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The Endocytic Protein Numb Regulates App Metabolism And Notch Signaling: Implications For Alzheimer's DiseaseKyriazis, George 01 January 2008 (has links)
Increased production of amyloid beta (A-beta) peptide, via altered proteolytic cleavage of amyloid protein precursor (APP), and abnormalities in neuronal calcium homeostasis play central roles in the pathogenesis of Alzheimer's disease (AD). Notch1, a membrane receptor that controls cell fate decisions during development of the nervous system, has been linked to AD because it is a substrate for the gamma-secretase protein complex in which mutations cause early-onset inherited AD. Numb is an evolutionarily conserved endocytic adapter involved in the internalization of transmembrane receptors. Mammals produce four Numb isoforms that differ in two functional domains, a phosphotyrosine-binding domain (PTB) and a proline-rich region (PRR). Recent studies showed that the PTB domain of Numb interacts with the cytoplasmic tails of APP and Notch but the functional relevance of these interactions with respect to AD pathogenesis is not clear. In the current studies, we proposed to investigate the biological consequences of the interaction of the Numb proteins with APP and Notch in neural cells stably overexpressing each of the four human Numb proteins. In the first part of our studies, we found that expression of the Numb isoforms lacking the insert in the PTB (SPTB-Numb) caused the abnormal accumulation of cellular APP in the early endosomes, and increased the levels of C-terminal APP fragments and A-beta. By contrast, expression of the Numb isoforms with the insert in PTB (LPTB-Numb) leads to the depletion of cellular APP and coincides with significantly lower production of APP derivatives and A-beta. The contrasting effects of the Numb isoforms on APP metabolism were not attributed to differences in the expression of APP nor the activities of the various APP-processing secretases. In the second part of our studies, we found that expression of SPTB-Numb protein enhances neuronal vulnerability to serum deprivation-induced cell death by a mechanism involving the dysregulation of cellular calcium homeostasis. Neural cells expressing SPTB-Numb exhibited enhanced Notch activity, which markedly upregulated the expression of transient receptor potential canonical 6 (TRPC6) channels enhancing calcium entry in response to store depletion. We also found that serum deprivation increased TRPC6 expression, mediating the serum deprivation-induced death in neural cells. Interestingly, expression of LPTB-Numb protein suppressed serum deprivation-induced activation of Notch and the subsequent upregulation of TRPC6 and cell death. Finally, we showed that the Numb proteins differentially impact Notch activation by altering the endocytic trafficking and processing of Notch. Taken together, these studies demonstrate that aberrant expression of the Numb proteins may influence APP metabolism and Notch-mediated cellular responses to injury by altering their endocytic trafficking and processing.
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<b>ELUCIDATION OF THE INITIAL SUBSTRATE ACCESS PATHWAY IN STE24, YEAST HOMOLOG OF ZMPSTE24</b>Eric Leonard Glasser (17605800) 11 December 2023 (has links)
<p dir="ltr">Premature aging disorders such as mandibuloacral dysplasia disorder (MAD) can be caused by improper maturation of nuclear scaffolding protein lamin A from its precursor prelamin A. ZMPSTE24 is responsible for both the earlier C-terminal CAAX cleavage and a subsequent N-terminal upstream cleavage during the posttranslational processing of prelamin A to lamin A. Although ZMPSTE24’s structure and function are well characterized, the role of the 4 apparent openings into its hollow inner chamber remains unknown. We hypothesize that one of these entrances, portal 1, is the initial substrate entry point based on its proximity to the zinc-coordinating active site. Unfortunately, ZMPSTE24 is difficult to express and purify. Fortunately, the yeast