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Research Interests: Chemistry, Chromatography, Biology, Medicine, Biological Sciences, and 15 moreLipids, Endocytosis, Animals, Male, Iron, Endosome, CHEMICAL SCIENCES, Electromagnetic phenomena, Rats, Phosphatidylcholine, Endosomes, Phosphatidylglycerol, Biochemistry and cell biology, Peritoneal macrophages, and Medical and Health Sciences
Accumulation of lipofuscin in retinal pigmented epithelial (RPE) cell, results from incomplete degradation of phagocytic and autophagic material by lysosomes. Both increased lipofuscin and enhanced cytokine release are associated with... more
Accumulation of lipofuscin in retinal pigmented epithelial (RPE) cell, results from incomplete degradation of phagocytic and autophagic material by lysosomes. Both increased lipofuscin and enhanced cytokine release are associated with macular degeneration in these cells, although the link between the two is unclear. Since stimulation of the P2X7 receptor (P2X7R) activates IL-1β release in some cell types, and triggers lysosomal exocytosis in others, we investigated the effect of P2X7R activation on lysosomal pH in, and cytokine release from, RPE cells. Surprisingly, stimulation of the P2X7R with agonist BzATP did not lead to a release of IL-1β but did trigger release of both IL-6 and MCP-1. BzATP led to a rise in intracellular Ca2+, and extracellular Ca2+ was necessary for IL-6 release. Lysosomal pH was elevated by BzATP and this alkalinization was inhibited by P2X7R antagoinsts A438079 and Brilliant Blue G. BzATP on its own did not significantly increase expression of IL-6, MCP-1 or the ectoATPase NTPDas...
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Lipocalins are a broad family of proteins identified initially in eukaryotes and more recently in Gram-negative bacteria. The functions of lipocalin or lipid-binding proteins are often elusive and very diverse. Recently, we have... more
Lipocalins are a broad family of proteins identified initially in eukaryotes and more recently in Gram-negative bacteria. The functions of lipocalin or lipid-binding proteins are often elusive and very diverse. Recently, we have determined the structure of GrlR (global regulator of LEE repressor), which plays a key role in the regulation of LEE (locus of enterocyte effacement) proteins. GrlR adopts a lipocalin-like fold that is composed of an eight-stranded β-barrel followed by an α-helix at the C-terminus. GrlR has a highly hydrophobic cavity region and could be a potential transporter of lipophilic molecules. To verify this hypothesis, we carried out structure-based analysis of GrlR, determined the structure of the lipid–GrlR complex and measured the binding of lipid to recombinant GrlR by ITC (isothermal titration calorimetry). In addition, we identified phosphatidylglycerol and phosphatidylethanolamine as the endogenously bound lipid species of GrlR using electrospray-ionization...
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Intracellular protein degradation is a universal feature of eukaryotic cells and vital for nutrition, protein turnover, intracellular signaling, development and other major physiological processes like antigen presentation and immunity.... more
Intracellular protein degradation is a universal feature of eukaryotic cells and vital for nutrition, protein turnover, intracellular signaling, development and other major physiological processes like antigen presentation and immunity. One of the major compartments of intracellular proteolysis is the endosome-lysosome system. The latter offers a highly orchestrated, vesicular pathway for protein transport and ultimate degradation in lysosomes. Though lysosomes are the classical organelles of complex, multi-enzymatic degradation, it is increasingly evident that endosomes conduct much more than mere transport functions. Endosomes contain significant levels of proteases like cathepsins and are sites of potent intracellular proteolysis. Further, discrete classes of endosomes harbor specific cathepsins and perform selective and exclusive functions. Hence, extra-lysosomal proteolytic machinery within the endocytic pathway enjoys spatial and temporal control over proteolytic functions. Th...
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Cyclic adenosine monophosphate (3',5'-cAMP) is a multifunctional second messenger which controls extremely diverse and physiologically important biochemical pathways. Among its myriad roles, 3',5'-cAMP functions as an... more
Cyclic adenosine monophosphate (3',5'-cAMP) is a multifunctional second messenger which controls extremely diverse and physiologically important biochemical pathways. Among its myriad roles, 3',5'-cAMP functions as an intracellular regulator of lysosomal pH, which is essential for the activity of acidic lysosomal enzymes. Defects in lysosomal acidification are attributed to many diseases like macular degeneration, Parkinson's, Alzheimer's, and cystic fibrosis. Strategic re-acidification of defective lysosomes by pharmacological increase of intracellular cAMP offers exciting therapeutic potential in these diseases. Modular assays for accurate assessment of intracellular cAMP and lysosomal pH are a critical component of this research. We describe label-free targeted metabolomics for quantitating intracellular cAMP and integrated assays for measuring lysosomal pH. These hybrid assays offer fast, unbiased information on intracellular cAMP concentrations and lysosomal pH that can be applied to many cell types and putative drug screening strategies.
