scholarly journals Identification of EBP50: A PDZ-containing Phosphoprotein that Associates with Members of the Ezrin-Radixin-Moesin Family

1997 ◽  
Vol 139 (1) ◽  
pp. 169-179 ◽  
Author(s):  
David Reczek ◽  
Mark Berryman ◽  
Anthony Bretscher
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Bovine Brain ◽  
Pdz Domains ◽  
Binding Partners ◽  
Phosphatase Treatment ◽  
Membrane Attachment ◽  
Polarized Epithelia ◽  
The Brain ◽  
Terminal Domains

Members of the ezrin-radixin-moesin (ERM) family of membrane–cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin–association domain (N-ERMAD). A collection of polypeptides at 50–53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50–53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an ∼2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1–like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

scholarly journals High-Cholesterol Diet Decreases the Level of Phosphatidylinositol 4,5-Bisphosphate by Enhancing the Expression of Phospholipase C (PLCβ1) in Rat Brain

2020 ◽  
Vol 21 (3) ◽  
pp. 1161 ◽  
Author(s):  
Yoon Sun Chun ◽  
Sungkwon Chung
Keyword(s):  
Neurodegenerative Diseases ◽  
Rat Brain ◽  
Phospholipase C ◽  
Cultured Cells ◽  
Cell Interaction ◽  
High Cholesterol Diet ◽  
Cholesterol Diet ◽  
High Cholesterol ◽  
Amyloid Aβ ◽  
The Brain

Cholesterol is a critical component of eukaryotic membranes, where it contributes to regulating transmembrane signaling, cell–cell interaction, and ion transport. Dysregulation of cholesterol levels in the brain may induce neurodegenerative diseases, such as Alzheimer’s disease, Parkinson disease, and Huntington disease. We previously reported that augmenting membrane cholesterol level regulates ion channels by decreasing the level of phosphatidylinositol 4,5-bisphosphate (PIP2), which is closely related to β-amyloid (Aβ) production. In addition, cholesterol enrichment decreased PIP2 levels by increasing the expression of the β1 isoform of phospholipase C (PLC) in cultured cells. In this study, we examined the effect of a high-cholesterol diet on phospholipase C (PLCβ1) expression and PIP2 levels in rat brain. PIP2 levels were decreased in the cerebral cortex in rats on a high-cholesterol diet. Levels of PLCβ1 expression correlated with PIP2 levels. However, cholesterol and PIP2 levels were not correlated, suggesting that PIP2 level is regulated by cholesterol via PLCβ1 expression in the brain. Thus, there exists cross talk between cholesterol and PIP2 that could contribute to the pathogenesis of neurodegenerative diseases.

scholarly journals Characterization of calcium-dependent membrane binding proteins of brain cortex

1985 ◽  
Vol 229 (3) ◽  
pp. 587-593 ◽  
Author(s):  
A R Rhoads ◽  
M Lulla ◽  
P B Moore ◽  
C E Jackson
Keyword(s):  
Brain Cortex ◽  
Peptide Mapping ◽  
Bovine Brain ◽  
Particulate Fraction ◽  
Structural Homology ◽  
One Dimensional ◽  
Calcium Dependent ◽  
Stokes Radius ◽  
The Brain

Proteins of Mr 68 000, 34 000 and 32 000 were selectively extracted by EGTA from brain cortex. The three proteins that were extracted along with calmodulin were acidic, monomeric, and did not exhibit structural homology, as demonstrated by one-dimensional peptide mapping. The Mr-68 000 protein was purified to homogeneity and had a Stokes radius of 3.54 nm and S20,W value of 5.1S. Purified calmodulin, Mr-68 000 protein and two proteins of Mr 34 000 and Mr 32 000, interacted with the brain particulate fraction, with half-maximal binding occurring at 3.5 microM, 8.3 microM and 150 microM-Ca2+ respectively. Proteins were bound independently of each other and calmodulin. Pretreatment of the particulate fraction with trypsin prevented the Ca2+-dependent binding of calmodulin; however, the binding of the Mr-68 000 protein or the Mr−32 000 and −34 000 proteins was unaffected. The Mr-68 000 protein of bovine brain did not cross-react immunologically with Mr-67 000 calcimedin from chicken gizzard.

scholarly journals VISUALIZATION OF EGYPT 101 VIRUS IN THE MOUSE'S BRAIN AND IN CULTURED HUMAN CARCINOMA CELLS BY MEANS OF FLUORESCENT ANTIBODY

1955 ◽  
Vol 102 (3) ◽  
pp. 243-248 ◽  
Author(s):  
Wilbur Fiske Noyes
Keyword(s):  
Cultured Cells ◽  
Fluorescent Antibody ◽  
Carcinoma Cells ◽  
Fluorescent Antibody Technique ◽  
Human Carcinoma ◽  
Antibody Technique ◽  
Human Cell Cultures ◽  
Brain And Spinal Cord ◽  
The Brain ◽  
Human Epidermoid Carcinoma

