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Thomas C Sudhof - One of the best experts on this subject based on the ideXlab platform.
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ca 2 dependent conformational change in synaptotagmin i
Journal of Biological Chemistry, 1994Co-Authors: Bazbek Davletov, Thomas C SudhofAbstract:Abstract Synaptotagmin I is a Ca2+/Phospholipid Binding Protein of synaptic vesicles with a proposed function as a Ca2+ sensor in synaptic vesicle exocytosis. Using controlled partial proteolysis as an assay, we now show that synaptotagmin I undergoes a conformational change as a function of Ca2+ Binding. As observed for Phospholipid Binding, Ba2+ and Sr2+ but not Mg2+, substitute for Ca2+ in effecting this conformational change. The first C2 domain from synaptotagmin I that represents the Ca(2+)-dependent Phospholipid Binding domain of synaptotagmin also undergoes a Ca(2+)-dependent change in controlled partial proteolysis. In contrast, no effect of Ca2+ was observed with mutant C2 domains containing point mutations that abolish Ca2+ Binding. The Ca2+ concentration dependence of the effect of Ca2+ on proteolysis mirrors the Ca2+ dependence of Phospholipid Binding. The conformational shift in synaptotagmin I caused by Ca2+/Phospholipid Binding could be the basis for its Ca(2+)-regulated function in triggering neurotransmitter release.
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synaptic targeting of rabphilin 3a a synaptic vesicle ca2 Phospholipid Binding Protein depends on rab3a 3c
Neuron, 1994Co-Authors: Kohji Takei, Martin Geppert, Laurie Daniell, Katinka Stenius, Edwin R Chapman, Reinhard Jahn, Pietro De Camilli, Thomas C SudhofAbstract:Abstract rab3A, a low molecular weight GTP-Binding Protein of synaptic vesicles with a putative function in synaptic vesicle docking, interacts in a GTP-dependent manner with rabphilin-3A, a peripheral membrane Protein that binds Call and Phospholipids. We now show that rabphilin-3A is an evolutionarily conserved synaptic vesicle Protein that is attached to synaptic vesicle membranes via its N terminus and exhibits a heterogeneous distribution among synapses. In rab3A-deficient mice, rabphilin-3A is decreased in synapses belonging to neurons that primarily express rab3A and accumulates in the perikarya of these neurons. In contrast, neurons expressing significant levels of rab3C still contain normal levels of rabphilin-3A in a synaptic pattern, and rabphilin-3A binds rab3C in vitro. These results suggest that analogous to the membrane recruitment of raf by ras, rab3A and rab3C may function in recruiting .rabphilin-3A to the synaptic vesicle membrane in a GTP-dependent manner.
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dynamin i is a ca 2 sensitive Phospholipid Binding Protein with very high affinity for Protein kinase c
Journal of Biological Chemistry, 1994Co-Authors: Kate A Powell, Thomas C Sudhof, Phillip J. RobinsonAbstract:Abstract Depolarization-induced Ca2+ influx into rat brain synaptosomes induces dephosphorylation of dephosphin, a 96-94-kDa Protein kinase C (PKC) substrate recently identified as dynamin I, a Protein associated with endocytosis. We characterized purified dynamin I to better understand regulation of its phosphorylation in nerve terminals. Purified dynamin I possessed a very high affinity for PKC but did not fit Michaelis-Menten kinetics. It had an optimum phosphorylation rate of 1.42 +/- 0.02 mumol/mg/min and a concentration giving half-maximal activity (S0.5) of 0.14 +/- 0.02 microM, the highest affinity reported for a PKC substrate Protein. Concentrations of dynamin greater than 0.5 microM inhibited phosphorylation. The stoichiometry was 1.5, indicating more than one phosphorylation site. Dynamin was predominantly associated with the brain particulate fraction under conditions of low ionic strength, and this prevented its phosphorylation by PKC until released by moderate increases in ionic strength (Na+, K+, and Mg2+) or by GTP or ATP. In intact synaptosomes the largest dynamin pool was associated with the particulate fraction, while a smaller pool was cytosolic or extracted with 150 nM NaCl and contained all the phosphorylated Protein. Purified dynamin also bound to Phospholipid-coated controlled-pore glass beads, but poorly in the presence of NaCl, Mg2+, GTP, or ATP. Ca2+ induced a reversible translocation from the cytosol to the particulate fraction (50% at 183 microM Ca2+) in brain homogenates, and the purified Protein also underwent Ca(2+)-sensitive translocation to Phospholipid-coated controlled-pore glass beads. We conclude that dynamin I is a nerve terminal Ca(2+)-sensitive Phospholipid-Binding Protein with very high substrate affinity for PKC. We propose that phosphorylation by PKC occurs in the nerve terminal soluble compartment and that Ca2+ may mediate its Binding to the particulate fraction, thereby blocking the PKC phosphorylation sites. These properties may contribute to the lack of PKC phosphorylation during depolarization, despite the presence of activated PKC.
