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Inhibition of TFEB activation promotes Coxiella burnetii growthDas Ghatak, Piya 05 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Coxiella burnetii is the etiologic agent of Q fever, a zoonotic disease characterized by flu-like sickness in acute cases; endocarditis may occur and turn deadly if not treated correctly in chronic patients.
Coxiella, an obligate intracellular bacterium, requires establishment of a replicative niche in the host cell. After being phagocytosed by the eukaryotic cell, the bacterium resides in a tight-fitting nascent phagosome which matures through the host canonical endocytic pathway, acquiring endosomal/lysosomal markers as well as acidic pH. Initial acidification of the Coxiella containing vacuole (CCV) is central to the bacterium’s pathogenesis because translocation of bacterial effector proteins into the host cell by the type 4B secretion system (T4BSS) initiates only after it senses the acidic environment. The effector proteins are required for subverting different host cell functions in favor of Coxiella growth, CCV maturation and are crucial for bacterial virulence.
Contrary to the belief that since CCV matures through the host endocytic pathway, CCV is as acidic as lysosome, we found that CCV is significantly less acidic (pH~5.2) than lysosomes (pH~4.8) and inducing further CCV acidification causes Coxiella lysis. Furthermore, increasing lysosomal biogenesis in the host cell is detrimental for Coxiella growth. So, we hypothesized that Coxiella blocks lysosomal biogenesis in host cells to maintain the CCV pH just optimal for its growth.
Lysosomal biogenesis is regulated by the master transcription factor EB (TFEB). Its ability to act as a transcription factor depends on its subcellular localization, which relies on its phosphorylation state. TFEB, when phosphorylated is cytosolic and inactive, whereas dephosphorylated TFEB translocates to the nucleus and is active, binding to promoter regions of lysosomal genes of the CLEAR network, thus controlling lysosome biogenesis. Therefore, we hypothesized that Coxiella blocks TFEB translocation to the nucleus, thus inhibiting lysosome biogenesis.
We determined that Coxiella grows significantly better in TFEB-KO cells than they do in parentals. Also, using a torin-induced TFEB translocation model, we observed remarkably decreased TFEB activation in the Coxiella infected cells as was evident by less TFEB translocation to nucleus. Overall, data obtained from this work suggest that Coxiella inhibits lysosome biogenesis by blocking TFEB nuclear translocation.
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The physiological role of P2X4 receptors in lysosome functionTan, Sin Lih January 2017 (has links)
P2X4 receptors (P2X4R) are ligand-gated ion channels activated by ATP and with a high permeability to Ca2+. They are predominantly localised to lysosomes and from there can traffic to the cell surface. ATP levels within the lysosome are high but P2X4Rs are inhibited by the acidic pH. Previously, it was shown that the alkalinisation of lysosomes using pharmacological reagents was sufficient to activate P2X4Rs, which promoted homotypic lysosome fusion. The main aim of this study was to identify physiological regulators of lysosomal P2X4Rs and to examine their role in lysosome Ca2+ signalling and fusion. The first candidate I investigated was P2X7R, which is typically co-expressed with P2X4R in immune and epithelial cells, and which has already been shown to induce changes in lysosome properties upon activation. I co-expressed these two receptors in normal rat kidney (NRK) cells and in HeLa cells and looked for a synergistic interaction between them in promoting lysosome fusion, as assessed by measuring the size of lysosomes. My results showed a significant increase in lysosome size following activation of P2X7R but only in the presence of P2X4R. Neither receptor alone was sufficient to promote lysosome fusion in response to the agonist BzATP. LAMP-GECO was used to measure changes in cytosolic [Ca2+] within the vicinity of the lysosome. Fusion of the Ca2+ reporter (GECO) to the C-terminus of LAMP-1 targets GECO to the cytosolic surface of the lysosome. Co-expression of P2X4R with P2X7R augmented the P2X7R-induced Ca2+ signal suggesting that P2X4Rs mediate lysosomal Ca2+ efflux downstream of P2X7R stimulation. Next, I showed that the expression of P2X4R was sufficient to enhance the cytosolic Ca2+ response to the activation of endogenous histamine H1 receptors and to promote lysosome fusion. Similar results were obtained with P2Y2R stimulation, which also couples to the phospholipase C pathway. Further experiments were conducted to look at differences in the trafficking behaviour of human and rat P2X4Rs and to examine a role for P2X4Rs in autophagic flux. My results suggest a synergistic interaction between P2X4R and P2X7R which inhibits autophagic flux, similar to the effect of bafilomycin treatment. Therefore, the effect of P2X4/7R in autophagy may be mediated by the alkalinisation of lysosomes. Altogether the results of my project improve our understanding of how the P2X4R Ca2+ channel regulates lysosome function.
