Le plasmocyte représente le stade final de la différenciation lymphocytaire B. Il s’agit de la cellule productrice des immunoglobulines (Ig), ce qui en fait l’acteur majeur de la réponse immunitaire humorale. Toutefois, lors d’une prolifération plasmocytaire anormale, l’Ig produite en excès peut devenir pathogène pour l’organisme, s’agréger et conduire à une maladie de dépôt d’Ig monoclonales. Il existe une grande variété de ces maladies de dépôts dont la classification repose sur la nature des dépôts. Nous avons développé dans notre laboratoire un modèle murin pour l’une d’entre elle, la LCDD « Light Chain Deposition Disease », qui reproduit parfaitement les lésions rénales observées chez les patients. Nous montrons grâce à ce modèle qu’en supprimant la production de l’Ig pathogène, nous préservons la fonction rénale de nos souris. Nous montrons aussi grâce à des traitements réalisés avec des inhibiteurs du protéasome (IP) et par l’étude du transcriptome des PCs que ces cellules sont sensibilisées par l’Ig pathogène aux IP via une activation de la voie de réponse au stress du réticulum endoplasmique. Nous nous sommes également intéressés à un patient atteint d’une HCDD « Heavy Chain Deposition disease ». Les études moléculaires et les expériences in-vitro que nous avons réalisées à partir des prélèvements de ce patient nous ont permis de proposer un scénario expliquant la production d’une chaîne lourde tronquée par le clone plasmocytaire : l’apparition d’une mutation au niveau de la chaîne légère aurait conduit à la mutation de la chaîne lourde afin de surmonter un stress du réticulum endoplasmique et ainsi permettre la survie cellulaire. / The plasma cell represents the final stage of B-lymphocytes differentiation. It is the immunoglobulin (Ig) producing cell, making it the major player in the humoral immune response. However, during abnormal plasma cell proliferation, the Ig produced in excess can become pathogenic for the organism, aggregate and lead to a monoclonal Ig deposition disease. There is a wide variety of these deposition diseases whose classification is based on the nature of the deposits. In our laboratory, we have developed a mouse model for one of them, the Light Chain Deposition Disease (LCDD), perfectly reproducing the renal lesions observed in patients. We show by this model that by suppressing the production of the pathogenic Ig the renal function of our mice is preserved. Additionally, thanks to proteasome inhibitors (PI) treatment and plasma cell transcriptome studies, we prove that these cells are sensitized by the pathogen Ig to PI via the activation of the endoplasmic reticulum stress response pathway. We also studied a patient with Heavy Chain Deposition disease (HCDD). The molecular studies and in-vitro experiments carried out with the sample from this patient allowed us to propose a new scenario explaining the production of a truncated heavy chain by the plasma cell clone: the appearance of a mutation at the light chain level would led to the mutation of the heavy chain, in order to overcome the endoplasmic reticulum stress and thus allowing cell survival.

Cholestasis is an important liver pathology. During cholestasis bile acids accumulate in the bile canaliculus affecting hepatocyte viability. The actions of bile acids require changes in the release of Ca2+ from intracellular stores and in Ca2+ entry. The target(s) of the Ca2+ entry pathway affected by bile acids is, however, not known. The overall objective of the work described in this thesis was to elucidate the target(s) and mechanism(s) of bile acids-induced modulation of hepatocytes calcium homeostasis. First, it was shown that a 12 h pre-incubation with cholestatic bile acids (to mimic cholestasis conditions) induced the inhibition of Ca2+ entry through store-operated Ca2+ channels (SOCs), while the addition of choleretic bile acids to the incubation medium caused the reversible activation of Ca2+ entry through SOCs. Moreover, it was shown that incubation of liver cells with choleretic bile acids counteracts the inhibition of Ca2+ entry caused by pre-incubation with cholestatic bile acids. Thus, it was concluded that SOCs are the target of bile acids action in liver cells. Surprisingly, despite the effect of choleretic bile acids in activating SOCs, the Ca2+ dye fura-2 failed to detect choleretic bile acid-induced Ca2+ release from intracellular stores in the absence of extracellular Ca2+. However, under the same conditions, when the sub-plasma membrane Ca2+ levels were measured using FFP-18 Ca2+ dye, choleretic bile acid induced a transient increase in FFP-18 fluorescence. This evidence suggested that choleretic bile acids-induced activation of Ca2+ entry through SOCs, involving the release of Ca2+ from a region of the endoplasmic reticulum (ER) located in the vicinity of the plasma membrane.

