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By admin, on April 5th, 2012
J Biol Chem. 2012 Apr 3;
Singh AK, McMillan J, Bukiya AN, Burton B, Parrill AL, Dopico AM
Large conductance, Ca(2+)- and voltage-gated K(+) (BK) channel proteins are ubiquitously expressed in cell membranes and control a wide variety of biological processes. Membrane cholesterol regulates the activity of membrane-associated proteins, including BK channels. Cholesterol modulation of BK channels alters action potential firing, colonic ion transport, smooth muscle contractility, endothelial function, and the channel alcohol response. The structural bases underlying cholesterol-BK channel interaction are unknown. Such interaction is determined by strict chemical requirements for the sterol molecule, suggesting cholesterol recognition by a protein surface. Here, we demonstrate that cholesterol action on BK channel-forming cbv1 proteins is mediated by their cytosolic C-tail domain (CTD), where we identified seven cholesterol recognition/interaction amino acid consensus motifs (CRACs 4-10), a distinct feature of BK proteins. Cholesterol-sensitivity is provided by the membrane-adjacent CRAC4 where Val444, Tyr450 and Lys453 are required for cholesterol-sensing, with hydrogen bonding and hydrophobic interactions participating in cholesterol location and recognition. However, cumulative truncations or Tyr-to-Phe substitutions in CRACs 5-10 progressively blunt cholesterol-sensitivity, documenting involvement of multiple CRACs in cholesterol-BK channel interaction. In conclusion, our study provides for the first time the structural bases of BK channel cholesterol sensitivity; the presence of membrane-adjacent CRAC4 and the long CTD with several other CRAC motifs, which are not found in other members of the TM6 superfamily of ion channels, very likely explains the unique cholesterol sensitivity of BK channels.
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Multiple cholesterol recognition/interaction amino acid consensus (CRAC) motifs in the cytosolic C tail of the slo1 subunit determine cholesterol sensitivity of Ca2+- and voltage-gated K+ (BK) channels.
By admin, on March 30th, 2012
Reprod Biol Endocrinol. 2012 Mar 29; 10(1): 25
Kunal SB, Killivalavan A, Medhamurthy R
ABSTRACT: BACKGROUND: In higher primates, during non-pregnant cycles, it is indisputable that circulating LH is essential for maintenance of corpus luteum (CL) function. On the other hand, during pregnancy, CL function gets rescued by the LH analogue, chorionic gonadotropin (CG). The molecular mechanisms involved in the control of luteal function during spontaneous luteolysis and rescue processes are not completely understood. Emerging evidence suggests that LH/CGR activation triggers proliferation and transformation of target cells by various signaling molecules as evident from studies demonstrating participation of Src family of tyrosine kinases (SFKs) and MAP kinases in hCG-mediated actions in Leydig cells. Since circulating LH concentration does not vary during luteal regression, it was hypothesized that decreased responsiveness of luteal cells to LH might occur due to changes in LH/CGR expression dynamics, modulation of SFKs or interference with steroid biosynthesis. METHODS: Since, maintenance of structure and function of CL is dependent on the presence of functional LH/CGR its expression dynamics as well as mRNA and protein expressions of SFKs were determined throughout the luteal phase. Employing well characterized luteolysis and CL rescue animal models, activities of SFKs, cAMP phosphodiesterase (cAMP-PDE) and expression of SR-B1 (a membrane receptor associated with trafficking of cholesterol ester) were examined. Also, studies were carried out to investigate the mechanisms responsible for decline in progesterone biosynthesis in CL during the latter part of the non-pregnant cycle. RESULTS AND DISCUSSION: The decreased responsiveness of CL to LH during late luteal phase could not be accounted for by changes in LH/CGR mRNA levels, its transcript variants or protein. Results obtained employing model systems depicting different functional states of CL revealed increased activity of SFKs [pSrc (Y-416)] and PDE as well as decreased expression of SR-B1correlating with initiation of spontaneous luteolysis. However, CG, by virtue of its heroic efforts, perhaps by inhibition of SFKs and PDE activation, prevents CL from undergoing regression during pregnancy. CONCLUSIONS: The results indicated participation of activated Src and increased activity of cAMP-PDE in the control of luteal function in vivo. That the exogenous hCG treatment caused decreased activation of Src and cAMP-PDE activity with increased circulating progesterone might explain the transient CL rescue that occurs during early pregnancy.