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The dynamics and function of the endolysosomal/lysosomal systemDavis, Luther John January 2019 (has links)
Lysosomes are intracellular organelles that were considered for a long time to be simply an acidic and hydrolytically active end point of trafficking routes for degradation, in the last 20 years, light has been shed on their functional heterogeneity and striking role in signalling and nutrient homeostasis. While the dynamic nature and variety of lysosomal functions are now better appreciated, the mechanisms governing lysosomal fusion, reformation, signalling, and homeostasis remain to be fully elucidated, and are investigated here. In this study, endolysosomes which formed by fusion of late endosomes with lysosomes and are thought to be the predominant site of hydrolytic activity, were further characterised. Using live cell imaging and fluorescent labelling, the proportion of endolysosomes in the total pool of lysosomes was estimated using probes to their acidity and cathepsin activity, and their larger size compared to storage lysosomes was observed. The endolysosomal membrane was also shown to be marked by Rab7, Rab9, PI(3,5)P2 supporting the role of endolysosomes a highly active and dynamic principal site of hydrolase activity. The contributions of VAMP7 and VAMP8 to endolysosome fusion, measured by delivery of endocytosed cargo from late endosomes to endolysosomes, were analysed by CRISPR-Cas9 mediated knockout. Cells lacking VAMP7 and VAMP8 had no effect on delivery to endolysosomes, however at EM level, they displayed extensive tethering between late endocytic organelles, and accumulated small tethered vesicles. YKT6 knockdown impeded delivery to endolysosomes in VAMP7+VAMP8 knockout cells, which was rescued by VAMP7 expression, suggesting YKT6 substituted for VAMP7 in lysosome fusion. Following the hypothesis that reversible dissociation of V1 and Vo sectors of the V- ATPase may control the increase in pH of reforming storage lysosomes, cells expressing tagged V1G1 and Voa3 were generated. These markers of both sectors are present on endolysosomal membranes, and on the emerging endolysosomal tubules, suggesting the V1 and Vo sectors remain associated at this earliest stage of lysosome reformation, but these markers are still in development. IV Two assays were developed to give a readout of, and assess lysosomal stress. Firstly, an assay measuring TFEB-GFP translocation to the nucleus gave a robust and quantifiable readout of lysosomal perturbation. Secondly, a qPCR assay was developed to measure lysosomal gene upregulation as a downstream reporter of TFEB-activating lysosomal perturbations, however this assay, despite being more lysosome-specific, lacked the consistency and dynamic range of the TFEB translocation quantification. In summary, lysosomes are a heterogeneous collection of organelles, which have been better characterised primarily according to their acidity and hydrolytic capacity. Additionally, more SNAREs appear to be involved in lysosome fusion in cells than suggested by cell free assays, and I have developed tools to trace the V-ATPase during reformation of lysosomes after fusion to form endolysosomes. Lastly, I have developed a robust, reporter for a range of lysosomal stress-inducing conditions, providing a broad indication of their effects on lysosomal signalling and homeostasis.