homolog, Ste24, not only shares many structural and functional similarities to ZMPSTE24 but is also much easier to express and purify in an active state. Therefore, we will use Ste24 and its substrate <b>a</b>-factor as a model system for ZMPSTE24 and its substrate prelamin A to deduce whether portal 1 acts as the primary substrate entry point. We examined portal 1’s function in primary substrate entry by observing how the incubation of portal 1 mutants engineered with cysteine residues around the portal with cysteine-reactive bismaleimide crosslinkers affects the activity of the C-terminal CAAX cleavage. If crosslinking of the cysteine residues occludes the portal, we hypothesize that activity will decrease because substrate cannot enter. The cysteine-less enzyme Ste24 (QA), which cannot react with the crosslinkers, was engineered by mutagenesis to contain 1 or 2 new cysteines at specific positions around this portal. We hypothesize that portal 1 occlusion with cysteine reactive bismaleimide crosslinkers will inactivate the enzyme by preventing substrate entry. We monitored changes in CAAX cleavage activity with a radioactive endoprotease-coupled CAAX assay.</p><p dir="ltr">In crude membranes derived from yeast expressing QA Ste24, activity was not inhibited in the presence of either BMH or BMOE crosslinker. For the single cysteine-containing mutants M210C, T267C, I307C, and V311C, each crosslinker similarly decreased activity over 50%. For the double cysteine-containing mutants M210C-I307C, T267C-I307C, and T267C-V311C, we found between 20-60% decreased activity in the presence of the crosslinker which has a length most similar to the distance between the two cysteines. These results closely reflect previous data and further suggest that CAAX activity of the enzyme may be decreased due to the occlusion of the primary entry site, portal 1.</p><p dir="ltr">With purified QA Ste24, changes in activity were less apparent. Activity for purified QA was not decreased in the presence of either crosslinker. Single cysteine-containing mutants did not show decreased activity in the presence of either crosslinker. Unlike what was observed in crude membrane preparations, the double-cysteine containing mutants exhibited minimal decrease in activity in the presence of the crosslinker that has a length most similar to the distance between the two cysteines.</p><p dir="ltr">In crude membrane preparations, the cysteine-containing QA Ste24 mutants have diminished activity in the presence of crosslinkers. This may be due to the occlusion of the primary entry point, portal 1. However, we recognize the possibility that the decrease in activity was the result of the occlusion of the exit portal site. It is imperative that further experiments confirm that exit portal occlusion is not occurring. For purified cysteine-containing QA Ste24 mutants, the negligible decrease in activity suggests either that the portal 1 is not the primary substrate entry point or that the conditions of the assay were not optimized to generate inhibition. For example, the concentration of crosslinker was not sufficient in the presence of excess lipid and the reconstitution mixture sequestered the crosslinker. Further optimization of the reaction conditions is warranted. The cysteine-containing QA Ste24 mutants must be assessed for free thiols to determine how successful the reaction with the crosslinkers is. A more developed understanding of how all four portals function in the Ste24 CAAX processing of <b>a</b>-factor will be very insightful towards the mechanism of ZMPSTE24 in lamin A CAAX processing and may catalyze new targets of study for ZMPSTE24-related diseases like MAD-B.</p>
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Sorting Signals, Domain Conformation and Interdomain Interactions in CFTR Misprocessing and RescueBhrigu, Gargi 19 May 2010 (has links)
No description available.
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SORTING AND SECRETION OF SURFACTANT PROTEIN CJohnson Conkright, Juliana j. 11 October 2001 (has links)
No description available.