Beta-cell failure is a key step in the progression from metabolic disorder to overt type 2 diabetes (T2D). This failure is characterised by both secretory defects and loss of beta-cell mass, the latter most likely through increases in the rate of apoptosis. Although the mechanisms underlying these beta-cell defects are unclear, evidence suggests that chronic exposure of beta-cells to elevated fatty acid (FA) plays a role in disease development in genetically susceptible individuals. Furthermore, it has been postulated that endoplasmic reticulum (ER) stress signalling pathways (the unfolded protein response; UPR) play a role in FA-induced beta-cell dysfunction. The broad aim of this thesis was to explore the nature of these relationships. Experiments detailed in this thesis demonstrate that MIN6 beta-cells mount a comprehensive ER stress response with exposure to elevated saturated fatty acid palmitate, but not the unsaturated fatty acid, oleate, within the low elevated physiological range. This response was time-dependent and involved both transcriptional and translational changes in UPR transducers and targets. The differential activation of ER stress in MIN6 beta-cells by saturated, but not unsaturated FA species may represent a mechanism of differential beta-cell death described in many studies with these FA. Furthermore, these experiments describe defects in ER to Golgi trafficking with chronic palmitate treatment, but not oleate or thapsigagin treatment, identifying this as a potential mechanism by which palmitate treatment induces ER stress. Moreover, these studies have shown the relevance to ER stress to a whole body model of T2D by demonstrating UPR activation in the islets of the db/db mouse. In conclusion, studies detailed in this thesis have demonstrated that ER stress occurs in in vitro and in vivo models of beta-cell lipotoxicity and apoptosis. In addition, these studies have identified defects in ER to Golgi trafficking as a mechanism by which palmitate treatment induces ER stress. These studies highlight the importance of ER stress in the development of T2D.

Dysregulation of protein activity, caused by alterations in protein sequence, expression, or localization, is associated with numerous diseases. In order to control the activity of harmful protein entities, affinity ligands such as proteins, oligonucleotides or small molecules can be engineered to specifically interact with them to modulate their function. In this thesis, non-immunoglobulin based affinity proteins known as affibody molecules are used to functionally inhibit proteins important for signaling through pathways that are overactive in different cancers.   In Paper I and Paper II, affibody molecules with high affinity for the receptor tyrosine kinases HER2 or EGFR are expressed in the secretory compartments of model cancer cell lines SKOV3 or A431 using a retrovirus-based gene delivery system. Equipping the affinity proteins with an ER retention tag, the affibody molecules together with their target protein are retained in the secretory compartments as shown by confocal fluorescence imaging. Flow cytometric analysis showed a 60 % or 80 % downregulation of surface located HER2 or EGFR in these cell lines, respectively. A significant decreased in proliferation rate of the cells was also observed, which for EGFR retention could be correlated with inhibition of phosphorylation in the kinase domain. In Paper III, novel affibody molecules interacting with the hormone binding site of the insulin growth factor-1 receptor were generated. One variant had high (1.2 nM) affinity for the receptor and could be used for immunofluorescence analysis and for receptor pull-out from cell lysates. Addition of this affibody molecule to MCF-7 cells had a dose dependent growth inhibitory effect on the cells. In Paper IV, novel affibody molecules against the intracellular oncoproteins H-Ras and Raf-1 were selected and characterized, and they proved to be specific for their target proteins. Mapping experiments showed that the affibody molecules selected against H-Ras interacted at over-lapping epitopes not affecting the interaction between Ras and Raf. In contrast, the predominant variant isolated during selection against Raf-1 could completely inhibit the Ras/Raf interaction in a real-time biospecific interaction analysis.   Taken together, the affibody molecules presented here and the strategies by which they are used to interfere with cancer related proteins and pathways may be valuable tools for further investigation of these systems and may possibly also be used to generate molecules suitable for cancer therapy. / QC 20100818

