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Proteomic Analysis of S-Palmitoylated Proteins in Ocular Lens Reveals Palmitoylation of AQP5 and MP20.
Wang Z, Schey KL
(2018) Invest Ophthalmol Vis Sci 59: 5648-5658
MeSH Terms: Animals, Aquaporin 5, Blotting, Western, Cattle, Chromatography, Liquid, Electrophoresis, Polyacrylamide Gel, Eye Proteins, Immunoblotting, Lens, Crystalline, Lipoylation, Membrane Proteins, Palmitates, Proteomics, Tandem Mass Spectrometry
Show Abstract · Added April 4, 2019
Purpose - The purpose of this study was to characterize the palmitoyl-proteome in lens fiber cells. S-palmitoylation is the most common form of protein S-acylation and the reversible nature of this modification functions as a molecular switch to regulate many biological processes. This modification could play important roles in regulating protein functions and protein-protein interactions in the lens.
Methods - The palmitoyl-proteome of bovine lens fiber cells was investigated by combining acyl-biotin exchange (ABE) chemistry and mass-spectrometry analysis. Due to the possibility of false-positive results from ABE experiment, a method was also developed for direct detection of palmitoylated peptides by mass spectrometry for validating palmitoylation of lens proteins MP20 and AQP5. Palmitoylation levels on AQP5 in different regions of the lens were quantified after iodoacetamide (IAA)-palmitate exchange.
Results - The ABE experiment identified 174 potential palmitoylated proteins. These proteins include 39 well-characterized palmitoylated proteins, 92 previously reported palmitoylated proteins in other tissues, and 43 newly identified potential palmitoylated proteins including two important transmembrane proteins in the lens, AQP5 and MP20. Further analysis by direct detection of palmitoylated peptides confirmed palmitoylation of AQP5 on C6 and palmitoylation of MP20 on C159. Palmitoylation of AQP5 was found to only occur in a narrow region of the inner lens cortex and does not occur in the lens epithelium, in the lens outer cortex, or in the lens nucleus.
Conclusions - AQP5 and MP20 are among 174 palmitoylated proteins found in bovine lens fiber cells. This modification to AQP5 and MP20 may play a role in their translocation from the cytoplasm to cell membranes during fiber cell differentiation.
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14 MeSH Terms
Acute Nitric Oxide Synthase Inhibition Accelerates Transendothelial Insulin Efflux In Vivo.
Williams IM, McClatchey PM, Bracy DP, Valenzuela FA, Wasserman DH
(2018) Diabetes 67: 1962-1975
MeSH Terms: Animals, Biological Transport, Blood Pressure, Blotting, Western, Glucose, Insulin, Male, Mice, Inbred C57BL, NG-Nitroarginine Methyl Ester, Nitric Oxide, Nitric Oxide Synthase, Transendothelial and Transepithelial Migration
Show Abstract · Added March 26, 2019
Before insulin can stimulate glucose uptake in muscle, it must be delivered to skeletal muscle (SkM) through the microvasculature. Insulin delivery is determined by SkM perfusion and the rate of movement of insulin across the capillary endothelium. The endothelium therefore plays a central role in regulating insulin access to SkM. Nitric oxide (NO) is a key regulator of endothelial function and stimulates arterial vasodilation, which increases SkM perfusion and the capillary surface area available for insulin exchange. The effects of NO on transendothelial insulin efflux (TIE), however, are unknown. We hypothesized that acute reduction of endothelial NO would reduce TIE. However, intravital imaging of TIE in mice revealed that reduction of NO by l--nitro-l-arginine methyl ester (l-NAME) enhanced the rate of TIE by ∼30% and increased total extravascular insulin delivery. This accelerated TIE was associated with more rapid insulin-stimulated glucose lowering. Sodium nitroprusside, an NO donor, had no effect on TIE in mice. The effects of l-NAME on TIE were not due to changes in blood pressure alone, as a direct-acting vasoconstrictor (phenylephrine) did not affect TIE. These results demonstrate that acute NO synthase inhibition increases the permeability of capillaries to insulin, leading to an increase in delivery of insulin to SkM.
© 2018 by the American Diabetes Association.
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Adenosine triphosphate as a molecular mediator of the vascular response to injury.
Guth CM, Luo W, Jolayemi O, Chadalavada KS, Komalavilas P, Cheung-Flynn J, Brophy CM
(2017) J Surg Res 216: 80-86
MeSH Terms: Adenosine Triphosphate, Animals, Aorta, Abdominal, Biomarkers, Biomechanical Phenomena, Blotting, Western, Female, Muscle Contraction, Rats, Rats, Sprague-Dawley, Receptors, Purinergic P2X7, Stress, Mechanical, Vascular System Injuries, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added May 22, 2018
BACKGROUND - Human saphenous veins used for arterial bypass undergo stretch injury at the time of harvest and preimplant preparation. Vascular injury promotes intimal hyperplasia, the leading cause of graft failure, but the molecular events leading to this response are largely unknown. This study investigated adenosine triphosphate (ATP) as a potential molecular mediator in the vascular response to stretch injury, and the downstream effects of the purinergic receptor, P2X7R, and p38 MAPK activation.