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Substrate deprivation as a novel therapy for the mucopolysaccharidoses.Roberts, Ainslie Lauren Kemp January 2007 (has links)
Reduction of an enzyme required for the lysosomal degradation of glycosaminoglycan (gag) chains will result in a mucopolysaccharidosis (MPS) disorder. Substrate deprivation therapy (SDT), a new therapy option for MPS, aims to reduce the synthesis of gag chains, the natural substrate for the deficient enzyme. Reduced substrate levels would balance the reduced level of enzyme in patient cells resulting in normalised gag turnover. Rhodamine B, a non-specific inhibitor, reduced gag synthesis in a range of normal and MPS cells and also decreased lysosomal storage of gag in MPS VI (72%) and MPS IIIA (60%) cells. This positive response in vitro was extended to an in vivo therapy trial in the MPS IIIA mouse. Bodyweight gain of male MPS IIIA mice treated with 1 mg/kg rhodamine B was reduced compared to untreated MPS IIIA mice and was indistinguishable to that of normal mice. Liver size, total gag content and lysosomal gag was reduced in treated MPS IIIA animals as was urinary gag excretion. The alteration in MPS IIIA clinical pathology by rhodamine B combined with the observation that treatment had no effect on the health of normal animals demonstrates the potential for this type of therapy for MPS disorders. The water cross maze was found to be the only learning and memory test capable of detecting differences in learning behaviour in MPS IIIA and normal untreated mice. MPS IIIA mice treated with SDT rhodamine B showed an improved outcome with better learning capabilities than MPS IIIA untreated mice observed using this test. This means that rhodamine B is likely to cross the blood-brain barrier. These results are the first evidence of a positive response by the CNS to a systemic therapy for MPS IIIA. Rhodamine B administration over 4 generations did not produce any deleterious side effects in MPS IIIA. In utero therapy over four generations did not cause a reduction in litter size or bodyweight profile demonstrating that reduction of gag over a combined timeframe of two years was safe. A higher dose of 5 mg/kg rhodamine B did not produce any additional benefits on MPS IIIA pathology and no signs of hepatoxicity were noted. Rhodamine B proved to be a general inhibitor of gag synthesis and had a positive outcome on a number of clinical parameters in MPS IIIA mice. SDT in MPS IIIA mice improved learning capabilities as detected by the water cross maze which has not been previously reported. This provides evidence that small molecules such as rhodamine B, that are able to cross the blood-brain barrier, can have some effect on neurological pathology. This proof of principle study showed that SDT can be used to have a positive outcome on MPS pathology. Additional inhibitors of gag synthesis can also be investigated before this type of therapy can be translated into clinical use in MPS patients. Although it may be feasible to use rhodamine B as a SDT agent in vivo, other inhibitors may be more practical. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1287050 / Thesis (PhD) -- School of Paediatrics and Reproductive Health, 2007.
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Caractérisation de p40, une protéine identifiée dans une préparation de membranes lysosomalesBoonen, Marielle 20 December 2007 (has links)
Le lysosome est un organite acide qui contient de nombreuses hydrolases responsables de la dégradation d’une grande variété de molécules issues majoritairement des voies d’endocytose et d’autophagie. La membrane de l’organite contient, elle aussi, un certain nombre de protéines qui assurent diverses fonctions telles que la génération d’un gradient de pH intralysosomal, la translocation de produits de dégradation vers le cytosol, la régulation des contacts membranaires, etc. Cependant, au vu de la grande diversité des produits générés par l’hydrolyse intralysosomale et susceptibles d’être transportés vers le cytosol, et de la complexité des mécanismes de contact/fusion/fission qui impliquent la membrane lysosomale, il est hautement probable que de nombreuses protéines membranaires restent à être identifiées. Nous avons mis en évidence un nouveau candidat membranaire lysosomal au cours d’une analyse protéomique réalisée sur des fractions enrichies en membranes de lysosomes. Cette protéine de 372 acides aminés, que nous avons dénommée p40, présente de nombreux segments transmembranaires prédits, quatre motifs potentiels d’adressage aux lysosomes et une homologie de séquence partielle avec des transporteurs de sucres nucléotidylés. Nous avons recherché la localisation de p40 dans la cellule par des techniques de fractionnement subcellulaire, appliquées sur le foie de souris. Nous avons comparé la distribution de cette protéine au sein des fractions obtenues par centrifugation différentielle et par centrifugation isopycnique sur un gradient continu de saccharose (avec et sans traitement préalable de la souris avec du Triton WR-1339) à celles de protéines marqueurs des organites. Nous avons combiné cette approche à une analyse morphologique par microscopie à fluorescence confocale de la localisation de p40 dans des cellules eucaryotes transfectées. Nos résultats nous ont permis de démontrer la localisation lysosomale de p40. D’autre part, nous avons mis en évidence que le temps de demi-vie de p40 est d’environ 10 heures et que p40 est une protéine dépourvue de chaînes oligosaccharidiques, fortement associée aux membranes. Nous avons ensuite entrepris de rechercher une possible activité de transport pour p40, suggérée par l’homologie de séquence détectée avec des transporteurs connus. Pour cela, nous avons utilisé une technique d’électrophysiologie perfectionnée, bien adaptée à la réalisation d’un large criblage de substrats potentiels d’une hypothétique activité de transport. Bien que nous ayons testé près de 70 composés au total, nous n’avons pas mis en évidence la fonction de p40 par cette approche. Dans un troisième temps, nous nous sommes intéressés à la façon dont la protéine p40 néosynthétisée est spécifiquement ciblée vers les lysosomes. Nous avons muté les quatre motifs potentiels d’adressage aux lysosomes présents dans la séquence de p40 et nous avons observé l’impact de ces mutations sur sa localisation subcellulaire. Ces travaux ont révélé que l’envoi de p40 vers les lysosomes est assuré par un signal de type « dileucine » situé 11 résidus en amont de l’extrémité C-terminale de la protéine : le motif EQERL360L361. En effet, la substitution des deux résidus leucine critiques par d’autres acides aminés abolit le transport de la protéine p40 vers les lysosomes et induisait son envoi vers la membrane plasmique.
