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Investigation of the Effects of Inhibiting N-glycosylation in CancerBeheshti Zavareh, Reza 06 December 2012 (has links)
Glycosylation, the addition of sugar moieties to nascent proteins, is one of the most common posttranslational modifications. Glycosylation regulates protein structure, function and localization. Most cell surface proteins and secreted proteins are glycosylated by the addition of Asparagine(N)-linked glycans (N-glycans). Aberrant N-glycosylation is a well-accepted feature of malignancy and is a potential prognostic marker for some types of cancer. For example, increased expression of complex N-glycans has been detected in cancers of breast, colon and has been correlated with reduced survival of the patients. Therefore, understanding the role of N-glycosylation in malignancy could be beneficial for developing novel therapeutic and prognostic strategies.
To examine the role of N-glycosylation in malignancy, we applied chemical biology and genetic approaches. First, we conducted a high throughput screen to identify compounds that could block L-PHA-induced cell death. Our screen identified the cardiac glycoside Na+/K+-ATPase inhibitors as novel inhibitors of N-glycosylation. Further analysis of N-glycans consistently confirmed that inhibition of Na+/K+-ATPase impairs the N-glycosylation, as well as migration and invasion. Interestingly, other studies have shown antimetastatic effects of cardiac glycosides in patients. Thus, our high throughput screen identified Na+/K+-ATPase inhibition as a novel strategy to target the N-glycosylation pathway.
In addition, we used a genetic approach to investigate the role of N-acetylglucosaminyltransferase I (GlcNAc-TI/Mgat1) in malignancy. Knockdown of GlcNAc-TI decreased the cell-surface expression of complex N-glycans. By confocal microscopy, knockdown of GlcNAc-TI decreased cell surface expression of β1 integrins and increased their localization around the nucleus. Moreover, GlcNAc-TI knockdown decreased the migration and invasion of malignant cells. Next, we investigated the effect of GlcNAc-TI in an orthotopic xenograft mouse model of metastasis. GlcNAc-TI knockdown significantly decreased the lung colony formation of the highly metastatic PC3N7 human prostate cancer cell line in mice. Our results suggest an important role for GlcNAc-TI in tumor metastasis. Interestingly, breast cancer patients with lower expression levels of Mgat1 had lower risk of disease relapse after therapy. Thus, GlcNAc-TI plays an important role in cancer progression and metastasis and GlcNAc-TI inhibitors could have therapeutic benefits for cancer patients. Moreover, expression levels of GlcNAc-TI could be used as a prognostic marker in patients with cancer.
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Identification of Genes Involved in the Assembly and Biosynthesis of the N-linked Flagellin Glycan in the Archaeon, Methanococcus maripaludisWu, JOHN 07 July 2009 (has links)
N-glycosylation is a metabolic process found in all three domains of life. It is the attachment of a polysaccharide glycan to asparagine (Asn) residues within the amino acid motif, Asn-Xaa-Ser/Thr. In the archaeon, Methanococcus maripaludis, a tetrasaccharide glycan was isolated from purified flagella and its structure determined by mass spectrometry analysis. The linking sugar to the protein is surprisingly, N-acetylgalactosamine (β-GalNAc), with the next proximal sugar a derivative of N-acetylglucosamine (β-GlcNAc), being named β-GlcNAc3Ac, and the third sugar a derivative of N-acetylmannosamine (β-ManNAc), with an attached threonine residue on the C6 carbon (β-ManNAc3NAm). The terminal sugar is an unusual diglycoside of aldulose ((5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose). Previous genetic analyses identified the glycosyltransferases (GTs) responsible for the transfer of the second and third sugars of the glycan, as well as the oligosaccharyltransferase (OST) which attaches the glycan to protein. Left unidentified were the first and fourth GTs, the flippase as well as any genes involved in glycan sugar biosynthesis and modification. In this work, genes suspected to be involved in the biosynthesis of N-linked sugars, as well as those that might encode the missing GTs and flippase were targeted for in-frame deletion. Mutants with a deleted annotated GT gene (MMP1088) had a small decrease in flagellin molecular weight as determined by immunoblotting. Mass spectrometry (MS) analysis confirmed that the N-linked glycan was missing the terminal sugar as well as the threonine found on the third sugar of wildtype cells. Mutants with a deleted gene annotated to be involved in acetamidino synthesis (a functional group that is present on the third sugar), also had a decrease in flagellin molecular weight. MS analysis determined that the N-linked glycan was missing the acetamidino group on the third sugar as well as its attached threonine, along with the terminal sugar. Both mutants were able to assemble functional flagella but had impaired motility compared to wildtype cells in mini-swarm agar. Deletions were also constructed in four other GT genes considered candidates in assembly of the linking sugar. However, none of these mutants had the expected decrease in flagellin molecular weight.