MATERIALS AND METHODS - A subfailure stretch rat aorta model was used to determine the effect of stretch injury on release of ATP and vasomotor responses. Stretch-injured tissues were treated with apyrase, the P2X7R antagonist, A438079, or the p38 MAPK inhibitor, SB203580, and subsequent contractile forces were measured using a muscle bath. An exogenous ATP (eATP) injury model was developed and the experiment repeated. Change in p38 MAPK phosphorylation after stretch and eATP tissue injury was determined using Western blotting. Noninjured tissue was incubated in the p38 MAPK activator, anisomycin, and subsequent contractile function and p38 MAPK phosphorylation were analyzed.
RESULTS - Stretch injury was associated with release of ATP. Contractile function was decreased in tissue subjected to subfailure stretch, eATP, and anisomycin. Contractile function was restored by apyrase, P2X7R antagonism, and p38-MAPK inhibition. Stretch, eATP, and anisomycin-injured tissue demonstrated increased phosphorylation of p38 MAPK.
CONCLUSIONS - Taken together, these data suggest that the vascular response to stretch injury is associated with release of ATP and activation of the P2X7R/P38 MAPK pathway, resulting in contractile dysfunction. Modulation of this pathway in vein grafts after harvest and before implantation may reduce the vascular response to injury.
Copyright © 2017 Elsevier Inc. All rights reserved.
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Inflammation-dependent cerebrospinal fluid hypersecretion by the choroid plexus epithelium in posthemorrhagic hydrocephalus.
Karimy JK, Zhang J, Kurland DB, Theriault BC, Duran D, Stokum JA, Furey CG, Zhou X, Mansuri MS, Montejo J, Vera A, DiLuna ML, Delpire E, Alper SL, Gunel M, Gerzanich V, Medzhitov R, Simard JM, Kahle KT
(2017) Nat Med 23: 997-1003
MeSH Terms: Acetazolamide, Animals, Antioxidants, Blotting, Western, Bumetanide, Cerebral Hemorrhage, Cerebral Ventricles, Cerebrospinal Fluid, Choroid Plexus, Diuretics, Gene Knockdown Techniques, Gene Knockout Techniques, Hydrocephalus, Immunoblotting, Immunohistochemistry, Immunoprecipitation, Inflammation, NF-kappa B, Proline, Protein-Serine-Threonine Kinases, Rats, Rats, Wistar, Salicylanilides, Solute Carrier Family 12, Member 2, Sulfonamides, Thiocarbamates, Toll-Like Receptor 4
Show Abstract · Added April 3, 2018
The choroid plexus epithelium (CPE) secretes higher volumes of fluid (cerebrospinal fluid, CSF) than any other epithelium and simultaneously functions as the blood-CSF barrier to gate immune cell entry into the central nervous system. Posthemorrhagic hydrocephalus (PHH), an expansion of the cerebral ventricles due to CSF accumulation following intraventricular hemorrhage (IVH), is a common disease usually treated by suboptimal CSF shunting techniques. PHH is classically attributed to primary impairments in CSF reabsorption, but little experimental evidence supports this concept. In contrast, the potential contribution of CSF secretion to PHH has received little attention. In a rat model of PHH, we demonstrate that IVH causes a Toll-like receptor 4 (TLR4)- and NF-κB-dependent inflammatory response in the CPE that is associated with a ∼3-fold increase in bumetanide-sensitive CSF secretion. IVH-induced hypersecretion of CSF is mediated by TLR4-dependent activation of the Ste20-type stress kinase SPAK, which binds, phosphorylates, and stimulates the NKCC1 co-transporter at the CPE apical membrane. Genetic depletion of TLR4 or SPAK normalizes hyperactive CSF secretion rates and reduces PHH symptoms, as does treatment with drugs that antagonize TLR4-NF-κB signaling or the SPAK-NKCC1 co-transporter complex. These data uncover a previously unrecognized contribution of CSF hypersecretion to the pathogenesis of PHH, demonstrate a new role for TLRs in regulation of the internal brain milieu, and identify a kinase-regulated mechanism of CSF secretion that could be targeted by repurposed US Food and Drug Administration (FDA)-approved drugs to treat hydrocephalus.
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Prominin-1 Is a Novel Regulator of Autophagy in the Human Retinal Pigment Epithelium.