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Lysosomes contribute to a multitude of cellular processes, and the pH of the lysosomal lumen plays a central mechanistic role in many of these functions. In addition to controlling the rate of enzymatic degradation for material delivered... more
Lysosomes contribute to a multitude of cellular processes, and the pH of the lysosomal lumen plays a central mechanistic role in many of these functions. In addition to controlling the rate of enzymatic degradation for material delivered through autophagic or phagocytotic pathways, lysosomal pH regulates events such as lysosomal fusion with autophagosomes and the release of lysosomal calcium into the cytoplasm. Disruption of either the steady state lysosomal pH or of the regulated manipulations to lysosomal pH may be pathological. For example, chloroquine elevates the lysosomal pH of retinal pigmented epithelial (RPE) cells and triggers a retinopathy characterized by the accumulation of lipofuscin-like material in both humans and animals. Compensatory responses to restore lysosomal pH are observed; new data illustrate that chronic chloroquine treatment increases mRNA expression of the lysosomal/autophagy master transcription factor TcFEB and of the vesicular proton pump vHATPase in ...
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Research Interests: Physiology, Calcium, Retinal Pigment Epithelium, Cell line, Humans, and 14 moreMice, Animals, Retinal Degeneration, Lipid metabolism, Medical Physiology, Cattle, Lysosomes, Hydrogen-Ion Concentration, Cytoplasm, Oxidation-Reduction, Biochemistry and cell biology, Adenosine Triphosphate, Cell Membrane, and binding sites
Optimal neuronal activity requires that supporting cells provide both efficient nutrient delivery and waste disposal. The incomplete processing of engulfed waste by their lysosomes can lead to accumulation of residual material and... more
Optimal neuronal activity requires that supporting cells provide both efficient nutrient delivery and waste disposal. The incomplete processing of engulfed waste by their lysosomes can lead to accumulation of residual material and compromise their support of neurons. As most degradative lysosomal enzymes function best at an acidic pH, lysosomal alkalinization can impede enzyme activity and increase lipofuscin accumulation. We hypothesize that treatment to reacidify compromised lysosomes can enhance degradation. Here, we demonstrate that degradation of ingested photoreceptor outer segments by retinal pigmented epithelial cells is increased by stimulation of D5 dopamine receptors. D1/D5 receptor agonists reacidified lysosomes in cells alkalinized by chloroquine or tamoxifen, with acidification dependent on protein kinase A. Knockdown with siRNA confirmed acidification was mediated by the D5 receptor. Exposure of cells to outer segments increased lipofuscin-like autofluorescence, but S...
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Cytokine release from non-inflammatory cells is a key step in innate immunity, and agonists triggering cytokine release are central in coordinating responses. P2X7 receptor (P2X7R) stimulation by extracellular ATP is best known to active... more
Cytokine release from non-inflammatory cells is a key step in innate immunity, and agonists triggering cytokine release are central in coordinating responses. P2X7 receptor (P2X7R) stimulation by extracellular ATP is best known to active the NLRP3 inflammasome and release IL-1β, but stimulation also leads to release of other cytokines. As cytokine signaling by retinal pigmented epithelial (RPE) cells is implicated in retinal neurodegeneration, the role of P2X7R in release of cytokine IL-6 from RPE cells was investigated. P2X7R stimulation triggered IL-6 release from primary mouse RPE, human iPS-RPE and human ARPE-19 cells. IL-6 release was polarized, with predominant rise across apical membranes. IL-6 release was inhibited by P2X7R antagonists A438079, A839977, and AZ10606120, but not the NRTI lamivudine (3TC), P2X1R antagonist NF279, or P2Y1R antagonist MRS2179. P2X7R-mediated IL-6 release required extracellular Ca2+ and was blocked by Ca2+ chelator BAPTA. IL-6 release and Ca2+ ele...
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The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent... more
The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE) cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4 mouse model of Stargardt's disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells , but identification of an appropriate receptor is crucial for manipulating this pathway . As the P2Y receptor for ADP is coupled to the inhibitory G protein, we asked whether blocking the P2Y receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cell...
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The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent... more
The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE) cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4 mouse model of Stargardt's disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells , but identification of an appropriate receptor is crucial for manipulating this pathway . As the P2Y receptor for ADP is coupled to the inhibitory G protein, we asked whether blocking the P2Y receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cell...