Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CaV2.2 channels play a key role in the gating of transmitter release sites (TRS) at presynaptic terminals. Physiological studies predict that the channels are linked directly to the TRS but the molecular composition of this complex remains poorly understood. We have used a high-affinity anti-CaV2.2 antibody, Ab571, to test a range of proteins known to contribute to TRS function for both an association in situ and a link in vitro. CaV2.2 clusters were isolated intact on immunoprecipitation beads and coprecipitated with a number of these proteins. Quantitative staining covariance analysis (ICA/ICQ method) was applied to the transmitter release face of the giant calyx terminal in the chick ciliary ganglion to test for TRS proteins with staining intensities that covary in situ with CaV2.2, resulting in a covariance sequence of NSF>RIM>spectrin>Munc18>VAMP>alpha-catenin, CASK>SV2>Na+-K+ approximately 0. A high-NaCl dissociation challenge applied to the immunoprecipitated complex, using the fractional recovery (FR) method [Khanna, R., Li, Q. & Stanley, E.F. (2006) PLoS.ONE., 1, e67], was used to test which proteins were most intimately associated with the channel, generating an FR sequence for CaV2.2 of: VAMP>or=actin>tubulin, NSF, Munc18, syntaxin 1>spectrin>CASK, SNAP25>RIM, Na+-K+ pump, v-ATPase, beta-catenin approximately 0. Proteins associated with endocytosis are considered in a companion paper [Khanna et al. (2007)Eur. J. Neurosci., 26, 560-574]. With the exception of VAMP and RIM, the ICQ and FR sequences were consistent, suggesting that proteins that covary the most strongly with CaV2.2 in situ are also the most intimately attached. Our findings suggest that the CaV2.2 cluster is an integral element of a multimolecular vesicle-fusion module that forms the core of a multifunctional TRS.
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PMID:The presynaptic CaV2.2 channel-transmitter release site core complex. 1768 36

Recycling of H(+)-ATPase to the apical plasma membrane, mediated by vesicular exocytosis and endocytosis, is an important mechanism for controlling H(+) secretion by the collecting duct. We hypothesized that SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins) may be involved in the targeting of H(+)-ATPase-coated vesicles. Using a tissue culture model of collecting duct H(+) secretory cells (inner medullary collecting duct (IMCD) cells), we demonstrated that they express the proteins required for SNARE-mediated exocytosis and form SNARE-fusion complexes upon stimulation of H(+)-ATPase exocytosis. Furthermore, exocytic amplification of apical H(+)-ATPase is sensitive to clostridial toxins that cleave SNAREs and thereby inhibit secretion. Thus, SNAREs are critical for H(+)-ATPase cycling to the plasma membrane. The process in IMCD cells has a feature distinct from that of neuronal cells: the SNARE complex includes and requires the vesicular cargo (H(+)-ATPase) for targeting. Using chimeras and truncations of syntaxin 1, we demonstrated that there is a specific cassette within the syntaxin 1 H3 domain that mediates binding of the SNAREs and a second distinct H3 region that binds H(+)-ATPase. Utilizing point mutations of the B1 subunit of the H(+)-ATPase, we document that this subunit contains specific targeting information for the H(+)-ATPase itself. In addition, we found that Munc-18-2, a regulator of exocytosis, plays a multifunctional role in this system: it regulates SNARE complex formation and the affinity of syntaxin 1 for H(+)-ATPase.
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PMID:Role of SNAREs and H+-ATPase in the targeting of proton pump-coated vesicles to collecting duct cell apical membrane. 1780 41

Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of alpha-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of alpha-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of alpha-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of alpha-SNAP potently inhibits Ca(2+)-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an alpha-SNAP mutant defective in NSF activation is used. We conclude that alpha-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for alpha-SNAP in the SNARE cycle that drives exocytotic membrane fusion.
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PMID:A novel site of action for alpha-SNAP in the SNARE conformational cycle controlling membrane fusion. 1809 56