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MOLECULAR MECHANISMS OF PHOSPHOLIPASE C β AND ε REGULATIONKaushik Muralidharan (13169904) 29 July 2022 (has links)
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<p>The phospholipase C (PLC) family of enzymes canonically hydrolyzes the inner plasma membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG are crucial secondary messengers that activate multiple signaling pathways and modulate gene expression to control cellular function and behavior. The PLCe subfamily is essential for normal cardiovascular function, where it is activated through direct interactions with the RhoA and Rap1A small GTPases, linking lipase activity to the stimulation of G protein-coupled receptors (GPCRs. RhoA activates PLCe at the plasma membrane, whereas Rap1A translocates and activates PLCe at the perinuclear membrane, where phosphatidylintol-4-phosphate (PI4P) is hydrolyzed. The domains of PLCe involved in G protein binding, activation, and translocation to different subcellular membranes are largely unknown. In this work, we use cell-based activity assays, epifluorescence, and confocal microscopy to identify the domains of PLCe involved in basal activity, subcellular localization, and regulation by RhoA and Rap1A GTPases. Our preliminary studies demonstrate that the unique N- and C-terminal regulatory domains of PLCe dictate its location within the cell and contribute differently to basal and G protein-dependent activity. These studies will provide needed insights into the regulation and localization of PLCe in cells, which is critical for its roles in cardiovascular function. </p>
<p>Lipids on membranes are known to regulate the function of lipases in cells, however their contribution towards this phenomenon is not well understood. It is difficult to explore this mechanism due to the dynamic behavior of lipid bilayers in cells and most importantly the unknown variable of the amount of lipids present locally when lipases are activated. This study utilizes an approach, to use in vitro reconstitution methods to understand the contribution of lipids in activation of PLCb3 which is known to hydrolyze PIP2 in cell to generate IP3 and DAG. We also use single enzyme kinetics using total internal reflection microscopy to understand recruitment of single enzyme to the membrane, its association and disassociation rates at the membrane. </p>
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Investigations into the Nature of the Endosomal System in Plasmodium falciparumKrai, Priscilla M. 27 August 2013 (has links)
The parasite Plasmodium falciparum causes the most virulent form of human malaria and is responsible for the vast majority of malaria-related deaths. During the asexual intraerythrocytic stage, the parasite must transport newly synthesized proteins and endocytosed cargo to a variety of organelles, many of which are formed de novo and have no human equivalent. This process in mammalian cells would utilize an endosomal protein trafficking system, but no endosomal structures or proteins have been described in the parasite. Prior work on the parasite genome indicated that several proteins, which could potentially coordinate an endosomal network, were encoded in the genome and expressed during the asexual parasite stages. In this study, we have localized and attempted to further characterize these proteins in the context of the endosomal system. Two well-conserved protein components of the late endosome, the retromer cargo-selective complex and Rab7, were found on a previously un-described inherited structure adjacent to the parasite Golgi apparatus and in close opposition to nascent rhoptries (specialized secretory organelles required for invasion). The retromer cargo-selective complex was also in close proximity to its putative cargo, a P. falciparum homolog of the sortilin family of protein sorting receptors, PfSortilin. Another protein, PfFCP, the sole FYVE domain-containing protein in the P. falciparum genome, was localized to the membrane of a specialized acidic organelle, known as the food vacuole, where the parasite catabolizes the majority of its host cell hemoglobin. We analyzed the effects of a PfFCP dominant negative mutant and found that it altered food vacuole morphology and trafficking. A previous report localized the early endosome phosphoinositide, phosphatidylinositol 3-phosphate, to the food vacuole membrane, and in conjunction with our studies on PfFCP, this has raised doubts about the food vacuole as a lysosome equivalent in the parasite. The combination of both early and late endosome protein homologs in the parasite, and their potential function, has led to a new model of protein trafficking within the parasite that includes the food vacuole as a terminal early endosome and the apical organelles as lysosome equivalents. / Ph. D.