Bhattacharya S, Yin J, Winborn CS, Zhang Q, Yue J, Chaum E
(2017) Invest Ophthalmol Vis Sci 58: 2366-2387
MeSH Terms: AC133 Antigen, Adult, Aged, Animals, Autophagy, Blotting, Western, Cells, Cultured, Female, Flow Cytometry, Gene Expression Regulation, Humans, Immunoprecipitation, Macular Degeneration, Male, Microscopy, Confocal, Middle Aged, RNA, Rabbits, Real-Time Polymerase Chain Reaction, Retinal Pigment Epithelium, Signal Transduction, Young Adult
Show Abstract · Added June 11, 2018
Purpose - Prominin-1 (Prom1) is a transmembrane glycoprotein, which is expressed in stem cell lineages, and has recently been implicated in cancer stem cell survival. Mutations in the Prom1 gene have been shown to disrupt photoreceptor disk morphogenesis and cause an autosomal dominant form of Stargardt-like macular dystrophy (STGD4). Despite the apparent structural role of Prom1 in photoreceptors, its role in other cells of the retina is unknown. The purpose of this study is to investigate the role of Prom1 in the highly metabolically active cells of the retinal pigment epithelium (RPE).
Methods - Lentiviral siRNA and the genome editing CRISPR/Cas9 system were used to knockout Prom1 in primary RPE and ARPE-19 cells, respectively. Western blotting, confocal microscopy, and flow sight imaging cytometry assays were used to quantify autophagy flux. Immunoprecipitation was used to detect Prom1 interacting proteins.
Results - Our studies demonstrate that Prom1 is primarily a cytosolic protein in the RPE. Stress signals and physiological aging robustly increase autophagy with concomitant upregulation of Prom1 expression. Knockout of Prom1 increased mTORC1 and mTORC2 signaling, decreased autophagosome trafficking to the lysosome, increased p62 accumulation, and inhibited autophagic puncta induced by activators of autophagy. Conversely, ectopic overexpression of Prom1 inhibited mTORC1 and mTORC2 activities, and potentiated autophagy flux. Through interactions with p62 and HDAC6, Prom1 regulates autophagosome maturation and trafficking, suggesting a new cytoplasmic role of Prom1 in RPE function.
Conclusions - Our results demonstrate that Prom1 plays a key role in the regulation of autophagy via upstream suppression of mTOR signaling and also acting as a component of a macromolecular scaffold involving p62 and HDAC6.
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Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis.
Chandrasekaran R, Kenworthy AK, Lacy DB
(2016) PLoS Pathog 12: e1006070
MeSH Terms: Adaptor Proteins, Signal Transducing, Animals, Bacterial Toxins, Blotting, Western, Caco-2 Cells, Clathrin, Clostridium Infections, Clostridium difficile, Endocytosis, Enterotoxins, Fluorescent Antibody Technique, Gene Knockdown Techniques, HEK293 Cells, Humans, Image Processing, Computer-Assisted, Mice, Microscopy, Confocal, Protein Transport, Reverse Transcriptase Polymerase Chain Reaction, Transfection, Virulence Factors
Show Abstract · Added April 26, 2017
Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. Toxicity depends on receptor binding and subsequent endocytosis. TcdB has been shown to enter cells by clathrin-dependent endocytosis, but the mechanism of TcdA uptake is still unclear. Here, we utilize a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches to investigate the endocytic mechanism(s) that contribute to TcdA uptake and subsequent cytopathic and cytotoxic effects. We show that TcdA uptake and cellular intoxication is dynamin-dependent but does not involve clathrin- or caveolae-mediated endocytosis. Confocal microscopy using fluorescently labeled TcdA shows significant colocalization of the toxin with PACSIN2-positive structures in cells during entry. Disruption of PACSIN2 function by RNAi-based knockdown approaches inhibits TcdA uptake and toxin-induced downstream effects in cells indicating that TcdA entry is PACSIN2-dependent. We conclude that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells.
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Molecular Basis for Subtype Specificity and High-Affinity Zinc Inhibition in the GluN1-GluN2A NMDA Receptor Amino-Terminal Domain.
Romero-Hernandez A, Simorowski N, Karakas E, Furukawa H
(2016) Neuron 92: 1324-1336
MeSH Terms: 2-Hydroxyphenethylamine, Animals, Binding Sites, Blotting, Western, Crystallography, Hydrogen Bonding, Piperidines, Protein Structure, Quaternary, Receptors, N-Methyl-D-Aspartate, Sf9 Cells, Spodoptera, Xenopus laevis, Zinc
Show Abstract · Added April 3, 2018
Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors containing a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concentration of zinc that binds to an extracellular domain called an amino-terminal domain (ATD). Despite physiological importance, the molecular mechanism underlying the high-affinity zinc inhibition has been incomplete because of the lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc-binding site and reveal distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability, supporting the general model in which the bi-lobe motion in ATD regulates the channel activity in NMDA receptors.
