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:3.6.3.1 (Mg2+-ATPase)
1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lipids in biological membranes are asymmetrically distributed across the bilayer; the amine-containing phospholipids are enriched on the cytoplasmic surface of the plasma membrane, while the choline-containing and sphingolipids are enriched on the outer surface. The maintenance of transbilayer lipid asymmetry is essential for normal membrane function, and disruption of this asymmetry is associated with cell activation or pathologic conditions. Lipid asymmetry is generated primarily by selective synthesis of lipids on one side of the membrane. Because passive lipid transbilayer diffusion is slow, a number of proteins have evolved to either dissipate or maintain this lipid gradient. These proteins fall into three classes: 1) cytofacially-directed, ATP-dependent transporters ("flippases"); 2) exofacially-directed, ATP-dependent transporters ("floppases"); and 3) bidirectional, ATP-independent transporters ("scramblases"). The flippase is highly selective for phosphatidylserine and functions to keep this lipid sequestered from the cell surface. Floppase activity has been associated with the ABC class of transmembrane transporters. Although they are primarily nonspecific, at least two members of this class display selectivity for their substrate lipid. Scramblases are inherently nonspecific and function to randomize the distribution of newly synthesized lipids in the endoplasmic reticulum or plasma membrane lipids in activated cells. It is the combined action of these proteins and the physical properties of the membrane bilayer that generate and maintain transbilayer lipid asymmetry.
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PMID:Regulation of transbilayer plasma membrane phospholipid asymmetry. 1257 5

Acetaminophen hepatotoxicity is mediated by an initial metabolic activation and covalent binding of drug metabolites to liver proteins. Acetaminophen metabolites have been shown to affect rat liver microsomal Ca2+ stores, but the mechanism is not well understood. The aim of the current work was to find out if the metabolism of acetaminophen by CYP2E1 affects ryanodine-sensitive Ca2+ stores in the endoplasmic reticulum of transduced HepG2 cells. Five millimoles acetaminophen decreased proliferation of CYP2E1-overexpressing HepG2 cells, increased cytosolic Ca2+ levels and produced significant cytotoxicity, while only little, mostly anti-proliferative effects were found in HepG2 cells lacking CYP2E1. CYP2E1 inhibitor-4-methylpyrazole decreased drug cytotoxicity in transduced cells and normalized elevated Ca2+ levels. Acetaminophen cytotoxicity was significantly higher in CYP2E1 expressing cells with depleted glutathione. In the cells engineered to overexpress CYP2E1, an increased [3H]ryanodine affinity (by 45%) and increased ligand maximal binding to ryanodine receptors (by 64%) was observed, most probably due to increased association rate of [3H]ryanodine. Ca2+ loading was decreased by about 53% in microsomal fractions isolated from transduced cells treated with acetaminophen and by 92% in glutathione depleted transfected cells treated with the drug. Ca2+/Mg2+-ATPase activity was unchanged in all microsomal fractions. Such effects were not observed in cells lacking CYP2E1. Our results confirm significant role of CYP2E1 in metabolic activation of acetaminophen and indicate that ryanodine receptors located in the liver endoplasmic reticulum are sensitive targets for acetaminophen metabolites.
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PMID:Acetaminophen alters microsomal ryanodine Ca2+ channel in HepG2 cells overexpressing CYP2E1. 1524 17

Transbilayer flipping of glycerophospholipids in the endoplasmic reticulum (ER) is a key feature of membrane biogenesis. Flipping appears to be an ATP-independent, bidirectional process facilitated by specific proteins or flippases. Although a phospholipid flippase has yet to be identified, evidence supporting the existence of dedicated flippases was recently obtained through biochemical reconstitution studies showing that certain chromatographically resolved fractions of detergent-solubilized ER proteins were enriched in flippase activity, whereas others were inactive. We now extend these studies by describing two convenient assays of flippase activity utilizing fluorescent phospholipid analogues as transport reporters. We use these assays to show that (i) proteoliposomes generated from a flippase-enriched Triton X-100 extract of ER can flip analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; (ii) flipping of all three phospholipids is likely due to the same flippase(s) rather than distinct, phospholipid-specific transport proteins; (iii) functional flippases represent approximately 1% (w/w) of ER membrane proteins in the Triton extract; and (iv) glycerophospholipid flippase activity in the ER can be attributed to two functionally distinct proteins (or classes of proteins) defined by their sensitivity to the cysteine and histidine modification reagents N-ethylmaleimide and diethylpyrocarbonate, respectively. Analyses of the N-ethylmaleimide-sensitive class of flippase activity revealed that the functionally critical sulfhydryl group in the flippase protein is buried in a hydrophobic environment in the membrane but becomes reactive on extraction of the protein into Triton X-100. This observation holds considerable promise for future attempts to isolate the flippase via an affinity approach.
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PMID:Chemical modification identifies two populations of glycerophospholipid flippase in rat liver ER. 1531 32