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Involvement of Src family of kinases and cAMP phosphodiesterase in the luteinizing hormone/ chorionic gonadotropin receptor-mediated signaling in the corpus luteum of monkey.
By admin, on March 30th, 2012
Reprod Biol Endocrinol. 2012 Mar 29; 10(1): 25
Kunal SB, Killivalavan A, Medhamurthy R
ABSTRACT: BACKGROUND: In higher primates, during non-pregnant cycles, it is indisputable that circulating LH is essential for maintenance of corpus luteum (CL) function. On the other hand, during pregnancy, CL function gets rescued by the LH analogue, chorionic gonadotropin (CG). The molecular mechanisms involved in the control of luteal function during spontaneous luteolysis and rescue processes are not completely understood. Emerging evidence suggests that LH/CGR activation triggers proliferation and transformation of target cells by various signaling molecules as evident from studies demonstrating participation of Src family of tyrosine kinases (SFKs) and MAP kinases in hCG-mediated actions in Leydig cells. Since circulating LH concentration does not vary during luteal regression, it was hypothesized that decreased responsiveness of luteal cells to LH might occur due to changes in LH/CGR expression dynamics, modulation of SFKs or interference with steroid biosynthesis. METHODS: Since, maintenance of structure and function of CL is dependent on the presence of functional LH/CGR its expression dynamics as well as mRNA and protein expressions of SFKs were determined throughout the luteal phase. Employing well characterized luteolysis and CL rescue animal models, activities of SFKs, cAMP phosphodiesterase (cAMP-PDE) and expression of SR-B1 (a membrane receptor associated with trafficking of cholesterol ester) were examined. Also, studies were carried out to investigate the mechanisms responsible for decline in progesterone biosynthesis in CL during the latter part of the non-pregnant cycle. RESULTS AND DISCUSSION: The decreased responsiveness of CL to LH during late luteal phase could not be accounted for by changes in LH/CGR mRNA levels, its transcript variants or protein. Results obtained employing model systems depicting different functional states of CL revealed increased activity of SFKs [pSrc (Y-416)] and PDE as well as decreased expression of SR-B1correlating with initiation of spontaneous luteolysis. However, CG, by virtue of its heroic efforts, perhaps by inhibition of SFKs and PDE activation, prevents CL from undergoing regression during pregnancy. CONCLUSIONS: The results indicated participation of activated Src and increased activity of cAMP-PDE in the control of luteal function in vivo. That the exogenous hCG treatment caused decreased activation of Src and cAMP-PDE activity with increased circulating progesterone might explain the transient CL rescue that occurs during early pregnancy.
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Involvement of Src family of kinases and cAMP phosphodiesterase in the luteinizing hormone/ chorionic gonadotropin receptor-mediated signaling in the corpus luteum of monkey.
By admin, on March 29th, 2012
Mol Med Report . 2012 Mar 20; Li WY, Yao CX, Zhang SF, Wang SL, Wang TQ, Xiong CJ, Li YB, Zang MX We recently demonstrated that fenofibrate induces the activities of citrate synthase and NADH oxidase in cardiac mitochondria. To further determine the molecular mechanisms underlying fenofibrate action, 8-week-old mice were administered fenofibrate (100 mg/kg/day) for 7 and 14 days, and the expression of genes involved in cardiac mitochondrial function, such as nuclear respiratory factor 1 transcript variant 2 (NRF-1-L) and 6 (NRF-1-S), mitochondrial outer membrane protein 40 (Tom40), lipoic acid synthetase (Lias), cytochrome b, medium-chain acyl-coenzyme A dehydrogenase (MCAD) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) was determined
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Improvement of myocardial lipid accumulation and prevention of PGC-1α induction by fenofibrate.