With the work done in this study, the glycosyl transferase that attaches the last sugar of the M. maripaludis N-linked assembly pathway has been identified as well as a gene involved in the biosynthesis and modification of the glycan sugars. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-07-07 15:45:19.052
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Investigation of the Effects of Inhibiting N-glycosylation in CancerBeheshti Zavareh, Reza 06 December 2012 (has links)
Glycosylation, the addition of sugar moieties to nascent proteins, is one of the most common posttranslational modifications. Glycosylation regulates protein structure, function and localization. Most cell surface proteins and secreted proteins are glycosylated by the addition of Asparagine(N)-linked glycans (N-glycans). Aberrant N-glycosylation is a well-accepted feature of malignancy and is a potential prognostic marker for some types of cancer. For example, increased expression of complex N-glycans has been detected in cancers of breast, colon and has been correlated with reduced survival of the patients. Therefore, understanding the role of N-glycosylation in malignancy could be beneficial for developing novel therapeutic and prognostic strategies.
To examine the role of N-glycosylation in malignancy, we applied chemical biology and genetic approaches. First, we conducted a high throughput screen to identify compounds that could block L-PHA-induced cell death. Our screen identified the cardiac glycoside Na+/K+-ATPase inhibitors as novel inhibitors of N-glycosylation. Further analysis of N-glycans consistently confirmed that inhibition of Na+/K+-ATPase impairs the N-glycosylation, as well as migration and invasion. Interestingly, other studies have shown antimetastatic effects of cardiac glycosides in patients. Thus, our high throughput screen identified Na+/K+-ATPase inhibition as a novel strategy to target the N-glycosylation pathway.
In addition, we used a genetic approach to investigate the role of N-acetylglucosaminyltransferase I (GlcNAc-TI/Mgat1) in malignancy. Knockdown of GlcNAc-TI decreased the cell-surface expression of complex N-glycans. By confocal microscopy, knockdown of GlcNAc-TI decreased cell surface expression of β1 integrins and increased their localization around the nucleus. Moreover, GlcNAc-TI knockdown decreased the migration and invasion of malignant cells. Next, we investigated the effect of GlcNAc-TI in an orthotopic xenograft mouse model of metastasis. GlcNAc-TI knockdown significantly decreased the lung colony formation of the highly metastatic PC3N7 human prostate cancer cell line in mice. Our results suggest an important role for GlcNAc-TI in tumor metastasis. Interestingly, breast cancer patients with lower expression levels of Mgat1 had lower risk of disease relapse after therapy. Thus, GlcNAc-TI plays an important role in cancer progression and metastasis and GlcNAc-TI inhibitors could have therapeutic benefits for cancer patients. Moreover, expression levels of GlcNAc-TI could be used as a prognostic marker in patients with cancer.