En conclusion, notre recherche visant à caractériser p40 montre que cette protéine nouvellement identifiée est une protéine membranaire lysosomale non glycosylée, ciblée vers les lysosomes à partir du TGN par un signal d’adressage EQERL360L361 localisé dans sa queue C-terminale. Des études complémentaires sont nécessaires afin de cerner la fonction assurée par p40 au sein de la membrane des lysosomes.
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Changes in the Lysosomal Proteome Under Stressed ConditionsO'Connor, Kaela 07 September 2022 (has links)
Autophagy is a critical cellular process that is implicated in a vast array of human diseases, including amyotrophic lateral sclerosis (ALS). The targets of this degradative mechanism are frequently altered depending upon the inducing condition, however exactly how these targets change has rarely been explored on a proteomic level. To assess the targets of autophagy, this project analyzed the proteome of lysosomes collected by immunoprecipitation from cells undergoing starvation or recovering from oxidative stress. Oxidative stress induces stress granule formation and is frequently used as a cellular model for ALS. Our results show that while lysosomes from these two conditions retain a similar array of traditionally lysosomal proteins, they nevertheless display substantial differences indicating that their autophagic targets differ. Using Ingenuity Pathway Analysis (IPA) as well as gene ontology (GO) term analysis, the purine biosynthesis pathway was identified as being significantly enriched in lysosomes collected from sodium arsenite treated cells. The enrichment of the purine biosynthesis pathway was heavily dependent on ATG7, indicating these proteins localized to the lysosome primarily through macroautophagy. IMPDH2, the rate-limiting enzyme for guanylate production in the purine biosynthesis pathway drew particular attention due to its significant enrichment in sodium arsenite lysosomes, and its partial dependence on ATG7. Due to this discovery, and the presence of KFERQ-like sequences within the protein, our data suggests that IMPDH2 may localize to the lysosome through both macroautophagy and chaperone-mediated autophagy. Furthermore, numerous ALS-linked proteins were identified to be enriched in sodium arsenite lysosomes including SOD1, DCTN1, PFN1, TUBA4A, SQSTM1, VCP, CHMP2B, TDP-43, VAPB and TMEM106B. Their presence within the lysosome strengthens arguments that autophagy plays a key role in ALS. Overall, this project confirms the changing substrates of autophagy depending on the environmental condition and highlights the purine biosynthesis pathway as being involved in the cellular response to oxidative stress.
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Identifying Regulators of Lysosome Reformation: Inhibitor Screen in Mammalian Cell CultureLiu, Ian January 2016 (has links)
Lysosomes are membrane-bound organelles that have diverse functions in eukaryotic cells. Malfunctions in lysosomes result in a range of diseases known as Lysosomal Storage Disorders. After fusing with late endosomes to form hybrid organelles, lysosomes bud off and are reformed in a poorly characterized process known as lysosome formation or reformation. Only one mammalian regulator of lysosome formation has been identified, the non-selective cation channel TRPML1. In the highly similar process of Autophagic Lysosome Reformation (ALR), three known regulators have also been identified, the vesicle-coating protein clathrin and two phosphatidylinositol kinases that catalyze the formation of the membrane phospholipid PI(4,5)P₂. Here, we use an inhibitor screen coupled with a live imaging assay to identify the actin microfilament as a novel regulator of lysosome formation.