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a single c2 domain from synaptotagmin i is sufficient for high affinity ca2 Phospholipid Binding
Journal of Biological Chemistry, 1993Co-Authors: Bazbek Davletov, Thomas C SudhofAbstract:Abstract Synaptotagmin I is a Ca(2+)- and Phospholipid-Binding Protein of synaptic vesicles with an essential function in neurotransmission. Ca2+/Phospholipid Binding by synaptotagmin I may be mediated by its C2 domains, sequence motifs that have been implicated in the Ca2+ regulation of a variety of Proteins. However, it is currently unknown if C2 domains are sufficient for Ca2+/Phospholipid Binding or if they even directly participate in Ca2+/Phospholipid Binding. In order to address this question, we have studied the Ca2+/Phospholipid-Binding properties of the first C2 domain of synaptotagmin I. Our results show that this C2 domain by itself binds Ca2+ and Phospholipids with high affinity (half-maximal Binding at 4-6 microM free Ca2+) and exhibits strong positive cooperativity. The C2 domain is specific for negatively charged Phospholipids and for those divalent cations that are known to stimulate synaptic vesicle exocytosis (Ca2+ > Sr2+, Ba2+ > Mg2+). These studies establish that C2 domains can serve as independently folding Ca2+/Phospholipid-Binding domains. Furthermore, the cation specificity and the cooperativity of Ca2+ Binding by the C2 domain from synaptotagmin I support a role for this Protein in mediating the Ca2+ signal in neurotransmitter release.
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A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/Phospholipid Binding.
Journal of Biological Chemistry, 1993Co-Authors: Bazbek Davletov, Thomas C SudhofAbstract:Abstract Synaptotagmin I is a Ca(2+)- and Phospholipid-Binding Protein of synaptic vesicles with an essential function in neurotransmission. Ca2+/Phospholipid Binding by synaptotagmin I may be mediated by its C2 domains, sequence motifs that have been implicated in the Ca2+ regulation of a variety of Proteins. However, it is currently unknown if C2 domains are sufficient for Ca2+/Phospholipid Binding or if they even directly participate in Ca2+/Phospholipid Binding. In order to address this question, we have studied the Ca2+/Phospholipid-Binding properties of the first C2 domain of synaptotagmin I. Our results show that this C2 domain by itself binds Ca2+ and Phospholipids with high affinity (half-maximal Binding at 4-6 microM free Ca2+) and exhibits strong positive cooperativity. The C2 domain is specific for negatively charged Phospholipids and for those divalent cations that are known to stimulate synaptic vesicle exocytosis (Ca2+ > Sr2+, Ba2+ > Mg2+). These studies establish that C2 domains can serve as independently folding Ca2+/Phospholipid-Binding domains. Furthermore, the cation specificity and the cooperativity of Ca2+ Binding by the C2 domain from synaptotagmin I support a role for this Protein in mediating the Ca2+ signal in neurotransmitter release.