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Role of cholesterol and N-glycosylation in apical sorting of GPI- APs in polarized epithelial FRT cells / Rôle du cholestérol et de la N-glycosylation dans le tri apical de GPI-APs dans les cellules polarisées épithéliales FRTImjeti, Naga Sailaja 01 July 2011 (has links)
Les cellules épithéliales sont capable de se polariser avec un domaine apical et un basolatéral qui diffèrent nettement en leurs protéines, leurs composition en lipides et donc en fonction. Cette asymétrie reflète la capacité des cellules épithéliales à affecter les protéines nouvellement synthétisées et les lipides à chacune des surfaces de la cellule. Alors que les signaux responsables pour la sélection basolatérale des protéines ont été clairement identifiés, la situation en ce qui concerne la sélection apicale des protéines demeure mal comprise. Nous avons précédemment montré que contrairement aux GPI-APs basolatérales l’oligoisomérisation des protéines ancrées glycosylphosphatidylinositol (GPI-AP) dans l'appareil de Golgi est requise pour le tri apical. Il est intéressent de noter que ce mécanisme est conservé dans deux types de cellules épithéliales, les cellules MDCK et les cellules FRT, qui présentent des différences de tri des GPI-APs. Cependant, le mécanisme précis menant à cet événement n'est pas compris. Nos données précédentes ont démontré que le simple ajout de cholestérol aux cellules MDCK est nécessaire et suffisant pour induire l'oligomérisation et le tri apical d'une GPI-AP basolatérale. Alors que dans l’étude présente sur les cellules FRT, nous avons montré que contrairement aux cellules MDCK, le cholestérol ne joue pas un rôle majeur dans la régulation du tri apicale des GPI-APs. De plus, nous avons également montré que les GPI-APs apicales et basolatérales ne sont pas séparées dans le Golgi en fonction de leur teneur en cholestérol entourant l'environnement membranaire. Par ailleurs, nous avon démontré que la N-glycosylation des protéines de l’ectodomaine est indispensable à l’oligomérisation et au tri apical de GPI-APS. Nos données indiquent qu’il existe au moins deux mécanismes, l’un dépendant du taux de cholestérol et l’autre de la N-glycosylation, qui déterminent l’oligomérisation dans l'appareil de Golgi et le tri des GPI-APs vers la membrane apicale. / Epithelial cells represent the ability to polarize with an apical and basolateral domains which differ markedly in proteins, lipid composition and therefore in function. This asymmetry reflects the ability of epithelial cells to sort newly synthesized proteins and lipid to either cell surface. While the signals responsible for basolateral targeting of the proteins have been clearly understood, the situation regarding the apical sorting of proteins is more obscure. We have previously shown that differently from basolateral GPI-APs oligomerization in the Golgi apparatus is necessary for apical sorting of Glycosylphosphatidylinositol- anchored proteins (GPI-APs). Interestingly this mechanism is conserved in two different kinds of epithelial cells, MDCK and FRT cells, which exhibits a difference in the sorting of GPI-APs. However the precise mechanism leading to this event is not understood. Our previous data demonstrated that simple addition of cholesterol to MDCK cells is necessary and sufficient to induce the oligomerization and apical sorting of a basolateral GPI-AP. Whereas, in this present study in FRT cells we showed that in contrast with MDCK cells cholesterol is not an active player in the regulation of GPI- APs apical sorting. In addition, we also showed that apical and basolateral GPI-APs are not segregated in the Golgi on the bases of the cholesterol content of the surrounding membrane environment. Furthermore, we demonstrated that N- glycosylation of the protein ectodomain is critical for oligomerization and apical sorting of GPI-APs. Our data indicates that at least two mechanisms depending either on cholesterol or on N-glycosylation exist to determine oligomerization in the Golgi and sorting to the apical membrane of GPI-APs.
