recently completed genome sequence of Physcomitrella was used in a comparative approach to study to what extent key enzyme and gene families involved in transport and metabolism of sugars and in regulation of the energy and carbon metabolism are conserved between mosses and vascular plants. It has long been known that transformed DNA can replicate episomally in Physcomitrella. We have now shown that such DNA can be rescued back into E. coli. Surprisingly, we found that the original plasmid can be recovered from moss transformants obtained with circular DNA. Plasmids rescued from transformants obtained with linearized DNA had been repaired either by homologous recombination or by cohesive end re-ligation. These findings suggest that methods using shuttle plasmids are feasible in Physcomitrella. Hexokinase, a key enzyme in the carbon metabolism, catalyzes the first step in hexose metabolism, but is also involved in sugar sensing and signaling. We have now made an initial characterization of the complete hexokinase family in Physcomitrella which is encoded by 11 genes. Two new types of plant hexokinases, types C and D, were found in addition to the previously described types A and B.

Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.

Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.

Until recently, the main mechanism of argininosuccinate synthase (AS) regulation was described to exist mainly at the level of transcription. Transcriptional regulation of AS has been shown to be coordinate with eNOS in response to shear stress, hypoxia, tumor necrosis factor á (TNF-á), and PPAR ã agonist troglitizone. However, it is now understood that one level of NO regulation is cellular control of arginine availability to eNOS via post-translational modifications of AS such as phosphorylation. The purpose of this investigation was to determine under what conditions AS is phosphorylated at S328, identify the pathway that AS phosphorylation at S328 plays a role, and how phosphorylation affects AS function in endothelial cells. We developed a phospho-specific antibody directed against pS328 AS and assayed for increases or decreases in phosphorylation relative to physiological factors. We found that AS phosphorylation at S328 occurred when endothelial cells were stimulated with physiological factors that stimulate nitric oxide production through calcium-dependent stimulation of eNOS. Furthermore, by utilizing kinase inhibitors and kinase knockdown experiments, we showed that phosphorylation at S328 significantly decreased when PKCá was knocked down, suggesting that S328 phosphorylation of AS is involved in PKCá signaling. In addition, by confocal microscopy, immunoprecipitation, and membrane fractionation, we showed that phosphorylation at S328 of AS promotes its co-localization with eNOS in the perinuclear region. These findings describe a novel pathway involving AS regulation of nitric oxide production, and may serve as a novel drug target in the restoration of vascular nitric oxide homeostasis.

The fundamental goal of proteomics is to gain an understanding of the expression and function of the proteome on the level of individual proteins, on the level of defined cell types and on the level of the entire organism. In this thesis, the human proteome is explored using membrane protein topology prediction methods to define the human membrane proteome and by global protein expression profiling, which relies on a complex study of the location and expression levels of proteins in tissues and cells. A whole-proteome analysis was performed based on the predicted protein-coding genes of humans using a selection of membrane protein topology prediction methods. The study used a majority decision-based method, which estimated that approximately 26% of the human genes encode for a membrane protein. The prediction results are displayed in a visualization tool to facilitate the selection of antigens to be used for antibody generation. Global protein expression profiles in a large number of cells and tissues in the human body were analyzed for more than 4000 protein targets, based on data from the antibody-based immunohistochemistry and immunofluorescence methods within the framework of the Human Protein Atlas project. The results revealed few cell-type specific proteins and a high fraction of human proteins expressed in most cells, suggesting that cell and tissue specificity is attained by a fine-tuned regulation of protein levels. The expression profiles were also used to analyze the relationship between 45 cell lines by hierarchical clustering and principal component analysis. The global protein expression patterns overall reflected the tumor origin of the cells, and also allowed for identification of proteins of importance for distinguishing different categories of cell lines, as defined by phenotype of progenitor cell. In addition, the protein distribution in 16 subcellular compartments in three of the human cell lines was mapped. A large fraction of proteins were localized in two or more compartments and, in line with previous results, a majority of proteins were detected in all three cell lines. Finally, mass spectrometry-based protein expression levels were compared to RNA-seq-based transcript expression levels in three cell lines. Highly ubiquitous mRNA expression was found and the changes of expression levels between the cell lines showed high correlations between proteins and transcripts. Large general differences in abundance of proteins from various functional classes were observed. A comparison between categories based on expression levels revealed that, in general, genes with varying expression levels between the cell lines or only expressed in one cell line were highly enriched for cell-surface proteins. These studies show a path for a systematic analysis to characterize the proteome in human cells, tissues and organs. / QC 20110317 / The Human Protein Atlas project