The antigens of the Egypt virus have been detected by means of the fluorescent antibody technique in the neurons of the brain and spinal cord, in both sensory and motor areas, of infected mice. The antigens have also been observed in infected human epidermoid carcinoma cells in culture. In both cases the antigens occurred in the cytoplasm of the cells exclusively. In the early stages of infection the antigen was localized about the nucleus of the human cultured cells. In some cases the staining of the antigens produced a sponge-like effect, probably because of the numerous vacuoles of lipid material. The antigens were observed in the protoplasmic connections between cells and they probably served as a main route of spread of the agent in these human cell cultures. The number of cells initially infected and showing antigen was proportional to the amount of virus added, and titration with one substrain of cells proved much more sensitive than intracerebral titration in the mouse.

scholarly journals Centrosomal protein centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos

Reproduction ◽  
2006 ◽  
Vol 132 (3) ◽  
pp. 423-434 ◽  
Author(s):  
G Manandhar ◽  
D Feng ◽  
Y-J Yi ◽  
L Lai ◽  
J Letko ◽  
...  
Keyword(s):  
Western Blotting ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
De Novo ◽  
Germinal Vesicle ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Embryonic Cells ◽  
Perinuclear Area

Centrin is an evolutionarily conserved 20 kDa, Ca+2-binding, calmodulin-related protein associated with centrioles and basal bodies of phylogenetically diverse eukaryotic cells. Earlier studies have shown that residual centrosomes of non-rodent mammalian spermatozoa retain centrin and, in theory, could contribute this protein for the reconstruction of the zygotic centrosome after fertilization. The present work shows that CEN2 and CEN3 mRNA were detected in germinal vesicle-stage (GV) oocytes, MII oocytes, and pre-implantation embryos from the two-cell through the blastocyst stage, but not in spermatozoa. Boar ejaculated spermatozoa possess centrin as revealed by immunofluorescence microscopy and western blotting. Immature, GV oocytes possess speckles of centrin particles in the perinuclear area, visualized by immunofluorescence microscopy and exhibit a 19 kDa band revealed by western blotting. Mature MII stage oocytes lacked centrin that could be detected by immunofluorescence or western blotting. The sperm centrin was lost in zygotes afterin vitrofertilization. It was not detectable in embryos by immunofluorescence microscopy until the late blastocyst stage. Embryonic centrin first appeared as fine speckles in the perinuclear area of some interphase blastocyst cells and as putative centrosomes of the spindle poles of dividing cells. The cells of the hatched blastocysts developed centrin spots comparable with those of the cultured cells. Some blastomeres displayed undefined curved plate-like centrin-labeled structures. Anti-centrin antibody labeled interphase centrosomes of cultured pig embryonic fibroblast cells as distinct spots in the juxtanuclear area. Enucleated pig oocytes reconstructed by electrofusion with pig fibroblasts displayed centrin of the donor cell during the early stages of nuclear decondensation but became undetectable in the late pronuclear or cleavage stages. These observations suggest that porcine zygotes and pre-blastocyst embryonic cells lack centrin and do not retain exogenously incorporated centrin. The early embryonic centrosomes function without centrin. Centrin in the blastocyst stage embryos is likely a result ofde novosynthesis at the onset of differentiation of the pluripotent blastomeres.

scholarly journals N-Terminal Motifs in Some Plant Disease Resistance Proteins Function in Membrane Attachment and Contribute to Disease Resistance

2012 ◽  
Vol 25 (3) ◽  
pp. 379-392 ◽  
Author(s):  
Daigo Takemoto ◽  
Maryam Rafiqi ◽  
Ursula Hurley ◽  
Greg J. Lawrence ◽  
Maud Bernoux ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Disease Resistance ◽  
Golgi Apparatus ◽  
Plant Disease Resistance ◽  
Plant Disease ◽  
Protein Accumulation ◽  
Resistance Proteins ◽  
Membrane Attachment ◽  
Signal Anchor ◽  
Terminal Domains

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.

scholarly journals Molecular cloning of the gene encoding the bovine brain ribonucelase and its expression in different regions of the brain

1991 ◽  
Vol 19 (23) ◽  
pp. 6469-6474 ◽  
Author(s):  
M.P. Sasso ◽  
A. Carsana ◽  
E. Confalone ◽  
C. Cosi ◽  
S. Sorrentino ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Bovine Brain ◽  
Gene Encoding ◽  
The Brain

scholarly journals Entry of polyunsaturated fatty acids into the brain: evidence that high-density lipoprotein-induced methylation of phosphatidylethanolamine and phospholipase A2 are involved