Glycerophospholipid flip-flop across biogenic membranes such as the endoplasmic reticulum (ER) is a fundamental feature of membrane biogenesis. Flip-flop requires the activity of specific membrane proteins called flippases. These proteins have yet to be identified in biogenic membranes and the molecular basis of their action is unknown. It is generally believed that flippase-facilitated glycerophospholipid flip-flop across the ER is governed by the stereochemistry of the glycerolipid, but this important issue has not been resolved. Here we investigate whether the ER flippase stereochemically recognizes the glycerophospholipids that it transports. To address this question we selected phosphatidylinositol (PI), a biologically important molecule with chiral centres in both its myo-inositol headgroup and its glycerol-lipid tail. The flip-flop of PI across the ER has not been previously reported. We synthesized fluorescence-labeled forms of all four diastereoisomers of PI and evaluated their flipping in rat liver ER vesicles, as well as in flippase-containing proteoliposomes reconstituted from a detergent extract of ER. Our results show that the flippase is able to translocate all four PI isomers and that both glycerol isomers of PI flip-flop across the ER membrane at rates similar to that measured for fluorescence-labeled phosphatidylcholine. Our data have important implications for recent hypotheses concerning the evolution of distinct homochiral glycerophospholipid membranes during the speciation of archaea and bacteria/eukarya from a common cellular ancestor.
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PMID:New fluorescent probes reveal that flippase-mediated flip-flop of phosphatidylinositol across the endoplasmic reticulum membrane does not depend on the stereochemistry of the lipid. 1578 18

The antitumor drug miltefosine has been recently approved as the first oral drug active against visceral leishmaniasis. We have previously identified the L. donovani miltefosine transporter (LdMT) as a P-type ATPase involved in phospholipid translocation at the plasma membrane of Leishmania parasites. Here we show that this protein is essential but not sufficient for the phospholipid translocation activity and, thus, for the potency of the drug. Based on recent findings in yeast, we have identified the putative beta subunit of LdMT, named LdRos3, as another protein factor required for the translocation activity. LdRos3 belongs to the CDC50/Lem3 family, proposed as likely beta subunits for P4-ATPases. The phenotype of LdRos3-defective parasites was identical to that of the LdMT-/-, including a defect in the uptake of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)-phosphatidylserine, generally considered as not affected in Lem3p-deficient yeast. Both LdMT and LdRos3 normally localized to the plasma membrane but were retained inside the endoplasmic reticulum in the absence of the other protein or when inactivating point mutations were introduced in LdMT. Modulating the expression levels of either protein independently, we show that any one of them could behave as the protein limiting the level of flippase activity. Thus, LdMT and LdRos3 seem to form part of the same translocation machinery that determines flippase activity and miltefosine sensitivity in Leishmania, further supporting the consideration of CDC50/Lem3 proteins as beta subunits required for the normal functioning of P4-ATPases.
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PMID:Phospholipid translocation and miltefosine potency require both L. donovani miltefosine transporter and the new protein LdRos3 in Leishmania parasites. 1678 29

Drs2p, a P-type adenosine triphosphatase required for a phosphatidylserine (PS) flippase activity in the yeast trans Golgi network (TGN), was first implicated in protein trafficking by a screen for mutations synthetically lethal with arf1 (swa). Here, we show that SWA4 is allelic to CDC50, encoding a membrane protein previously shown to chaperone Drs2p from the endoplasmic reticulum to the Golgi complex. We find that cdc50Delta exhibits the same clathrin-deficient phenotypes as drs2Delta, including delayed transport of carboxypeptidase Y to the vacuole, mislocalization of resident TGN enzymes and the accumulation of aberrant membrane structures. These trafficking defects precede appearance of cell polarity defects in cdc50Delta, suggesting that the latter are a secondary consequence of disrupting Golgi function. Involvement of Drs2p-Cdc50p in PS translocation suggests a role in restricting PS to the cytosolic leaflet of the Golgi and plasma membrane. Annexin V binding and papuamide B hypersensitivity indicate that drs2Delta or cdc50Delta causes a loss of plasma membrane PS asymmetry. However, clathrin and other endocytosis null mutants also exhibit a comparable loss of PS asymmetry, and studies with drs2-ts and clathrin (chc1-ts) conditional mutants suggest that loss of plasma membrane asymmetry is a secondary consequence of disrupting protein trafficking.
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PMID:Roles for the Drs2p-Cdc50p complex in protein transport and phosphatidylserine asymmetry of the yeast plasma membrane. 1695 84