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Caractérisation fonctionnelle des transporteurs lysosomaux orphelins / Towards the Elucidation of Orphan Lysosomal TransportersVerdon, Quentin 29 June 2016 (has links)
Les lysosomes contiennent environ soixante hydrolases différentes, qui peuvent dégrader une grande variété de macromolécules. L’activité de ces enzymes est dépendante du pH, maintenu dans les lysosomes entre 4.5 et 5.0 par une pompe à protons : la v-ATPase. Les produits de dégradation sont recyclés dans le cytoplasme par des transporteurs actifs secondaires de la membrane des lysosomes.Les maladies de surcharges lysosomales sont causées par des mutations de gènes codant pour des protéines lysosomales, souvent des enzymes. Elles sont caractérisées par un engorgement des lysosomes avec des agrégats ou des cristaux. Les symptômes associés à ces maladies sont variés, mais la moitié d’entre elles induisent des défauts neurologiques. L’étude de ces maladies a permis d’élucider la fonction de nombreuses enzymes, mais la connaissance des transporteurs lysosomaux reste parcellaire. Peu de ces transporteurs sont ainsi caractérisés au niveau moléculaire.Je me suis intéressé à deux gènes dont la mutation provoque une maladie de surcharge particulière : CLN3 et CLN7. Leur mutation provoque des céroïdes lipofuscinoses neuronales, des maladies de surcharge lysosomales caractérisées par une neurodégénérescence précoce et par l’accumulation dans les lysosomes d’un pigment autofluorescent, la lipofuscine. La mutation de 14 gènes différents cause une céroïde lipofuscinose neuronale. J’ai étudié CLN3 et CLN7 car ils codaient pour des protéines membranaires du lysosome, qui pourraient donc être des transporteurs.Sur CLN7, j’ai effectué des tests de transport en utilisant les acides aminés comme substrats potentiels, sans résultats probants. Concernant CLN3, le contenu métabolique de lysosomes a été étudié par spectrométrie de masse dans des souris WT ou de souris où le gène CLN3 était déficient. Les lysosomes des cellules déficientes contenaient moins de produits de la protéolyse, ce qui suggérait que CLN3 était important pour la protéolyse lysosomale. Cela a été confirmé par des mesures plus directes sur des neurones et des fibroblastes primaires, et sur des fibroblastes immortalisés. Ces résultats pourraient aider à comprendre les premières étapes de la physiopathologie dans les cellules où des gènes CLN sont déficients.Pour accroître le nombre de transporteurs lysosomaux potentiels, j’ai participé à la finalisation d’une étude par protéomique de la membrane lysosomale. Elle a révélé 46 potentiels transporteurs de fonction encore inconnue. Dans cette liste, j’ai étudié TMEM104, SPINSTER, MFSD1, SLC37A2, TTYH3 et SNAT7. Pour ce faire, j’ai d’abord muté les motifs d’adressage de ces protéines pour les rediriger, lors de leur synthèse, vers la membrane plasmique, afin de faciliter leur étude. Aucune fonction claire n’a pu être identifiée par cette approche.SNAT7 appartenait cependant à une famille de transporteurs de glutamine, ce qui était suffisamment encourageant pour envisager d’autres approches. Sa fonction a ainsi été étudiée en développement un nouveau test indirect basé sur la détection d’une surcharge artificielle des lysosomes en acides aminés. Un test fonctionnel plus direct a ensuite été mis au point sur des fractions enrichies en lysosomes en utilisant des acides aminés radiomarqués. Ces deux tests ont montré que SNAT7 était un transporteur spécifique de l’asparagine et de la glutamine.J’ai enfin étudié l’hypothèse suggérant que SNAT7 pourrait jouer dans la nutrition de cellules cancéreuses. En effet, certaines utilisent la glutamine comme nutriment principal à la place du glucose ; mais les apports sanguins en glutamine, dans les tumeurs, sont parfois insuffisants. La glutamine est donc obtenue par macropinocytose de protéines extracellulaires et dégradation lysosomale de ces protéines, avant un recyclage vers le cytoplasme. J’ai montré qu’en l’absence de SNAT7, ce phénomène était bloqué. SNAT7 est donc une cible thérapeutique intéressante pour tenter de bloquer l’approvisionnement des cellules cancéreuses en glutamine. / Within lysosomes, about sixty different hydrolases degrade macromolecules. This degradation is dependent on the acidity of the lysosomal lumen, which pH ranges between 4.5 and 5.0. The lysosomal pH is maintained by the v-ATPase, a proton pump. Lysosomal degradation generates catabolites, which can be recycled to cytosol by secondary active transporters: lysosomal transporters.The dysfunction of lysosomal proteins leads to lysosomal storage disorders (LSDs), rare inherited metabolic diseases characterised by accumulation of material inside lysosomes. Depending on the mutated gene, symptoms of LSDs vary