Jacob H. Rand - One of the best experts on this subject based on the ideXlab platform.
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resistance to annexin a5 anticoagulant activity a thrombogenic mechanism for the antiPhospholipid syndrome
Lupus, 2008Co-Authors: Jacob H. Rand, Anthony S Quinn, Douglas J TaatjesAbstract:The Phospholipid Binding Protein, annexin A5 (AnxA5), has potent anticoagulant properties that result from its forming 2-dimensional crystals over Phospholipids, blocking the availability of the Phospholipids for critical coagulation enzyme reactions. This article reviews the evidence that antiPhospholipid antibodies can disrupt this anticoagulant shield and unmask thrombogenic anionic Phospholipids, which may thereby contribute to thrombosis in patients with the antiPhospholipid syndrome (APS). This mechanism for thrombosis in APS can be monitored with coagulation assays for resistance to anticoagulant activity of AnxA5.
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pregnancy loss in the antiPhospholipid antibody syndrome a possible thrombogenic mechanism
The New England Journal of Medicine, 1997Co-Authors: Jacob H. Rand, Harry A M Andree, Charles J Lockwood, Seth Guller, Jonathan Scher, Peter C HarpelAbstract:Background The mechanisms of vascular thrombosis and pregnancy loss in the antiPhospholipid-antibody syndrome are unknown. Levels of annexin V, a Phospholipid-Binding Protein with potent anticoagulant activity, are markedly reduced on placental villi from women with this syndrome. Hypercoagulability in such women may therefore be due to the reduction of surface-bound annexin V by antiPhospholipid antibodies. To test this idea, we studied how antiPhospholipid antibodies affect levels of annexin V on cultured trophoblasts and human umbilical-vein endothelial cells and how they affect the procoagulant activity of these cells. Methods We isolated IgG fractions from three patients with the antiPhospholipid-antibody syndrome and from normal controls. These antibodies were incubated with cultured BeWo cells (a placental-trophoblast cell line), primary cultured trophoblasts, and human umbilical-vein endothelial cells. Annexin V on the cell surfaces was measured by an enzyme-linked immunosorbent assay. The coagula...
Volker Gerke - One of the best experts on this subject based on the ideXlab platform.
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Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator
Journal of visualized experiments : JoVE, 2018Co-Authors: Anna Livia Linard Matos, Volker Gerke, David Grill, Sergej Kudruk, Nicole Heitzig, Hans-joachim Galla, Ursula RescherAbstract:The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of Proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of Phospholipid-Binding Proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the Binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-Binding Proteins and bilayer compositions.
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Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles.
Molecular biology of the cell, 2005Co-Authors: Sylvette Chasserot-golaz, Volker Gerke, Nicolas Vitale, Emeline Umbrecht-jenck, D. E. Knight, Marie-france BaderAbstract:Annexin 2 is a calcium-dependent Phospholipid-Binding Protein that has been implicated in a number of membrane-related events, including regulated exocytosis. In chromaffin cells, we previously reported that catecholamine secretion requires the translocation and formation of the annexin 2 tetramer near the exocytotic sites. Here, to obtain direct evidence for a role of annexin 2 in exocytosis, we modified its expression level in chromaffin cells by using the Semliki Forest virus expression system. Using a real-time assay for individual cells, we found that the reduction of cytosolic annexin 2, and the consequent decrease of annexin 2 tetramer at the cell periphery, strongly inhibited exocytosis, most likely at an early stage before membrane fusion. Secretion also was severely impaired in cells expressing a chimera that sequestered annexin 2 into cytosolic aggregates. Moreover, we demonstrate that secretagogue-evoked stimulation triggers the formation of lipid rafts in the plasma membrane, essential for exocytosis, and which can be attributed to the annexin 2 tetramer. We propose that annexin 2 acts as a calcium-dependent promoter of lipid microdomains required for structural and spatial organization of the exocytotic machinery.
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characterization of the cell cycle regulated Protein calcyclin from ehrlich ascites tumor cells identification of two Binding Proteins obtained by ca2 dependent affinity chromatography
FEBS Journal, 1991Co-Authors: Anna Filipek, Volker Gerke, Klaus Weber, Jacek KuznickiAbstract:The nearly complete amino acid sequence obtained for murine calcyclin from Ehrlich ascites tumor cells reveals a very strong similarity with the rat and human sequences previously deduced from corresponding cDNA clones. While mouse and rat calcyclins are identical, the human Protein shows at three positions a conservative amino acid replacement. Using a mouse calcyclin affinity matrix, two Proteins with molecular masses of about 36 kDa have been purified from Ehrlich ascites tumor cells. The interaction between these two Proteins and the immobilized calcyclin is strictly Ca2+-dependent. Immunological criteria and partial sequence data identify the two calcyclin-Binding Proteins as the Phospholipid-Binding Protein annexin II (p36) and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. These observations suggest that calcyclin may exert its physiological function by a Ca2+-dependent interaction with cellular targets, e.g. annexin II or glyceraldehyde-3-phosphate dehydrogenase.
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identification of a homologue for annexin vii synexin in dictyostelium discoideum
Journal of Biological Chemistry, 1991Co-Authors: Volker GerkeAbstract:Abstract Immunological and biochemical data have been used to show that the slime mold Dictyostelium discoideum expresses a Ca2+/Phospholipid-Binding Protein related to vertebrate annexins. The Dictyostelium Protein (apparent molecular mass 46 kDa) is recognized by an antibody directed against an annexin consensus peptide and exhibits the properties characteristic for annexins, i.e. it interacts in a Ca2(+)-dependent manner with negatively charged Phospholipids. Limited proteolysis converts the 46-kDa Protein into a 32-kDa derivative which retains the Ca2+/Phospholipid-Binding properties of the 46-kDa polypeptide. Partial Protein sequence data identify the Dictyostelium Protein as the typical annexin and indicate that the 46-kDa Protein is an annexin VII (synexin) homologue. The identification of an annexin in a simple eucaryote should lead to the introduction of genetic approaches to analyze the physiological role of the annexins.
Helena R Chang - One of the best experts on this subject based on the ideXlab platform.
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decreased expression of annexin a1 is correlated with breast cancer development and progression as determined by a tissue microarray analysis
Human Pathology, 2006Co-Authors: Dejun Shen, Farzad Nooraie, Yahya Elshimali, Victor Lonsberry, Jianbo He, David Chia, Shikha Bose, David Seligson, Helena R ChangAbstract:Summary Annexin A1 (ANXA1) is a calcium- and Phospholipid-Binding Protein and a known mediator of glucocorticoid-regulated inflammatory responses. Using a combined multiple high-throughput approach, we recently identified a reduced expression of ANXA1 in human breast cancer. The finding was confirmed at the gene level by quantitative reverse transcription-polymerase chain reaction and at the Protein level by immunohistochemical staining of normal, benign, and malignant breast tissues. In this study, we constructed and used a high-density human breast cancer tissue microarray to characterize the expressional pattern of ANXA1 according to histopathologies. The tissue microarray contains 1158 informative breast tissue cores of different histologies including normal tissues, hyperplasia, in situ and invasive tumors, and lymph node metastases. Our results showed that there was a significant decrease in glandular expression of ANXA1 in ductal carcinoma in situ and invasive ductal carcinoma compared with either normal breast tissue or hyperplasia ( P
Alan R. Spitzer - One of the best experts on this subject based on the ideXlab platform.
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synexin and gtp increase surfactant secretion in permeabilized alveolar type ii cells
American Journal of Physiology-lung Cellular and Molecular Physiology, 2001Co-Authors: Avinash Chander, Alan R. SpitzerAbstract:We have previously suggested that synexin (annexin VII), a Ca2+-dependent Phospholipid Binding Protein, may have a role in surfactant secretion, since it promotes membrane fusion between isolated l...