Copyright © 2016 Elsevier Inc. All rights reserved.
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The L-Arginine Transporter Solute Carrier Family 7 Member 2 Mediates the Immunopathogenesis of Attaching and Effacing Bacteria.
Singh K, Al-Greene NT, Verriere TG, Coburn LA, Asim M, Barry DP, Allaman MM, Hardbower DM, Delgado AG, Piazuelo MB, Vallance BA, Gobert AP, Wilson KT
(2016) PLoS Pathog 12: e1005984
MeSH Terms: Animals, Blotting, Western, Cationic Amino Acid Transporter 2, Cell Line, Citrobacter rodentium, Disease Models, Animal, Enterobacteriaceae Infections, Host-Parasite Interactions, Humans, Immunophenotyping, Mice, Mice, Inbred C57BL, Mice, Knockout, Transfection
Show Abstract · Added October 27, 2016
Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with Citrobacter rodentium, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, Slc7a2-/-mice infected with C. rodentium had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected Slc7a2-/-mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1β, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking Slc7a2 exhibited reduced adherence of C. rodentium to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of C. rodentium and induction of inflammatory response was confirmed in vitro, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of Slc7a2 or Tln1. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of C. rodentium and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic Escherichia coli. Overexpression of SLC7A2 in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that C. rodentium enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.
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Frequent Use of the IgA Isotype in Human B Cells Encoding Potent Norovirus-Specific Monoclonal Antibodies That Block HBGA Binding.
Sapparapu G, Czakó R, Alvarado G, Shanker S, Prasad BV, Atmar RL, Estes MK, Crowe JE
(2016) PLoS Pathog 12: e1005719
MeSH Terms: Antibodies, Monoclonal, B-Lymphocytes, Blood Group Antigens, Blotting, Western, Caliciviridae Infections, Cell Line, Enzyme-Linked Immunosorbent Assay, Gastroenteritis, Humans, Hybridomas, Immunoglobulin A, Immunoglobulin G, Norwalk virus, Polymerase Chain Reaction
Show Abstract · Added April 13, 2017
Noroviruses (NoV) are the most common cause of non-bacterial acute gastroenteritis and cause local outbreaks of illness, especially in confined situations. Despite being identified four decades ago, the correlates of protection against norovirus gastroenteritis are still being elucidated. Recent studies have shown an association of protection with NoV-specific serum histo-blood group antigen-blocking antibody and with serum IgA in patients vaccinated with NoV VLPs. Here, we describe the isolation and characterization of human monoclonal IgG and IgA antibodies against a GI.I NoV, Norwalk virus (NV). A higher proportion of the IgA antibodies blocked NV VLP binding to glycans than did IgG antibodies. We generated isotype-switched variants of IgG and IgA antibodies to study the effects of the constant domain on blocking and binding activities. The IgA form of antibodies appears to be more potent than the IgG form in blocking norovirus binding to histo-blood group antigens. These studies suggest a unique role for IgA antibodies in protection from NoV infections by blocking attachment to cell receptors.
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Spatial distributions of phosphorylated membrane proteins aquaporin 0 and MP20 across young and aged human lenses.
Gutierrez DB, Garland DL, Schwacke JH, Hachey DL, Schey KL
(2016) Exp Eye Res 149: 59-65
MeSH Terms: Adolescent, Adult, Aging, Aquaporins, Blotting, Western, Chromatography, Liquid, Eye Proteins, Humans, Lens, Crystalline, Membrane Proteins, Middle Aged, Peroxiredoxins, Phosphorylation, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Young Adult
Show Abstract · Added May 6, 2017
In the human ocular lens it is now realized that post-translational modifications can alter protein function and/or localization in fiber cells that no longer synthesize proteins. The specific sites of post-translational modification to the abundant ocular lens membrane proteins AQP0 and MP20 have been previously identified and their functional effects are emerging. To further understand how changes in protein function and/or localization induced by these modifications alter lens homeostasis, it is necessary to determine the spatial distributions of these modifications across the lens. In this study, a quantitative LC-MS approach was used to determine the spatial distributions of phosphorylated AQP0 and MP20 peptides from manually dissected, concentric layers of fiber cells from young and aged human lenses. The absolute amounts of phosphorylation were determined for AQP0 Ser235 and Ser229 and for MP20 Ser170 in fiber cells from the lens periphery to the lens center. Phosphorylation of AQP0 Ser229 represented a minor portion of the total phosphorylated AQP0. Changes in spatial distributions of phosphorylated APQ0 Ser235 and MP20 Ser170 correlated with regions of physiological interest in aged lenses, specifically, where barriers to water transport and extracellular diffusion form.
Copyright © 2016 Elsevier Ltd. All rights reserved.
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15 MeSH Terms