By admin, on March 27th, 2012
One gene encoding surface membrane proteins,Annexin of Schis tosoma japonicum,have been cloned,and also the studies on the gene expression and biological function was carried out preliminarily in this paper.Using the …
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Surface Membrane Protein Annexin of Schistosoma Japonicum …
By admin, on March 27th, 2012
One gene encoding surface membrane proteins,Annexin of Schis tosoma japonicum,have been cloned,and also the studies on the gene expression and biological function was carried out preliminarily in this paper.Using the …
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Surface Membrane Protein Annexin of Schistosoma Japonicum …
By admin, on March 16th, 2012
Mol Biol Cell. 2012 Mar 14;
Rao S, Schmidt O, Harbauer AB, Schönfisch B, Guiard B, Pfanner N, Meisinger C
The preprotein translocase of the outer mitochondrial membrane (TOM) functions as the main entry gate for the import of nuclear-encoded proteins into mitochondria. The major subunits of the TOM complex are the three receptors Tom20, Tom22 and Tom70, and the central channel-forming protein Tom40. Cytosolic kinases have been shown to regulate the biogenesis and activity of the Tom receptors. Casein kinase 2 stimulates the biogenesis of Tom22 and Tom20, whereas protein kinase A (PKA) impairs the receptor function of Tom70. Here we report that PKA exerts an inhibitory effect on the biogenesis of the β-barrel protein Tom40. Tom40 is synthesized as precursor on cytosolic ribosomes and subsequently imported into mitochondria. We show that PKA phosphorylates the precursor of Tom40. The phosphorylated Tom40 precursor is impaired in import into mitochondria, whereas the non-phosphorylated precursor is efficiently imported. We conclude that PKA plays a dual role in the regulation of the TOM complex. Phosphorylation by PKA not only impairs the receptor activity of Tom70, but also inhibits the biogenesis of the channel protein Tom40.
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Biogenesis of the preprotein translocase of the outer mitochondrial membrane: protein kinase A phosphorylates the precursor of Tom40 and impairs its import.
By admin, on March 16th, 2012
Mol Biol Cell. 2012 Mar 14;
Rao S, Schmidt O, Harbauer AB, Schönfisch B, Guiard B, Pfanner N, Meisinger C
The preprotein translocase of the outer mitochondrial membrane (TOM) functions as the main entry gate for the import of nuclear-encoded proteins into mitochondria. The major subunits of the TOM complex are the three receptors Tom20, Tom22 and Tom70, and the central channel-forming protein Tom40. Cytosolic kinases have been shown to regulate the biogenesis and activity of the Tom receptors. Casein kinase 2 stimulates the biogenesis of Tom22 and Tom20, whereas protein kinase A (PKA) impairs the receptor function of Tom70. Here we report that PKA exerts an inhibitory effect on the biogenesis of the β-barrel protein Tom40. Tom40 is synthesized as precursor on cytosolic ribosomes and subsequently imported into mitochondria. We show that PKA phosphorylates the precursor of Tom40. The phosphorylated Tom40 precursor is impaired in import into mitochondria, whereas the non-phosphorylated precursor is efficiently imported. We conclude that PKA plays a dual role in the regulation of the TOM complex. Phosphorylation by PKA not only impairs the receptor activity of Tom70, but also inhibits the biogenesis of the channel protein Tom40.
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Biogenesis of the preprotein translocase of the outer mitochondrial membrane: protein kinase A phosphorylates the precursor of Tom40 and impairs its import.
By admin, on March 13th, 2012
J Neurochem. 2012 Mar 10;
Macdonald JI, Dietrich A, Gamble S, Hryciw T, Grant RI, Meakin SO
Vesicular transport in neurons plays a vital role in neuronal function and survival. Nesca is a novel protein that we previously identified and herein describe its pattern of expression, subcellular localization and protein-protein interactions both in vitro and in vivo. Specifically, a large proportion of Nesca is in tight association with both actin and microtubule cytoskeletal proteins. Nesca binds to F-actin, microtubules, βIII and acetylated α-tubulin, but not neurofilaments or the actin binding protein drebrin, in in vitro binding assays. Nesca co-immunoprecipitates with kinesin heavy chain (KIF5B) and kinesin light chain (KLC) motors as well as with the synaptic membrane precursor protein, syntaxin-1, and is a constituent of the post synaptic density. Moreover, in vitro binding assays indicate that Nesca directly binds KIF5B, KLC and syntaxin-1. In contrast, Nesca does not co-immunoprecipitate with the kinesin motors KIF1B, KIF3A nor does it bind syntaxin-4 or SNAP-25 in vitro. Nesca expression in neurons is highly punctuate, co-stains with syntaxin-1, and is found in fractions containing markers of early endosomes and Golgi suggesting that it is involved in vesicular transport. Collectively, these data suggest that Nesca functions as an adapter involved in neuronal vesicular transport including vesicles containing soluble N-ethylmaleimide sensitive factor attachment protein (SNAP) receptors (SNAREs) that are essential to exocytosis. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.
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Nesca, a Novel Neuronal Adapter Protein, Potentially Links the Molecular Motor Kinesin with the Pre-synaptic Membrane Protein, Syntaxin-1, in Hippocampal Neurons.