greatly, although about half of LSD patients display some kind of neurodegenerative symptoms. Studying the physiopathology of LSDs has led to a good understanding of the function of lysosomal enzymes, but the knowledge of lysosomal transporters remain poor, since only a few LSDs has been shown to be linked with a mutation in a lysosomal transporter gene.I focused on two proteins which dysfunction causes a special type of LSDs: CLN3 and CLN7. Mutations in CLN3 and CLN7 cause neuronal ceroid lipofuscinoses (NCLs), a special type of LSD which has mostly neurodegenerative symptoms and which is characterized by the accumulation of a specific pigment inside lysosomes: lipofuscin. There are fourteen NCL genes, but CLN3 and CLN7 are the two only proteins of the family which are resident proteins of the lysosomal membrane, suggesting they might be transporters.Amino acids were screened as possible substrates for CLN7, but none could be shown to be transported. For CLN3, the content in metabolites of lysosomes from Cln3-deficient mice and from WT mice were compared by mass spectrometry, revealing a specific decrease in the amount of catabolites of proteins in lysosomes from Cln3-deficient mice. This suggested a lack of lysosomal proteolysis, which was checked in neurons, in primary fibroblasts and in immortalized fibroblasts. These results suggested that CLN proteins could take part to a metabolic pathway important for lysosomal proteolysis and, more generally, for neuronal health. These results could help improve the understanding of the early steps of NCL physiopathology.To extend the number of candidates for lysosomal transporters, I took part to the validation step of an extensive proteomic study of the lysosomal membrane, which revealed forty-six new candidates for lysosomal transporters. I studied in more details TMEM104, SPINSTER, MFSD1, SLC37A2, TTYH3 and SNAT7. Proteins were overexpressed in HeLa cells to check for lysosomal localization. Then, their putative sorting motifs were mutated to misroute their expression to plasma membrane and to enable their functional study. No function could elucidate for the first five candidates.SNAT7 could not be misrouted to plasma membrane either, but, since it belonged to a family of transporters for glutamine, its function was studied by an indirect assay based on a lysosomal overload in amino acids and a direct transport measure on lysosome-enriched cellular fractions. Thus, SNAT7 was shown to be a lysosomal transporter selective for glutamine and asparagine.The function of SNAT7 is the nutrition of cancer cells was then studied. Many cancer cells use glutamine as their main source of carbon, nitrogen and energy. Because of insufficient blood supply, they use macropinocytosis to uptake extracellular proteins, which degradation in lysosomes generates glutamine. Then, glutamine is recycled to the cytosol. SNAT7 was shown to be critical in this process: in glutamine-dependent cancer cells, when SNAT7 expression is reduced, cells cannot obtain glutamine from extracellular proteins. Thus, blocking SNAT7 is a promising approach to target specifically the metabolism of cancer cells.
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Etude du rôle des lysosomes et du cholestérol au cours de la différenciation des kératinocytes épidermiques.Jans, Ralph 22 April 2004 (has links)
Etude du rôle des lysosomes et du cholestérol au cours de la différenciation des kératinocytes épidermiques (par Ralph Jans)
La majorité des rôles protecteurs de l’épiderme, couche superficielle de la peau, sont garantis par les kératinocytes qui se différencient de manière progressive et terminale dans les couches suprabasales de ce tissu, dont le renouvellement est assuré par la prolifération de cellules-souches dans la couche basale. Toute perturbation des mécanismes qui contrôlent la prolifération et/ou la différenciation des kératinocytes conduit à des dysfonctionnements. Comprendre ces mécanismes reste donc un défi majeur pour la biologie cutanée.
Plusieurs types cellulaires subissent l’exocytose de lysosomes quand il y a entrée d’ions calcium dans les cellules. Puisque les kératinocytes subissent une entrée de calcium au cours de leur différenciation, ces cellules pourraient exocyter des lysosomes dans ces circonstances. Un traitement de kératinocytes avec un ionophore induit en effet une sécrétion de la forme mature lysosomale de la cathepsine D, une libération de l’activité des enzymes lysosomales solubles cathepsine C et β-galactosidase, ainsi que l’apparition des protéines lysosomales membranaires Lamp-1 et Lamp-2 au niveau de la membrane plasmique. L’exocytose de lysosomes peut donc faire partie de la différenciation des kératinocytes, mais permet aussi à ces cellules de réparer des ruptures de la membrane plasmique.