Although it is known that proteins are delivered to and recycled from the plasma membrane (PM) via endosomes, the nature of the compartments and pathways responsible for cargo and vesicle sorting and cellular signaling is poorly understood. To define and dissect specific recycling pathways, chemical effectors of proteins involved in vesicle trafficking, especially through endosomes, would be invaluable. Thus, we identified chemicals affecting essential steps in PM/endosome trafficking, using the intensely localized PM transport at the tips of germinating pollen tubes. The basic mechanisms of this localized growth are likely similar to those of non-tip growing cells in seedlings. The compound endosidin 1 (ES1) interfered selectively with endocytosis in seedlings, providing a unique tool to dissect recycling pathways. ES1 treatment induced the rapid agglomeration of the auxin translocators PIN2 and AUX1 and the brassinosteroid receptor BRI1 into distinct endomembrane compartments termed "endosidin bodies"; however, the markers PIN1, PIN7, and other PM proteins were unaffected. Endosidin bodies were defined by the syntaxin SYP61 and the V-ATPase subunit VHA-a1, two trans-Golgi network (TGN)/endosomal proteins. Interestingly, brassinosteroid (BR)-induced gene expression was inhibited by ES1 and treated seedlings displayed a brassinolide (BL)-insensitive phenotype similar to a bri1 loss-of-function mutant. No effect was detected in auxin signaling. Thus, PIN2, AUX1, and BRI1 use interactive pathways involving an early SYP61/VHA-a1 endosomal compartment.
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PMID:Endosidin1 defines a compartment involved in endocytosis of the brassinosteroid receptor BRI1 and the auxin transporters PIN2 and AUX1. 1970 10

Proteomics technology was employed to profile host responses to rabies virus (RABV) infection in order to understand how RABV infection results in neuronal dysfunction. In mice infected with wild-type (wt) RABV, the expression of proteins involved in ion homeostasis was altered. H+ ATPase and Na+/K+ ATPase were up-regulated while Ca2+ ATPase was downregulated, which resulted in reduction of intracellular Na+ and Ca2+ concentrations. Furthermore, infection with wt RABV resulted in down-regulation of SNAREs such as alpha-SNAP, TRIM9, syntaxin, and pallidin, all of which are involved in docking and fusion of synaptic vesicles to and with the presynaptic membrane. As a consequence, the accumulation of synaptic vesicles was observed in the presynapses of mice infected with wt RABV. These data demonstrate that infection with wt RABV results in the alteration of host protein expression, particularly those involved in ion homeostasis and docking and the fusion of synaptic vesicles to the presynaptic membrane, which may lead to neuronal dysfunction.
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PMID:Pathogenic rabies virus alters host protein expression in the central nervous system: implications for neuronal dysfunction. 1863 69

Investigations of regulated S-nitrosylation and denitrosylation of vasorelevant proteins are a newly emergent area in vascular biology. We previously showed that monocrotaline pyrrole (MCTP)-induced megalocytosis of pulmonary arterial endothelial cells (PAECs), which underlies the development of pulmonary arterial hypertension, was associated with a Golgi blockade characterized by the trapping of diverse vesicle tethers, soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs), and soluble NSF-attachment proteins (SNAPs) in the Golgi; reduced trafficking of caveolin-1 (cav-1) and endotheial nitric oxide (NO) synthase (eNOS) from the Golgi to the plasma membrane; and decreased caveolar NO. We have investigated whether NSF, the ATPase involved in all SNARE disassembly, might be the upstream target of MCTP and whether MCTP might regulate NSF by S-nitrosylation. Immunofluorescence microscopy and Golgi purification techniques revealed the discordant decrease of NSF by approximately 50% in Golgi membranes after MCTP despite increases in alpha-SNAP, cav-1, eNOS, and syntaxin-6. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide failed to affect the initiation or progression of MCTP megalocytosis despite a reduction of 4,5-diaminofluorescein diacetate fluorescence and inhibition of S-nitrosylation of eNOS as assayed using the biotin-switch method. Moreover, the latter assay not only revealed constitutive S-nitrosylation of NSF, eNOS, cav-1, and clathrin heavy chain (CHC) in PAECs but also a dramatic 70-95% decrease in the S-nitrosylation of NSF, eNOS, cav-1, and CHC after MCTP. These data point to depletion of NSF from Golgi membranes as a mechanism for Golgi blockade after MCTP and to denitrosylation of vasorelevant proteins as critical to the development of endothelial cell megalocytosis.
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PMID:Depletion of the ATPase NSF from Golgi membranes with hypo-S-nitrosylation of vasorelevant proteins in endothelial cells exposed to monocrotaline pyrrole. 1877 48

Neuronal synapses are specialized sites for information exchange between neurons. Many diseases, such as addiction and mood disorders, likely result from altered expression of synaptic proteins, or altered formation of synaptic complexes involved in neurotransmission or neuroplasticity. A detailed description of native multiprotein complexes in synaptic plasma membranes (PM) is therefore essential for understanding biological mechanisms and disease processes. For the first time in this study, two-dimensional Blue Native/SDS-PAGE electrophoresis, combined with tandem mass spectrometry, was used to screen multiprotein complexes in synaptic plasma membranes from rat hippocampus. As a result, 514 unique proteins were identified, of which 36% were integral membrane proteins. In addition, 19 potentially novel and known heterooligomeric multiprotein complexes were found, such as the SNARE and ATPase complexes. A potentially novel protein complex, involving syntaxin, synapsin I and Na+/K+ ATPase alpha-1, was further confirmed by co-immunoprecipitation and immunofluorescence staining. As demonstrated here, Blue Native-PAGE is a powerful tool for the separation of hydrophobic membrane proteins. The combination of Blue Native-PAGE and mass spectrometry could systematically identify multiprotein complexes.
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PMID:Proteomic screen for multiprotein complexes in synaptic plasma membrane from rat hippocampus by blue native gel electrophoresis and tandem mass spectrometry. 1943 78

Life processes are governed at the chemical level, and therefore knowledge of how single molecules interact, provides a fundamental understanding of nature. The molecular mechanism of membrane fusion essential to vital cellular activities such as intracellular transport, hormone secretion, enzyme release, or neurotransmission, involve the assembly and disassembly of a specialized set of proteins present in opposing bilayers. Target membrane proteins at the cell plasma membrane SNAP-25 and syntaxin termed t-SNAREs, and secretory vesicle-associated protein VAMP or v-SNARE, are part of the conserved protein complex involved in fusion of opposing membranes. It has been demonstrated that in the presence of Ca2+, t-SNAREs and v-SNARE in opposing bilayers interact and self-assemble in a circular pattern, to form conducting channels. Such self-assembly of t-/v-SNAREs in a ring conformation occurs only when the respective SNAREs are in association with membrane. X-ray diffraction measurements further demonstrate that t-SNAREs in the target membrane and v-SNARE in the vesicle membrane overcome repulsive forces to bring opposing membranes close to within a distance of 2.8 A. Studies suggest that calcium bridging of the opposing bilayers, lead to release of water from hydrated Ca2+ ions as well as the loosely coordinated water at PO-lipid head groups, leading to membrane destabilization and fusion. The t-/v-SNARE is a tight complex, who's disassembly requires an ATPase called NSF, which functions as a right-handed molecular motor.
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PMID:Membrane fusion: role of SNAREs and calcium. 1960 99

Ouabain is both a cardiac glycoside used in therapy of congestive heart failure and an endogenous steroid hormone. It specifically binds to Na(+), K(+)-ATPase (NKA) and blocks its activity. Overdose of ouabain induces retinal damage. In different species ouabain-induced retinal degeneration affects different cell types. In fish and rabbit ouabain induces retinal cell death preferentially in the ganglion cell layer and outer photoreceptor segments respectively. In rats, the pattern of NKA expression has been studied with most detail among retinal neurons. In addition, ouabain selectively destroyed some types of neurons in rodents. However, ouabain-sensitive retinal neurons remain unclear in rats. We show here that injection of ouabain into the rat vitreous body induced dramatic cell death in the inner nuclear layer (INL). The cell death was time- and dose-dependent. Ouabain-induced dying cells in the INL were TUNEL-positive. Immunohistochemistry analysis revealed that there was a significant decrease in the number of calbindin D-28K- and syntaxin-1-positive horizontal and amacrine cells in the INL of ouabain-treated rat retinas. Thus our results revealed that the horizontal and amacrine cells are the most sensitive cell types to ouabain in the retina of Sprague-Dawley rat.
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PMID:The new targets of ouabain in retinal interneurons of Sprague-Dawley rats. 2010 55

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors)-mediated exocytotic release of neurotransmitters is a key process in neuronal communication, controlled by a number of molecular interactions. A synaptic vesicle v-SNARE protein (VAMP2 or synaptobrevin), in association with two plasma membrane t-SNAREs (syntaxin 1 and SNAP25), assemble to form a protein complex that is largely accepted as the minimal membrane fusion machine. Acidification of the synaptic vesicle lumen by the large multi-subunit vacuolar proton pump (V-ATPase) is required for loading with neurotransmitters. Recent data demonstrate a direct interaction between the c-subunit of the V-ATPase and VAMP2 that appears to play a role at a late step in transmitter release. In this review, we examine evidence suggesting that the V0 membrane sector of the V-ATPase not only participates in proton pumping, but plays a second distinct role in neurosecretion, downstream of filling and close to vesicle fusion.
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PMID:A role for V-ATPase subunits in synaptic vesicle fusion? 2137 31


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