The biogenesis and maintenance of the entire endomembrane system is dependent upon membrane fusion proteins. Mounting evidence indicates that the integral membrane GTPase Atlastin is a membrane fusion protein involved in the homotypic fusion of the endoplasmic reticulum (ER) membrane suggesting a role in the biogenesis and maintenance of ER structure. I helped show that recombinant Drosophila atlastin is able to promote the fusion of synthetic membranes in vitro and that this fusion is dependent upon atlastin GTPase activity. The structure-function experiments presented here assist in elucidating domains required in the mechanism of atlastin mediated membrane fusion. ER homotypic fusion is dependent upon the self-association of Atlastin subunits in adjacent membranes to bring the bilayers into close molecular contact. Atlastin dimerization occurs in the presence of GTPγS but not GDP and stable dimerization is dependent upon a juxtamembrane middle domain three-helix bundle (3HB). The atlastin GTPase domain and 3HB form a potent soluble domain inhibitor of atlastin homotypic fusion, while the GTPase domain alone shows little inhibition. Designed GTPase domain mutations show that GTP binding and atlastin dimerization is insufficient to support fusion without GTP hydrolysis. Additionally, domain analysis of atlastin reveals that the C-terminal cytoplasmic domain of atlastin is absolutely required for membrane fusion, possibly through a protein-lipid interaction of an amphipathic alpha-helix. Genetic lesions in the human Atlastin-1 gene, SPG3A, result in a form of autosomal dominant hereditary spastic paraplegia (HSP). A better understanding of Atlastin function should lend significant insight into normal ER biogenesis and maintenance, as well as the pathology of human disease.

Chronic hyperglycemia, a hallmark of type 2 diabetes, can decrease β-cell function and mass (β-cell glucotoxicity); however, the mechanisms are incompletely understood. The objective was to examine the mechanisms of β-cell glucotoxicity using in vivo and ex vivo models. The hypothesis is that oxidative stress plays a causal role in high glucose-induced β-cell dysfunction in vivo via pathways that involve endoplasmic reticulum (ER) stress and JNK. The model of β-cell glucotoxicity was achieved by prolonged i.v. glucose infusion (to achieve hyperglycemia). In Study 1, 48h glucose infusion increased total and mitochondrial superoxide levels in islets, and impaired β-cell function in vivo and ex vivo. Co-infusion of the superoxide dismutase mimetic Tempol decreased total and mitochondrial superoxide, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. These results suggest that increased superoxide generation plays a role in β-cell glucotoxicity. In Study 2, 48h glucose infusion increased activation of the unfolded protein response (XBP-1 mRNA splicing and phospho-eIF2α levels). This was partially prevented by Tempol. Co-infusion of the chemical chaperone 4-phenylbutyrate with glucose decreased spliced XBP-1 levels, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. Co-infusion of 4-phenylbutyrate also decreased total and mitochondrial superoxide induced by high glucose. These results suggest that 1) ER stress plays a causal role in high glucose-induced β-cell dysfunction, and 2) there is a link between oxidative stress and ER stress in high glucose-induced β-cell dysfunction in vivo. In Study 3, JNK inhibition using the inhibitor SP600125 in rats or JNK-1 null mice prevented high glucose-induced β-cell dysfunction ex vivo and in vivo. SP600125 prevented high-glucose-induced β-cell dysfunction without decreasing total and mitochondrial superoxide levels. Both Tempol and 4-phenylbutyrate prevented JNK activation induced by high glucose. These results suggest a role of JNK activation in high glucose-induced β-cell dysfunction downstream of increased superoxide generation and ER stress in vivo. Together, the results suggest that 1) oxidative stress, ER stress and JNK activation are causally involved in β-cell glucotoxicity, and 2) High glucose-induced oxidative stress and ER stress are linked, and both impair β-cell dysfunction via JNK activation in vivo.

Protein disulfide isomerases (PDIs) catalyze the oxidation and isomerization of disulfide bonds in proteins passing through the endoplasmic reticulum (ER). Although as many as 20 enzymes are classified as PDI family members, their relative contributions to protein folding have remained an open question. Additionally, Ero1 has been characterized as the ER oxidase that transfers oxidizing equivalents from oxygen to PDI enzymes. However, knockout mice lacking the mammalian Ero1 isoforms, Ero1Lα and Ero1Lβ, are viable, and the role of other potential ER oxidases in maintaining an oxidative ER environment is now an important issue. By systematic depletion of ER PDI family members and potential ER oxidases and assessment of disulfide bond formation of secreted endogenous substrates, I have outlined the functional relationships among some of these enzymes. PDI family member depletion revealed that PDI, although not essential for complete disulfide bond formation in client proteins, is the most significant catalyst of oxidative folding. In comparison, ERp57 acts preferentially on glycosylated substrates, ERp72 functions in a more supplementary capacity, and P5 has no detectable role in formation of disulfide bonds for the substrates assayed. Initially, no impact of depletion of Ero1 was observed under steady state conditions, suggesting that other oxidase systems are working in parallel to support normal disulfide bond formation. Subsequent experiments incorporating a reductive challenge revealed that Ero1 depletion produces the strongest delay in re-oxidation of the ER and oxidation of substrate. Depletion of two other potential ER oxidases, peroxiredoxin 4 (PRDX4) and Vitamin K epoxide reductase (VKOR), showed more modest effects. Upon co-depletion of Ero1 and other oxidases, additive effects were observed, culminating in cell death following combined removal of Ero1, PRDX4, and VKOR activities. These studies affirm the predominant roles of Ero1 in ER oxidation processes and, for the first time, establish VKOR as a significant contributor to disulfide bond formation.

Chronic hyperglycemia, a hallmark of type 2 diabetes, can decrease β-cell function and mass (β-cell glucotoxicity); however, the mechanisms are incompletely understood. The objective was to examine the mechanisms of β-cell glucotoxicity using in vivo and ex vivo models. The hypothesis is that oxidative stress plays a causal role in high glucose-induced β-cell dysfunction in vivo via pathways that involve endoplasmic reticulum (ER) stress and JNK. The model of β-cell glucotoxicity was achieved by prolonged i.v. glucose infusion (to achieve hyperglycemia). In Study 1, 48h glucose infusion increased total and mitochondrial superoxide levels in islets, and impaired β-cell function in vivo and ex vivo. Co-infusion of the superoxide dismutase mimetic Tempol decreased total and mitochondrial superoxide, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. These results suggest that increased superoxide generation plays a role in β-cell glucotoxicity. In Study 2, 48h glucose infusion increased activation of the unfolded protein response (XBP-1 mRNA splicing and phospho-eIF2α levels). This was partially prevented by Tempol. Co-infusion of the chemical chaperone 4-phenylbutyrate with glucose decreased spliced XBP-1 levels, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. Co-infusion of 4-phenylbutyrate also decreased total and mitochondrial superoxide induced by high glucose. These results suggest that 1) ER stress plays a causal role in high glucose-induced β-cell dysfunction, and 2) there is a link between oxidative stress and ER stress in high glucose-induced β-cell dysfunction in vivo. In Study 3, JNK inhibition using the inhibitor SP600125 in rats or JNK-1 null mice prevented high glucose-induced β-cell dysfunction ex vivo and in vivo. SP600125 prevented high-glucose-induced β-cell dysfunction without decreasing total and mitochondrial superoxide levels. Both Tempol and 4-phenylbutyrate prevented JNK activation induced by high glucose. These results suggest a role of JNK activation in high glucose-induced β-cell dysfunction downstream of increased superoxide generation and ER stress in vivo. Together, the results suggest that 1) oxidative stress, ER stress and JNK activation are causally involved in β-cell glucotoxicity, and 2) High glucose-induced oxidative stress and ER stress are linked, and both impair β-cell dysfunction via JNK activation in vivo.

The heat shock or stress response is a transient response to stressful stimuli that protects vital cellular proteins from damage and irreversible aggregation. Heat shock proteins (Hsps) are molecular chaperones that bind to unfolded protein and inhibit their aggregation, thereby maintaining their solubility until they can be refolded to their native conformation. Hsp47 is an endoplasmic reticulum (ER)-resident protein that serves as a molecular chaperone during collagen production. Collagen is the major class of insoluble fibrous protein found in the extracellular matrix and in connective tissues. It is the single most abundant protein of the animal kingdom; at least 14 different forms exist, each with distinct structures and binding properties. The various types of collagen all possess protein regions with the distinct triple helical conformation. This complex physical structure requires very organized assembly and HSP47 has been established as an integral component of this process for collagen types I-V. Most of the previous studies examining the expression and function of hsp47 have been conducted with mammalian cultured cells. The present study represented the first investigation of the expression of hsp47 in the poikilothermic vertebrate, <i>Xenopus laevis</i>. Full-length <i>Xenopus</i> hsp47 nucleotide and amino acid sequences were obtained from Genbank and compared with hsp47 from chicken, mouse, rat, human and zebrafish. <i>Xenopus</i> HSP47 protein had an identity of approximately 77% with chicken, 73% with mouse, 72% with rat and human, and 70% with zebrafish. Most of the sequence identity between HSP47 from all investigated organisms occurred centrally in the amino acid sequence and in several carboxyl terminal regions. Three key features were conserved between HSP47 proteins from most species investigated: a hydrophobic leader sequence, two potential glycosylation sites and the ER-retention signal, RDEL. A partial cDNA clone encoding <i>Xenopus</i> hsp47 was obtained from the American Type Culture Collection (ATCC) and used to generate hsp47 antisense riboprobe for the purpose of investigating hsp47 mRNA accumulation in <i>Xenopus</i> A6 kidney epithelial cells and embryos. Northern blot analysis detected hsp47 mRNA constitutively in A6 cells. The expression pattern for hsp47 mRNA was compared with two other <i>Xenopus</i> heat shock proteins that have been previously characterized in our laboratory: hsp70, a cystolic/nuclear hsp and BiP, an ER-resident hsp. The results of hsp47 mRNA accumulation in A6 cells suggested that the expression pattern for <i>Xenopus</i> hsp47 was unique but, with respect to some stressors, resembled that of a cytosolic hsp rather than an ER-resident hsp. HSP47 protein levels were also examined in A6 cells. Heat shock, sodium arsenite and b-aminopropionitrile fumerate treatments enhanced hsp47 accumulation. In some experiments, western blot analysis revealed the presence of two closely sized protein bands. It is possible that minor differences in HSP47 protein size may be due to post-translational modification, namely phosphorylation or glycosylation. The present study also examined the accumulation and spatial pattern of hsp47 mRNA accumulation during <i>Xenopus laevis</i> early development. Hsp47 was constitutively expressed throughout <i>Xenopus</i> early development. Constitutive levels of hsp47 mRNA in unfertilized eggs, fertilized eggs and cleavage stage embryos indicated that these transcripts were maternally inherited. Constitutive hsp47 mRNA accumulation was enhanced in neurula and tailbud embryos compared to earlier stages. This finding may be explained by the shift towards organogenesis during these stages. Whole mount <i>in situ</i> hybridization revealed hsp47 message along the dorsal region of the embryo, in the notochord and somites, as well as in the head region including the eye vesicle. Hsp47 mRNA induction in <i>Xenopus</i> embryos was also examined in response to heat shock. Hsp47 mRNA accumulated in response to heat shock immediately following the midblastula transition (MBT). In tailbud stages, hsp47 mRNA accumulated in the notochord, somites and head region. Northern blot analysis and whole mount <i>in situ</i> hybridization results revealed an expression pattern that coincided well with the development of collagen-rich tissues thereby substantiating the proposed role of HSP47 as a procollagen molecular chaperone.

<p>The proper modification and trafficking of plasma membrane proteins are essential for normal neuronal function, such as dendrite morphogenesis, spine formation and synaptic plasticity. The secretory organelles including endoplasmic reticulum and Golgi apparatus are critical for the trafficking of these molecules as shown in fibroblasts. Although these secretory organelles have been observed in neurons including dendritic branches, their spatial organization and function in protein trafficking, neuronal development and plasticity are not clear yet. Here, I used photobleaching and photoactivation approaches combined with electron microscopy to show that although rapidly diffusing within the continuous network of the somato-dendritic ER, membrane proteins such as nascent AMPA receptors are confined by ER spatial complexity. The spatial range of ER membrane protein mobility becomes progressively confined over neuronal development and is rapidly restricted by synaptic activity. Thus, constrained lateral mobility within the ER provides a novel mechanism for compartmentalized trafficking of nascent receptors throughout dendrites. I also identified an ER protein as a novel microtubule-associated protein regulating dendritic ER spatial complexity, neuronal dendrite elongation and spine formation. Together, these results describe the spatial organization of dendritic ER and its role in regulating membrane protein trafficking, neuronal morphogenesis and postsynaptic functions.</p> / Dissertation