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Interaction of hERG Channels and Syntaxin 1AMihic, Anton 14 July 2009 (has links)
The human ether-à-go-go related gene (hERG) encodes the pore-forming voltage-gated K+ channel that is essential for cardiac repolarization. Dr. Tsushima’s laboratory has previously characterized the endogenous expression of SNARE proteins in the mammalian heart, and the interaction of the SNARE protein syntaxin 1A (STX1A) with several cardiac ion channels. Here, we utilize a multi-disciplinary approach to describe the inhibitory effect of STX1A on hERG channel function. STX1A impairs hERG channel maturation and trafficking to the plasma membrane and induces a hyperpolarizing shift in the voltage-sensitivity of steady-state inactivation. We identify the residues involved in this protein-protein interaction through the use of hERG truncation mutations. We also describe the pharmacological and temperature-mediated rescue of hERG channel trafficking in the presence of STX1A. The regulation of cardiac ion channels by SNARE proteins represents a novel biological mechanism that may have universally intrinsic implications for normal and diseased heart function.
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Interaction of hERG Channels and Syntaxin 1AMihic, Anton 14 July 2009 (has links)
The human ether-à-go-go related gene (hERG) encodes the pore-forming voltage-gated K+ channel that is essential for cardiac repolarization. Dr. Tsushima’s laboratory has previously characterized the endogenous expression of SNARE proteins in the mammalian heart, and the interaction of the SNARE protein syntaxin 1A (STX1A) with several cardiac ion channels. Here, we utilize a multi-disciplinary approach to describe the inhibitory effect of STX1A on hERG channel function. STX1A impairs hERG channel maturation and trafficking to the plasma membrane and induces a hyperpolarizing shift in the voltage-sensitivity of steady-state inactivation. We identify the residues involved in this protein-protein interaction through the use of hERG truncation mutations. We also describe the pharmacological and temperature-mediated rescue of hERG channel trafficking in the presence of STX1A. The regulation of cardiac ion channels by SNARE proteins represents a novel biological mechanism that may have universally intrinsic implications for normal and diseased heart function.
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Cellular Uptake of DNA Nanoparticles and Regulation of Cell Surface NucleolinChen, Xuguang 30 July 2009 (has links)
No description available.
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Cornichon Proteins: Unexpected Roles in Plant Pathogen Infection, ER Morphology Maintenance and Pollen DevelopmentLi, Jianhui 17 May 2017 (has links)
Cornichon (CNI) proteins are a conserved family of proteins among eukaryotes, from Erv14 in the yeast Saccharomyces cerevisiae to CNI homologs (CNIHs) in mammals and plants. Erv14 functions as a cargo receptor of coat protein complex II (COPII) for protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, en route to their final destinations. By interacting with specific cargo proteins, CNI proteins regulate key steps of embryo polarity in Drosophila, budding in yeast, and synaptic transmission in the mammalian brain. However, we have very limited understanding of plant CNIHs. Positive-strand RNA viruses assemble their viral replication complexes (VRCs) at specific host organelle membranes. With a better understanding of host factors involved in targeting viral replication proteins to the preferred organelles, we expect to block trafficking of viral replication proteins and thus, viral infection, by manipulating the required host proteins. Brome mosaic virus (BMV) is a model of positive-strand RNA viruses and its replication can be recapitulated in yeast. Importantly, BMV replication protein 1a is the only required viral protein to form VRCs at the perinuclear ER membrane in yeast. I demonstrate that Erv14 and COPII coat proteins are required for targeting BMV 1a to the perinuclear ER in yeast, suggesting a novel function of COPII vesicles in protein trafficking to the perinuclear ER membrane and in the BMV VRC formation. As for cellular functions, I show that plant CNIHs complement the defective distribution of BMV 1a in yeast mutant lacking Erv14. Taking advantage of Arabidopsis thaliana knockout mutants and knockdown of gene expression in Nicotiana benthamina, I also discover that CNIHs unexpectedly play crucial roles in pollen development, infection of a bacterial pathogen, and maintenance of ER tubules. I further confirm that CNI proteins are also required for maintaining ER tubules in yeast, suggesting a novel and conserved role in shaping ER morphology. Therefore, these findings indicate the functional diversity and redundancy of CNI proteins in key cellular processes and suggest a novel strategy to control plant pathogenic viruses and bacteria by manipulating plant CNIHs. / Ph. D.
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