By admin, on March 13th, 2012
J Neurochem. 2012 Mar 10;
Macdonald JI, Dietrich A, Gamble S, Hryciw T, Grant RI, Meakin SO
Vesicular transport in neurons plays a vital role in neuronal function and survival. Nesca is a novel protein that we previously identified and herein describe its pattern of expression, subcellular localization and protein-protein interactions both in vitro and in vivo. Specifically, a large proportion of Nesca is in tight association with both actin and microtubule cytoskeletal proteins. Nesca binds to F-actin, microtubules, βIII and acetylated α-tubulin, but not neurofilaments or the actin binding protein drebrin, in in vitro binding assays. Nesca co-immunoprecipitates with kinesin heavy chain (KIF5B) and kinesin light chain (KLC) motors as well as with the synaptic membrane precursor protein, syntaxin-1, and is a constituent of the post synaptic density. Moreover, in vitro binding assays indicate that Nesca directly binds KIF5B, KLC and syntaxin-1. In contrast, Nesca does not co-immunoprecipitate with the kinesin motors KIF1B, KIF3A nor does it bind syntaxin-4 or SNAP-25 in vitro. Nesca expression in neurons is highly punctuate, co-stains with syntaxin-1, and is found in fractions containing markers of early endosomes and Golgi suggesting that it is involved in vesicular transport. Collectively, these data suggest that Nesca functions as an adapter involved in neuronal vesicular transport including vesicles containing soluble N-ethylmaleimide sensitive factor attachment protein (SNAP) receptors (SNAREs) that are essential to exocytosis. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.
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Nesca, a Novel Neuronal Adapter Protein, Potentially Links the Molecular Motor Kinesin with the Pre-synaptic Membrane Protein, Syntaxin-1, in Hippocampal Neurons.
By admin, on March 11th, 2012
Subcell Biochem. 2012; 58: 1-24
Minogue S, Waugh MG
Phosphatidylinositol 4-phosphate (PtdIns4P) is a quantitatively minor membrane phospholipid which is the precursor of PtdIns(4,5)P (2) in the classical agonist-regulated phospholipase C signalling pathway. However, PtdIns4P also governs the recruitment and function of numerous trafficking molecules, principally in the Golgi complex. The majority of phosphoinositides (PIs) phosphorylated at the D4 position of the inositol headgroup are derived from PtdIns4P and play roles in a diverse array of fundamental cellular processes including secretion, cell migration, apoptosis and mitogenesis; therefore, PtdIns4P biosynthesis can be regarded as key point of regulation in many PI-dependent processes.Two structurally distinct sequence families, the type II and type III PtdIns 4-kinases, are responsible for PtdIns4P synthesis in eukaryotic organisms. These important proteins are differentially expressed, localised and regulated by distinct mechanisms, indicating that the enzymes perform non-redundant roles in trafficking and signalling. In recent years, major advances have been made in our understanding of PtdIns4K biology and here we summarise current knowledge of PtdIns4K structure, function and regulation.
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The Phosphatidylinositol 4-Kinases: Don’t Call it a Comeback.
By admin, on March 8th, 2012
J Mol Biol . 2012 Mar 2; Naveed H, Jimenez-Morales D, Tian J, Pasupuleti V, Kenney LJ, Liang J Biogenesis of β-barrel membrane proteins is a complex, multi-step, and as yet incompletely characterized process. The bacterial porin family is perhaps the best studied protein family among the β-barrel membrane proteins that allows diffusion of small solutes across the bacterial outer membrane.
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Engineered oligomerization state of OmpF protein through computational design decouples oligomer dissociation from unfolding.
By admin, on March 8th, 2012
J Mol Biol . 2012 Mar 2; Naveed H, Jimenez-Morales D, Tian J, Pasupuleti V, Kenney LJ, Liang J Biogenesis of β-barrel membrane proteins is a complex, multi-step, and as yet incompletely characterized process.
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Engineered oligomerization state of OmpF protein through computational design decouples oligomer dissociation from unfolding.
By admin, on March 7th, 2012
is required. Experience with sphingolipid transfer protein structure-function and sphingolipid membrane … NMR is preferred including cloning, point mutagenesis, and protein and lipid purification; as well as a background in…
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Hormel Fellow at University of Minnesota (Minneapolis, MN)
By admin, on March 7th, 2012
Free Radic Biol Med. 2012 Feb 28;
Ziberna L, Tramer F, Moze S, Vrhovsek U, Mattivi F, