Résumé : Calnuc est une protéine ubiquitaire qui lie le calcium et qui est présente au réseau trans-golgien (TGN) ainsi qu'aux endosomes. Notre groupe a précédemment mis en évidence le rôle de Calnuc dans le transport de Low density lipoprotein receptor-related protein 9 (LRP9), un récepteur aux lipoprotéines de faible densité qui cycle entre le TGN et les endosomes. Les récepteurs lysosomiaux au mannose-6-phosphate (MPR) et Sortiline sont bien caractérisés et empruntent également cette voie. À l'image de LRP9, nous avons montré que Calnuc prévient leur dégradation aux lysosomes en participant à leur recyclage à partir des endosomes vers le TGN. En fait, Calnuc est importante pour l'activation et l'association membranaire de Rab7, une petite protéine G qui recrute ensuite le complexe Rétromère responsable du transport rétrograde des récepteurs. La glycoprotéine lysosomiale Ceroid lipofuscinosis neuronal 5 (CLN5) est également impliquée dans ce processus. La structure et la fonction de cette dernière n'étant pas clairement définies, nous avons établi qu'elle est synthétisée sous forme d’une glycoprotéine transmembranaire de type II, mais son domaine N-terminal cytoplasmique et son segment transmembranaire sont rapidement éliminés suivant le clivage du peptide signal de manière à former une protéine CLN5 mature fortement associée à la membrane par une hélice amphipathique (AH). La compréhension des propriétés de base de CLN5 est particulièrement pertinente puisque la protéine est impliquée dans certaines variantes de céroïdes-lipofuscinoses neuronales (NCL), une maladie neurodégénérative rare causée par une surcharge des lysosomes. D'ailleurs, nos données indiquent que les mutants pathologiques de CLN5 dépourvus de cette AH perdent leur association membranaire, sont retenus au réticulum endoplasmique et sont rapidement dégradés. En raison de la similitude des fonctions de Calnuc et de CLN5 au niveau du triage endosomial, nous avons exploré le lien entre les deux protéines. Calnuc cytosolique et CLN5 luminale semblent former un complexe, par l'intermédiaire de la protéine transmembranaire CLN3, de façon à influencer l'activité de Rab7. CLN3 étant aussi associée aux NCL, nous avons finalement exploré la potentielle implication de Calnuc dans la maladie. L'absence de Calnuc entraîne des phénotypes cellulaires typiques des NCL comme un engorgement des lysosomes, une accumulation de matériel autofluorescent et une augmentation de l'autophagie. Les niveaux protéiques de Calnuc sont diminués dans toutes les lignées de fibroblastes de patients atteints de NCL disponibles ce qui indique que Calnuc pourrait être impliquée dans certains types de NCL. La présente thèse couvre donc la découverte de la fonction de Calnuc dans le transport intracellulaire, jusqu'à son implication potentielle dans les NCL, de même qu'une étude topologique de CLN5. / Abstract : Calnuc is a ubiquitous Ca2+-binding protein present on the trans-Golgi network (TGN) and endosomes. We previously highlighted the role of Calnuc in the transport of Low density lipoprotein receptor-related protein 9 (LRP9), a low density lipoprotein (LDL) receptor that cycles between the TGN and endosomes. Lysosomal receptors mannose-6-phosphate receptor (MPR) and Sortilin are well-characterized and also use the TGN-to-endosome trafficking pathway. Similarly to LPR9, we showed that Calnuc prevent their degradation in lysosomes by acting in their recycling from endosomes to the TGN. In fact, Calnuc is a important for the activation and the membrane association of Rab7, a small G protein which then recruit the Retromer complex known to be responsible for the retrograde transport of receptors. Lysosomal glycoprotein Ceroid lipofuscinosis neuronal 5 (CLN5) is also involved in this process. Because its structure and function have not yet been clearly defined, we established that it is synthesized as a type II transmembrane (TM) glycoprotein, but its cytoplasmic N-terminus and TM segment are rapidly removed following signal-peptide cleavage to generate mature CLN5 which is tightly associated to membrane through an amphipathic helix (AH). The understanding of the basic properties of CLN5 is particularly important given that CLN5 is involved in some variants of neuronal ceroid lipofuscinosis (NCL), a rare neurodegenerative disease caused by lysosomal overload. Moreover, our data indicate that CLN5 pathological mutants deprived of AH lose their membrane association, are retained in the endoplasmic reticulum, and are rapidly degraded. Based on the similarity featured by Calnuc and CLN5 in endosomal sorting, we explored the link between these two proteins. Cytosolic Calnuc and luminal CLN5 seem to form a complex, through the transmembrane protein CLN3, in order to influence the activity of Rab7. As CLN3 is also associated with NCL, we finally explored the potential involvement of Calnuc in this disease. Canuc depletion leads to typical NCL phenotypes such as lysosome enlargement, accumulation of autofluorescent material and of an increased of autophagy induction. Canuc's levels are decreased in all fibroblasts cell lines of NCL patients available indicating that Calnuc could be involved in some types of NCL. This thesis thus covers the discovery of the function of Calnuc in intracellular transport up to its potential involvement in the NCL, as well as a topological study CLN5.

Master of Science / Department of Biology / Stella Y. Lee / CLN5 is one of several proteins that when mutated result in the lysosomal storage disorder (LSD) Neuronal Ceroid Lipofuscinosis (NCL). CLN5 is a soluble lysosomal protein that has no known function at this time. Previously we showed that eight asparagine residues in CLN5 are N-glycosylated, and that this modification is important for the protein’s transport and function. Now, we have identified a link between the activation of autophagy and CLN5 deficiency. The autophagy-lysosomal protein degradation system is one of the major pathways the cell uses to degrade intracellular material and recycle cellular building blocks. It was recently shown that other CLN proteins affect the relative level of autophagy, indicating a potential link between the autophagy pathway and the NCLs. By knocking down endogenous CLN5 in HeLa we showed that, upon stress induction, cells responded with higher levels of autophagy activation. Consistent with these knockdown experiments, there is a higher level of the autophagy marker protein, LC3-II, in CLN5 patient cells that are naturally deficient for the CLN5 protein. Pharmaceutical induction of autophagy through different means also showed higher LC3-II levels compared to control, though patterns differed in the type of autophagy induced. In summary, we discovered that the autophagy pathway is altered in CLN5 deficient cells, indicating a potential role for CLN5 in autophagy. Further analyses of the autophagy pathway will shed light on where CLN5 is acting and the mechanism by which defective CLN5 causes NCL.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Stella Yu-Chien Lee / Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative lysosomal storage disorders which is the most frequent group of inherited neurodegenerative disorders that affect children leading to severe pathological conditions such as progressive loss of motor neuron functions, loss of vision, mental retardation, epilepsy, ataxia and atrophy in cerebral, cerebella cortex and retina and eventually premature death. Among the many genes that cause NCL, mutations in CLN5 leads to different forms of NCL (infantile, late infantile, juvenile and adult) and mutations in CLN8 leads to progressive epilepsy with mental retardation (EPMR) and a variant late infantile form of NCL. The function(s) of both CLN5 and CLN8 proteins remain elusive. CLN5 is a glycosylated soluble protein that resides in the lysosome. We observed that endogenous CLN5 protein exist in two forms and identified a previously unknown C-terminal proteolytic processing event of CLN5. Using a cycloheximide chase experiment we demonstrated that the proteolytic processing of CLN5 is a post-translational modification. Furthermore treatment with chloroquine showed the processing occurs in low pH cellular compartments. After treatment with different protease inhibitors our results suggested the protease involved in the processing of CLN5 could be a cysteine protease. Using two glycosylation mutants of CLN5, retained in the endoplasmic reticulum (ER) or the Golgi we showed the proteolytic processing occurs in an organelle beyond the ER. This study contributes to understanding the characteristics of the CLN5 protein. CLN8 is an ER resident transmembrane protein that shuttles between the ER and the ER-Golgi intermediate compartment (ERGIC). In our study we identified a potential interaction between CLN8 and a PP2A holoenzyme complex consisting regulatory subunit A α isoform and regulatory subunit B α isoform. Using two CLN8 patient derived fibroblast cell lines we were able to show that the phosphorylated levels of PP2A target kinase Akt was reduced at both of its regulatory sites Ser473 and Thr308 and the activity of PP2A was increased. A delay of ceramide transport from ER to Golgi in CLN8 deficient patient cell lines was observed using BODIPY FL C5-Ceramide staining. Our results provide evidence for CLN8 protein being involved in the regulation of PP2A activity and trafficking of ceramide from ER to Golgi.

Master of Science / Division of Biology / Stella Y Lee / Neuronal Ceroid Lipofuscinoses (NCLs) are a group of lysosomal storage diseases that are characterized by accumulating autofluorescent lipopigments in cells. NCLs are a form of progressive neurodegenerative diseases with symptoms ranging from blindness, loss of speech and motor activities to ataxia and seizures. Patients do not live to adulthood in most cases, making it prevalent in children. Among the many genes that cause NCL, CLN5 leads to different forms of NCL (infantile, late infantile, juvenile, and adult). CLN5 protein resides in the lysosomes but its function has not been established. It is predicted to contain eight N-glycosylation sites, but the role of N-glycosylation on its function and trafficking has not been assessed. We analyzed the role of N-glycosylation on the transport and stability of human CLN5. We created N-glycosylation mutants of each site by changing the Asn to Gln and our analysis of these mutants show that all the eight N-glycosylation sites are used in vivo. We also report effects of abolishing individual N-glycosylation sites on the trafficking of CLN5. While the lack of glycosylation at some sites results in CLN5 being retained in the ER or Golgi, others do not affect CLN5 trafficking. Cycloheximide chase experiments show that one of the mutants (N401Q) in CLN5 leads to lower protein levels in cell pellets with an increased secretion compared to CLN5 wild type, while other mutations show differential stability in cell pellets. These results demonstrate that each N-glycosylation site plays a different role(s) in the stability, transport and/or function of CLN5.

No description available.

Le tri et le transport efficace des hydrolases acides vers le lysosome jouent un rôle critique pour la fonction des cellules. Plus de 50 maladies humaines sont dues à des mutations des enzymes lysosomales, des protéines régulant des processus-clés du transport vers le lysosome ou des enzymes effectuant des modifications posttraductionnelles importantes pour la fonction du lysosome. L’objectif de cette thèse est d’identifier des protéines et des mécanismes permettant à la cellule de réguler le transport des enzymes vers le lysosome. Nous avons formulé l’hypothèse que des protéines mutées dans des maladies lysosomales et dont les fonctions étaient inconnues pouvaient jouer un rôle dans le transport vers le lysosome. Les céroïdes-lipofuscinoses neuronales forment une famille de maladies lysosomales rares mais sont aussi les maladies neurodégénératives infantiles les plus fréquentes. Plusieurs gènes impliqués dans les NCL encodent des protéines aux fonctions inconnues. Les travaux présentés dans cette thèse ont identifié la protéine « ceroid lipofuscinosis neuronal-5 » (CLN5) qui est localisée à l’endosome et au lysosome comme élément nécessaire au recrutement et à l’activation de rab7. Rab7 est une protéine Rab-clé qui contrôle le trafic à l’endosome tardif. Cette petite GTPase est impliquée dans le recrutement de retromer, un complexe protéique qui régule le trafic de l’endosome vers l’appareil de Golgi des récepteurs de tri lysosomal comme sortilin et le récepteur du mannose-6-phosphate. Dans les cellules où CLN5 est déplété, les récepteurs de tri lysosomal sont moins recyclés plus rapidement dégradés. En utilisant des expériences de photomarquage nous avons aussi pu démontrer que Rab7 est moins activées en l’absence de CLN5. Pour exécuter leur fonction les protéines rabs doivent être recrutée à la membrane et activées par l’échange d’une molécule de GDP pour une molécule de GTP. Le recrutement des Rabs à la membrane nécessite une modification posttraductionnelle lipidique pour être facilités. En utilisant un modèle de levures nous avons démontré que l’homologue de Rab7, Ypt7 est palmitoylée. Nous avons aussi démontré que la palmitoyltransférase Swif1 est nécessaire au recrutement de Ypt7 à la membrane. Nous avons aussi remarqué que les sous- unités de retromer chez la levure sont moins recrutées lorsque les palmitoyltransférases sont déplétées. Dans les cellules de mammifères nous avons démontré que Rab7 est également palmitoylé et que cette palmitoylation est possiblement effectuée par les palmitoyltransférases DHHC1 et DHHC8. La palmitoylation de Rab7 a lieu sur les cystéines en C-terminal qui sont nécessaires au recrutement membranaire et qui auparavant étaient uniquement décrites comme prénylées. En utilisant la méthode de « click chemistry » nous avons découvert que lorsque la prénylation de Rab7 est bloquée le niveau de palmitoylation augmente. Pour caractériser l’interaction entre CLN5 et Rab7 nous avons performé des expériences afin d’établir définitivement la topologie de cette protéine. Nous avons ainsi démontré que CLN5 est une protéine hautement glycosylée qui est initialement traduite en protéine transmembranaire et subséquemment clivée par un membre de la famille des peptidase de peptide signal (SPP). Cette protéine soluble peut alors possiblement interagir avec CLN3 qui est aussi palmitoylée pour recruter et activer Rab7. Nos études suggèrent pour la première fois que CLN5 pourrait être un recruteur et un activateur de Rab7 qui agirait avec la protéine CLN3 pour séquestrer Rab7 avec les autres récepteurs palmitoylés et permettre leur recyclage vers l’appareil de Golgi. / The proper sorting and trafficking of acid hydrolases plays a critical role in the normal function of cells. Over 50 known human diseases are caused by mutations of lysosomal enzymes, of proteins that regulate key processes of transport to the lysosome or of enzymes that perform posttranslational modifications which are important for the function of the lysosome. The main objective of this thesis is to identify proteins and mechanisms that allow the cell to regulate the transport of enzymes toward the lysosome. We formulated the hypothesis that proteins mutated in lysosomal diseases and that have no known functions could play a role in transport toward the lysosome. Neuronal ceroid-lipofuscinoses form a family of lysosomal storage disorders that are very rare but are also the most frequent infantile neurodegenerative diseases. The work presented in this thesis identified ceroid-lipofuscinosis neuronal-5 (CLN5), which is located at the late-endosomal/lysosomal compartment as a necessary element for the recruitment and activation of Rab7. Rab7 is an important GTPase that controls traffic from the late-endosome to the trans-Golgi network. Rab7 has been implicated in the recruitment of the retromer complex, which regulates retrograde transport of the lysosomal sorting receptor such as sortilin and the mannose-6-phosphate receptor. In the cells where CLN5 is depleted, the lysosomal sorting receptors are less recycled and degraded more rapidly. Using photolabelling assays we were also able to show that Rab7 is less activated in the absence of CLN5. To perform their function, Rab proteins have to be recruited to membranes and activated by the exchange of a GDP nucleotide for GTP. The recruitment of Rabs to membranes necessitates a lipidic posttranslational modification to raise the affinity. Using yeast as a model we demonstrated that the Rab7 homolog, Ypt7 is palmitoylated. We have also showed that the yeast palmitoyltransferase Swif1 is required for Ypt7 membrane recruitment. We have also observed that retromer subunits in yeast are less recruited when palmitoyltranferases are depleted. In mammals we have shown that Rab7 is also palmitoylated and that this palmitoylation may be done by palmitoyltransferases DHHC1 and DHHC8. The palmitoylation of Rab7 occurs on the C-terminal cysteines that are required for membrane recruitment and were previously only shown to be prenylated. By using Click chemistry we have discovered that when Rab7 prenylation is blocked the level of palmitoylation is augmented. To characterize the interaction of Rab7 and CLN5 we performed experiments to definitively establish the topology of this latter protein. Our results show that CLN5 is a heavily glycosylated protein that is initially translated as a type II transmembrane protein and subsequently cleaved by a member of the signal-peptide peptidase (SPP) family. This protein can then possibly interact with another member of the CLN family, CLN3 that is predicted to be palmitoylated to recruit and activate Rab7. Our studies establish for the first time that CLN5 is required for the recruitment and activation of Rab7 and may cooperate with the possibly palmitoylated protein CLN3 to sequester Rab7 in specific membrane domains with sorting receptors to allow their recycling toward the trans-Golgi network.