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Impact de la matrice extracellulaire sur la migration des cellules souches de glioblastome : un modèle tridimensionnel de culture et une nouvelle stratégie thérapeutique / The impact of the extracellular matrix on glioblastoma stem cells migration : a tridimensional culture model and a new therapeutic strategySaleh, Ali 20 June 2017 (has links)
Les glioblastomes multiformes (GBM) comptent parmi les tumeurs au pronostic le plus sombre. L’extraordinaire capacité invasive des cellules tumorales rend toutes les interventions thérapeutiques actuelles totalement impuissantes. Une sous-population de Cellules Souches de Glioblastome (CSG) hautement invasive est responsable de la récurrence tumorale. Dans le cerveau, les GBM migrent principalement le long des vaisseaux sanguins au sein de l’espace périvasculaire riche en laminine, fibronectine et collagène ainsi qu’en suivant l’alignement des fibres myélinisées du corps calleux. La Matrice Extracellulaire (MEC) de ces régions joue un rôle important dans l’invasion des GBM, mais les mécanismes mis en jeu n’ont pas été complètement dévoilés. De plus, le développement de nouvelles thérapies anti-migratrices ciblant l’interaction des GBM avec la MEC reste encore limité. Dans le but de mimer la composition biochimique et les propriétés mécaniques de la MEC cérébrale et d’étudier leur rôle(s) dans la migration des CSG, nous avons développé un nouveau support de nanofibres (NF) alignées et fonctionnalisées avec de la laminine. Mes travaux de thèse ont montré que les NF génèrent un microenvironnement tridimensionnel (3D) favorisant l’adhésion et la migration des CSG. Cette adhésion est améliorée en comparaison avec les supports planaires (SP) conventionnels (2D) et récapitule mieux les mécanismes d’interaction des CSG avec la MEC au cours de l’invasion dans le modèle murin de tumeurs xénogreffées. Dans ces conditions physiologiques plus convenables générées par les NF, la variation des composantes biochimiques et mécaniques de la MEC affecte la migration des CSG. La présence ou l’absence de laminine régule le mode migratoire et l’orientation de fibres contrôle la direction de migration des CSG. D’un autre coté, l’altération de la glycosylation des protéines de la surface cellulaire module l’interaction des cellules tumorales du cerveau avec la MEC et augmente leur invasion. La deuxième partie de mes travaux de thèse a permis de démontrer que les glycomimétiques phostines « 3.1a » réorganisent le processus de la N-glycosylation des CSG diminuant leur invasivité in vitro et in vivo en inhibant les voies de signalisation de la kinase FAK et du récepteur de TGF-β impliqués dans l’interconnexion cellule-MEC. / Glioblastoma Multiforme (GBM) is a biologically aggressive tumor with an extremely poor prognosis. The highly invasive capacity of a subpopulation of Glioblastoma Initiating Cells (GIC) makes complete surgical resection impossible. GBM dissemination occurs along preexisting brain structures such as the perivascular space rich in laminin, fibronectine and collagen as well as the aligned myelinated fibers of the corpus callosum. The Extracellular Matrix (ECM) of these cerebral regions plays an important role during GBM invasion, but the underlying mechanisms remain largely unknown. Accordingly, the development of new anti-migratory therapies targeting the cell-ECM interactions is lacking. In order to mimic the compositional and physical properties of the cerebral ECM and to investigate their role(s) in GBM invasion, we have set up a new aligned nanofibers (NF)scaffold functionalized with laminin. My work demonstrated that the NFs constitute a tridimensional (3D) microenvironment supporting GIC adhesion and migration. The cell-ECM adhesion is improved on the NF in comparison to the conventional 2D planar surfaces (PS). Furthermore, the mechanisms of GIC interaction with the ECM on the NF are similar to those observed in the human GBM xenograft murine model. In this physiologically more relevant 3D microenvironment reproduced by the NF, the variation of the different biochemical and mechanical components of the ECM affects the migration of GIC. The presence or absence of laminin on the NF regulates the mode of migration and the orientation of the fibers dictates the direction of migration of GIC. On the other hand, the glycosylation that decorates cell surface proteins modulates the interaction of GBM tumor cells with the ECM and its alteration increases their invasion. The second part of my thesis demonstrated that the glycomimetics phostines « 3.1a » remodel the N-glycosylation of GIC and decrease their invasivity in vitro and in vivo via the inhibition of FAK and TGFβ-R signaling pathways known to be implicated in the cell-ECM intercommunication.