1996 ◽  
Vol 316 (3) ◽  
pp. 805-811 ◽  
Author(s):  
Valérie MAGRET ◽  
Latifa ELKHALIL ◽  
Françoise NAZIH-SANDERSON ◽  
Françoise MARTIN ◽  
Jean-Marie BOURRE ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Polyunsaturated Fatty Acids ◽  
High Density Lipoprotein ◽  
Phospholipase A2 ◽  
Density Lipoprotein ◽  
Bovine Brain ◽  
High Density ◽  
Close Relationship ◽  
The Brain

The conversion of phosphatidylethanolamine (PE) into phosphatidylcholine (PC) by a sequence of three transmethylation reactions is shown to be stimulated by the apolipoprotein E-free subclass of high-density lipoprotein (HDL3) in isolated bovine brain capillary (BBC) membranes. HDL3-induced stimulation of BBC membranes pulsed with [methyl-14C]methionine causes a transient increase in each methylated phospholipid, i.e. phosphatidyl-N-monomethylethanolamine (PMME), phosphatidyl-NN-dimethylethanolamine (PDME) and PC. PC substrate arising from the activation of PE N-methyltransferase (PEMT) is hydrolysed by a phospholipase A2 (PLA2), as demonstrated by the accumulation of lysophosphatidylcholine (lyso-PC). When PE containing [14C]arachidonic acid in the sn-2 position ([14C]PAPE) is incorporated into BBC membranes, HDL3 stimulation induces the formation of PMME, PDME, PC and lyso-PC and the release of [14C]arachidonic acid, which correlates with the previous production of lyso-PC, suggesting that HDL3 stimulates a PLA2 that can release polyunsaturated fatty acids (PUFA). Both PEMT and PLA2 activities depend on a HDL3 concentration in the range 0–50 μg/ml and are strictly dependent on HDL3 binding, because HDL3 modified by tetranitromethane is no longer able to bind to specific receptors and to trigger PEMT and PLA2 activation. Moreover, HDL3 prelabelled with [14C]PAPE can stimulate PDME and lyso-PC synthesis in BBC membranes in the presence of S-adenosylmethionine, suggesting that HDL3 can supply BBC membranes in polyunsaturated PE and can activate enzymes involved in PE N-methylation and PUFA release. The results support the hypothesis of a close relationship between HDL3 binding, PE methylation and PUFA release, and suggest that the PC pool arising from PE could be used as a pathway for the supply of PUFA to the brain.

scholarly journals Human tauopathy-derived tau strains determine the substrates recruited for templated amplification

Brain ◽  
2021 ◽  
Author(s):  
Airi Tarutani ◽  
Haruka Miyata ◽  
Takashi Nonaka ◽  
Kazuko Hasegawa ◽  
Mari Yoshida ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Corticobasal Degeneration ◽  
Biochemical Properties ◽  
Cellular Model ◽  
Cryo Electron Microscopy ◽  
Structural Differences ◽  
The Brain ◽  
Strain Dependent ◽  
Disease Specific ◽  
Prion Hypothesis

Abstract Tauopathies are a subset of neurodegenerative diseases characterized by abnormal tau inclusions. Specifically, three-repeat tau and four-repeat tau in Alzheimer’s disease (AD), three-repeat tau in Pick's disease (PiD) and four-repeat in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) form amyloid-like fibrous structures that accumulate in neurons and/or glial cells. Amplification and cell-to-cell transmission of abnormal tau based on the prion hypothesis are believed to explain the onset and progression of tauopathies. Recent studies support not only the self-propagation of abnormal tau, but also the presence of conformationally distinct tau aggregates, namely tau strains. Cryo-electron microscopy analyses of patient-derived tau filaments have revealed disease-specific ordered tau structures. However, it remains unclear whether the ultrastructural and biochemical properties of tau strains are inherited during the amplification of abnormal tau in the brain. In this study, we investigated template-dependent amplification of tau aggregates using a cellular model of seeded aggregation. Tau strains extracted from human tauopathies caused strain-dependent accumulation of insoluble filamentous tau in SH-SY5Y cells. The seeding activity towards full-length four-repeat tau substrate was highest in CBD-tau seeds, followed by PSP-tau and AD-tau seeds, while AD-tau seeds showed higher seeding activity than PiD-tau seeds towards three-repeat tau substrates. Abnormal tau amplified in cells inherited the ultrastructural and biochemical properties of the original seeds. These results strongly suggest that the structural differences of patient-derived tau strains underlie the diversity of tauopathies, and that seeded aggregation and filament formation mimicking the pathogenesis of sporadic tauopathy can be reproduced in cultured cells. Our results indicate that the disease-specific conformation of tau aggregates determines the tau isoform substrate that is recruited for templated amplification, and also influences the prion-like seeding activity.

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