Gene expression is controlled at several levels including mRNA decay. Sarco/endoplasmic reticulum Ca2+-Mg2+-ATPase isoform 2b (SERCA2b) is central to Ca2+ signalling and homeostasis in several tissues. SERCA2b mRNA decay involves interactions between cis-acting elements in its 3'-region and trans-acting nuclear protein factors. In the presence of the protein factors, the synthetic capped and polyadenylated RNA fragment 2b1 (3444-3753) decays faster than other SERCA2b 3'-region fragments. Here we determined the minimum cis-acting destabilizing element in the decay and its interactions with the nuclear protein factors. The in vitro decay required ATP hydrolysis and Mg2+ but not Ca2+. The decay was directional from 3' to 5', and involved a novel 35b GC rich domain designated 2b1-4 corresponding to 3521-3555. The decay of 2b1 RNA was decreased by (a) competition with 2b1-4, (b) mutation of 2b1 to delete 2b1-4, and (c) depleting the extracts of destabilizing trans-acting factors using immobilized 2b1-4. To determine the minimal destabilizing elements 2b1-4 was divided into 7b domains A-E. Deleting AB, BC, CD or DE inactivated the destabilizing cis-acting element but deleting A, B, C, D or E had no effect. In electrophoresis mobility shift assays the nuclear protein extracts retarded the mobility of labeled uncapped 2b1 RNA without a poly A+ tail. A positive co-operativity in the interactions was shown in protein concentration dependence of the shift and in the competition of 2b1-4 in inhibiting the mobility of 2b1 RNA. Based on further experiments, the domain CDE (3535-3555) was sufficient to compete with 2b1 RNA for the protein binding. Consistent with this competition, excess CDE RNA retarded the in vitro decay of 2b1 RNA. Thus the RNA decay required ATP hydrolysis and Mg2+ but not Ca2+, the minimum binding domain was in the sequence 3535-3555, and the decay may involve a multimeric protein complex.
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PMID:Characterization of SERCA2b Ca2+-Mg2+ ATPase mRNA decay by nuclear proteins. 1714 9

Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Insertion of lipids into the external leaflet created an area difference between the two leaflets that caused the formation of a bud-like structure. Under conditions of negligible flip-flop, the bud was stable. Upon reconstitution of the energy-independent flippase activity of the yeast endoplasmic reticulum into GUVs, the initial bud formation was reversible, and the shapes were recovered. This can be ascribed to a rapid flip-flop leading to relaxation of the monolayer area difference. Theoretical analysis of kinetics of shape changes provides self-consistent determination of the flip-flop rate and further kinetic parameters. Based on that analysis, the half-time of phospholipid flip-flop in the presence of endoplasmic reticulum proteins was found to be on the order of few minutes. In contrast, GUVs reconstituted with influenza virus protein formed stable buds. The results argue for the presence of specific membrane proteins mediating rapid flip-flop.
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PMID:Flippase activity detected with unlabeled lipids by shape changes of giant unilamellar vesicles. 1736 12

A phospholipid flippase activity from the endoplasmic reticulum (ER) of the model organism Saccharomyces cerevisiae has been characterized and functionally reconstituted into proteoliposomes. Analysis of the transbilayer movement of acyl-7-nitrobenz-2-oxa-1,3-diazol-4-yl (acyl-NBD)-labeled phosphatidylcholine in yeast microsomes using a fluorescence stopped-flow back exchange assay revealed a rapid, ATP-independent flip-flop (half-time, <2 min). Proteoliposomes prepared from a Triton X-100 extract of yeast microsomal membranes were also capable of flipping NBD-labeled phospholipid analogues rapidly in an ATP-independent fashion. Flippase activity was sensitive to the protein modification reagents N-ethylmaleimide and diethylpyrocarbonate. Resolution of the Triton X-100 extract by velocity gradient centrifugation resulted in the identification of a approximately 4S protein fraction enriched in flippase activity as well as of other fractions where flippase activity was depleted or undetectable. We estimate that flippase activity is due to a protein(s) representing approximately 2% (wt/wt) of proteins in the Triton X-100 extract. These results indicate that specific proteins are required to facilitate ATP-independent phospholipid flip-flop in the ER and that their identification is feasible. The architecture of the ER protein translocon suggests that it could account for the flippase activity in the ER. We tested this hypothesis using microsomes prepared from a temperature-sensitive yeast mutant in which the major translocon component, Sec61p, was quantitatively depleted. We found that the protein translocon is not required for transbilayer movement of phospholipids across the ER. Our work defines yeast as a promising model system for future attempts to identify the ER phospholipid flippase and to test and purify candidate flippases.
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PMID:Flip-flop of fluorescently labeled phospholipids in proteoliposomes reconstituted with Saccharomyces cerevisiae microsomal proteins. 1761 31

During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc(3)Man(9)GlcNAc(2)-P-P-Dol (DLO) biosynthesis.
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PMID:Recycling of dolichyl monophosphate to the cytoplasmic leaflet of the endoplasmic reticulum after the cleavage of dolichyl pyrophosphate on the lumenal monolayer. 1807 51


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