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Functional Characterization of the Membrane Glycoprotein CD133Mak, Anthony 17 December 2012 (has links)
The AC133 epitope of the pentaspan transmembrane glycoprotein CD133 has been used as a cell-surface marker for normal and cancer stem cells from a broad range of tissue types. Despite the utility of CD133 as a marker, little is known regarding its regulation and biological function. To study these poorly understood aspects of CD133, I took two main experimental approaches: RNA interference (RNAi) screening and affinity purification coupled with mass spectrometry (AP-MS) to identify CD133 regulatory genes and CD133 protein-protein interactions (PPIs), respectively. Both of these experimental approaches relied on a human embryonic kidney (HEK) 293 cell line that exogenously expresses affinity tagged CD133 (HEK293/AC133). This cell line allowed me to perform a large-scale RNAi screen to interrogate 11,248 genes for their involvement in cell-surface AC133 recognition. This resulted in the identification of the N-glycosylation pathway as a direct contributor to CD133 plasma membrane localization and cell-surface AC133 detection. I used the same RNAi screening approach on the colon adenocarcinoma cell line Caco-2, which express CD133 from its native promoter, to identify factors that regulate endogenous CD133 transcription. I was able to demonstrate that AF4 promotes CD133 transcription in a number of cancer cell lines. Furthermore, I showed that CD133 expression in an acute lymphoblastic leukemia (ALL) cell line SEM, which is dependent on the mixed-lineage leukemia (MLL)-AF4 gene fusion, is critical for the viability of these cells. To gain further insight into the function of CD133, I performed AP-MS using HEK293/AC133 cells to identify CD133 PPIs. I identified histone deacetylase 6 (HDAC6) as a CD133 protein interaction partner. I found that HDAC6 negatively regulates CD133 trafficking into the endosomal-lysosomal degradation pathway. CD133 binds HDAC6 to prevent inhibition of HDAC6 deacetylase activity by phosphorylation. Protection of HDAC6 from phosphorylation promotes HDAC6 deacetylation of β-catenin, which results in β-catenin dependent signalling and the suppression of cancer cell differentiation. My thesis provide functional roles for CD133 as a pro-proliferative protein and as a key signalling protein in certain cancer cell lines.
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Functional Characterization of the Membrane Glycoprotein CD133Mak, Anthony 17 December 2012 (has links)
The AC133 epitope of the pentaspan transmembrane glycoprotein CD133 has been used as a cell-surface marker for normal and cancer stem cells from a broad range of tissue types. Despite the utility of CD133 as a marker, little is known regarding its regulation and biological function. To study these poorly understood aspects of CD133, I took two main experimental approaches: RNA interference (RNAi) screening and affinity purification coupled with mass spectrometry (AP-MS) to identify CD133 regulatory genes and CD133 protein-protein interactions (PPIs), respectively. Both of these experimental approaches relied on a human embryonic kidney (HEK) 293 cell line that exogenously expresses affinity tagged CD133 (HEK293/AC133). This cell line allowed me to perform a large-scale RNAi screen to interrogate 11,248 genes for their involvement in cell-surface AC133 recognition. This resulted in the identification of the N-glycosylation pathway as a direct contributor to CD133 plasma membrane localization and cell-surface AC133 detection. I used the same RNAi screening approach on the colon adenocarcinoma cell line Caco-2, which express CD133 from its native promoter, to identify factors that regulate endogenous CD133 transcription. I was able to demonstrate that AF4 promotes CD133 transcription in a number of cancer cell lines. Furthermore, I showed that CD133 expression in an acute lymphoblastic leukemia (ALL) cell line SEM, which is dependent on the mixed-lineage leukemia (MLL)-AF4 gene fusion, is critical for the viability of these cells. To gain further insight into the function of CD133, I performed AP-MS using HEK293/AC133 cells to identify CD133 PPIs. I identified histone deacetylase 6 (HDAC6) as a CD133 protein interaction partner. I found that HDAC6 negatively regulates CD133 trafficking into the endosomal-lysosomal degradation pathway. CD133 binds HDAC6 to prevent inhibition of HDAC6 deacetylase activity by phosphorylation. Protection of HDAC6 from phosphorylation promotes HDAC6 deacetylation of β-catenin, which results in β-catenin dependent signalling and the suppression of cancer cell differentiation. My thesis provide functional roles for CD133 as a pro-proliferative protein and as a key signalling protein in certain cancer cell lines.