Puisque le cholestérol pourrait contrôler certaines voies de signalisation au cours de la différenciation des kératinocytes, nous avons analysé les effets d’une déplétion en cholestérol induite par un traitement avec la méthyl-β-cyclodextrine sur le phénotype et la signalisation du kératinocyte. Cette déplétion induit une augmentation de l’expression de l’involucrine, marqueur de différenciation tardive, et une répression de l’expression de la kératine 10, marqueur de la différenciation précoce, et de la kératine 14, marqueur des kératinocytes non-différenciés. Ce traitement active le récepteur de l’EGF et HER2, ainsi que la MAP kinase p38 dont l’activation (en particulier l’activation de p38α) est responsable de l’augmentation de l’expression de l’involucrine.
En résumé, nos observations suggèrent un nouveau rôle pour les lysosomes au cours de la différenciation des kératinocytes épidermiques, mais suggèrent surtout un rôle critique du cholestérol dans la régulation de ce processus.
Role of lysosomes and cholesterol during the differentiation process of epidermal keratinocytes (by Ralph Jans)
A major part of the protective role of the epidermis, superficial layer of the skin, is guaranteed by the keratinocytes that differentiate progressively and terminally in the suprabasal layers of this tissue. The renewal of the epidermis is performed by proliferating stem cells in the basal layer. Perturbation of the mechanisms that regulate the proliferation and/or the differentiation of keratinocytes leads to an invalid barrier function. Therefore, elucidating these mechanisms is a major challenge for skin researchers.
In several cell types, lysosomes undergo exocytosis upon entry of calcium into the cells. Since keratinocytes are subjected to an entry of calcium during their differentiation in vivo, these cells could exhibit an exocytosis of lysosomes under these circumstances. The results presented in this work show that an incubation of keratinocytes with the calcium ionophore ionomycin triggers the secretion of the enzymatic activities of the lysosomal enzymes cathepsin C and β-galactosidase as well as the release of the lysosomal form of cathepsin D. This treatment also induces the appearance of the lysosomal membrane proteins Lamp-1 and Lamp-2 at the plasma membrane of keratinocytes. Exocytosis of lysosomes could be part of the keratinocyte differentiation process, but could also allow these cells to repair their plasma membrane upon disruption due to mechanical stresses.
Since cholesterol could be involved in the regulation of several signal transduction pathways during keratinocyte differentiation, we have investigated the effects of a depletion of cholesterol on the keratinocyte phenotype and on selected signaling pathways. Cholesterol depletion was induced by incubating the cells with methyl-β-cyclodextrin. This treatment, followed by an inhibition of cholesterol neosynthesis using lovastatin, triggers an upregulation of the expression of involucrin, a late differentiation marker, and a downregulation of keratin 10 and keratin 14, which, respectively, are markers of early-differentiating and undifferentiated keratinocytes. Cholesterol depletion activates the membrane receptors EGFR and HER2 and the MAP kinase p38. Using the specific inhibitor PD169316, we demonstrate that the p38α isoform is responsible for the upregulation of involucrin during cholesterol depletion. In summary, our observations suggest a novel role for lysosomes during keratinocyte differentiation and indicate a critical role for cholesterol in the regulation of this process.
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Analýza lysosomů Trichomonas vaginalis / Analysis of lysosomes of Trichomonas vaginalisZimmann, Nadine January 2021 (has links)
Lysosomes represent the central degradative compartment of eukaryote cells. Harboring a variety of acid hydrolases at acidic pH, this organelle is designed for the degradation and recycling of material for cellular homeostasis and sustenance. Studies on mammalian lysosomes have been extensive and revealed a long list of lysosomal proteins. While the function of most of these remains elusive, it is not surprising that a large subset have been found to be hydrolases. However, little is known about the biogenesis and function of this organelle in parasitic protists, and even less about its role in secretion. This work aimed to shed light on the (phago-)lysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting, and involvement in hydrolase secretion. Our studies revealed a lysosomal proteome of 462 proteins in 21 functional classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. The identification of a large set of proteins involved in vesicular trafficking and cytoskeleton rearrangement indicates a dynamic phagolysosomal compartment. Our research, as well as the research of others, have identified several hydrolases also in the secretome, including the cysteine protease TvCP2. However, previously the mode...
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