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MOLECULAR FACTORS THAT INFLUENCE THE BINDING OF AGONISTS TO AMPA RECEPTORSMontgomery, Kyle Everett 01 January 2009 (has links)
AMPA receptors mediate excitatory synaptic transmission throughout the central nervous system via activation by their natural agonist glutamate. Several other molecules have been recognized as receptor agonist or antagonist, and recently allosteric modulators have been developed that potentiate the currents generated by these receptors. The goal of this thesis has been to address specific and as yet unresolved questions regarding the binding interactions between the AMPA receptors and these classes of molecules. For instance AMPA receptors are seemingly converted to have lower affinity for agonist as they move towards synapses and we evaluate two hypotheses put forward to explain the molecular mechanisms responsible for this. Additionally, guanine nucleotides competitively inhibit AMPA receptors and a second goal has been to further characterize guanine nucleotide binding, and to create mutations that selectively diminish this so that the function of the inhibition can be evaluated. A third goal has been to characterize the molecular factors that influence the effects of the allosteric modulators in order to explain why their efficacy differs greatly between brain regions. Experiments pertaining to these three goals were carried out sequentially and are described below as Projects 1 (guanine nucleotide inhibition), Project 2 (agonist affinity), and Project 3 (allosteric modulators). Project 1. Guanine nucleotides competitively inhibit AMPA-Rs (AMPA receptors) and because this inhibition is ubiquitous among virtually all types of glutamate receptors from fish to mammals, it likely serves a physiological function. Evaluation of this would be greatly facilitated if nucleotide binding could be eliminated through mutations without altering other aspects of receptor function, or if compounds were discovered that selectively prevent nucleotide binding. It was previously reported that a lysine in the chick kainate binding protein (cKBP) is specifically involved in guanine nucleotide binding. Therefore we mutated the homologous lysine (K445) in AMPA-R subunit GluR1 plus 12 additional residues around the glutamate binding pocket with the expectation that this would reduce nucleotide binding even further. Nucleotide affinity was determined by measuring the displacement of [3H]fluorowillardiine. As expected, the guanine nucleotide affinity was decreased about five-fold in R1-K445A mutants and the agonist affinity was seemingly unchanged. However, when tested by electrophysiology, characteristics of the mutant such as desensitization and the EC50 for glutamate were found to be altered. None of the other mutations were more successful at decreasing nucleotide affinity selectively. Nonetheless, these studies have given new insight into the docking mode of guanine nucleotides. The loss of binding in R1-K445A was much larger for GTP and GDP than for GMP, and guanosine binding, which is much lower, was unaffected by the mutation. These data suggest that the first phosphate of GMP determines the higher affinity of the phosphorylated nucleotides, and that K445 stabilizes the binding of the second and third phosphates of GDP and GTP. This along with various other observations suggest that the guanine base docks deep within the agonist binding pocket and that bulky additions, such as the phosphates, are accommodated by projecting out of the cleft in the vicinity of lysine 445. However, the exact docking mode of guanine nucleotides would have to be determined by crystallography. Project 2. Agonist binding to AMPA-R in brain consists of a high and low affinity components with KDs of 9-28 nM and 190-700 nM. Previous studies have suggested that newly synthesized receptors have high affinity and are converted to lower affinity by a secondary process. Two particular processes have been implicated, namely the conversion of receptor glycosylation from immature to complex, and modulation by receptor associated proteins. Both hypotheses were evaluated in this project using homomeric receptors GluR1-4 expressed in HEK 293 cells. The role of glycosylation was tested mostly with GluR4 receptors because they are expressed in distinct populations that exhibit either immature or complex glycans and their binding consists of high and low affinity components similar to those previously seen in brain receptors. Cells were treated with castanospermine or deoxymannojirimycin to decrease the proportion of receptors with complex glycosylation, or with cycloheximide plus chloroquine to increase the number of receptors with complex glycosylation. Although 70% of receptors from cells treated with cyloheximide/chloroquine exhibited complex glycans compared to <5% with other treatments, the affinity decreased at most 2-fold. Also, the low affinity component was nearly 80% of the total binding in receptors that exhibited virtually no complex glycans. Taken together these data indicate that complex glycosylation is not the key factor that confers low affinity. To test the second hypothesis GluR1i or GluR2i were co-expressed with stargazin which associates to receptors in neurons and affects their kinetics and trafficking. Considering the affinities of the two components seen in brain, we expected stargazin to cause a 20-fold or greater decrease in binding affinity. This was not the case, however our results did suggest that stargazin caused the appearance of a low affinity component but this was small and remained largely masked by the more abundant high affinity component. Recently, experiments with brain membranes have revealed preliminary evidence that an associated protein of ~85kDa may cause receptors to have low affinity. This hypothesis is currently under investigation. Project 3. Ampakines are cognitive enhancers that potentiate AMPA receptor currents at excitatory synapses. The efficacy of these drugs varies substantially among neurons in different brain regions, being for example about three times larger in the hippocampus than in the thalamus. Binding assays have shown that these compounds also increase the affinity of receptors for agonists. Importantly, the efficacy of these drugs to increase synaptic responses and agonist binding exhibit a positive correlation. Indeed, we have found that the increase in agonist binding (Emax) induced by the prototypical ampakine CX546 is highly variable across eight brain regions and that there is a 3-fold difference between the hippocampus and the thalamus which is similar to the difference reported for physiological efficacy. Therefore, binding assays or receptor autoradiography can potentially be used to predict the physiological efficacy of these drugs in a particular brain region. An important goal of this project has been to identify factors that may be responsible for the regionally different efficacies. Ampakines show some preference for receptor subunits but various considerations suggest that other factors must be involved. In this project we evaluated the role of a novel class of proteins called TARPs (transmembrane AMPA receptor regulatory proteins) that have recently been discovered to be tightly associated with AMPA receptors and to regulate their kinetics. Four of these proteins, named lambda;2(stargazin),λ3,λ4,and λ8 are abundant in the brain, but they exhibit highly selective regional distribution. We determined the maximum increase in agonist binding (Emax ) caused by saturating CX546 in three different AMPA receptor subunits, GluR1i, GluR2i, and GluR4i without and with co-expression of the four TARPs. Without TARPs, both Glu2i and GluR4i showed an Emax value of 100% over baseline binding. Co-expression of TARPs increased the Emax in GluR2i and this was largest for λ3 and λ8 (~130%). However, TARPs decreased the Emax of CX546 in GluR4i and this was most notable with λ2 and λ4 (~72%). Agonist binding in GluR1i was increased by only 15% and it was not significantly changed by TARPs. The expression patterns of TARPs and AMPA-R subunits in the brain have been partially characterized in the literature. Thus, it was previously reported that GluR4i transcripts are abundant in the thalamus but minor in the hippocampus. Using western blots we confirmed that this is also true for protein content; in the thalamus expression of GluR1, GluR2, GluR3, and GluR4 was 4%, 33%, 40%, and 147% respectively, of that in the hippocampus. When considering the known expression patterns of TARP variants, the hippocampus can be described as being enriched in GluR2, λ3 and λ8 while GluR4, λ2 and λ4 are prevalent in the thalamus. In comparison between these specific subunit/TARP combinations, the Emax values for those representative of the hippocampus (GluR2i/λ3 or λ8) were ~2-times larger than the Emax values of thalamic combinations (R4i/λ2 or λ4). Thus we can conclude that the differences in the expression of both TARP variants and AMPA-R subunits are critical factors for determining the variable efficacy of ampakines across brain regions.
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Beta-1,4-galactosyltransferase-3 deficiency suppresses the growth of immunogenic tumors in mice / ガラクトース転移酵素-3欠損マウスは高免疫原性腫瘍の増殖を抑制するWei, Heng 23 January 2024 (has links)
京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25008号 / 医科博第155号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 伊藤 貴浩, 教授 藤田 恭之, 教授 伊藤 能永 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Regulation of the orphan receptor Gpr176 activity via post-translational modifications in the central circadian clock / 概日時計中枢における翻訳後修飾を介したオーファン受容体Gpr176の活性調節Wang, Tianyu 23 March 2023 (has links)
京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24558号 / 薬科博第175号 / 新制||薬科||19(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 土居 雅夫, 教授 竹島 浩, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM
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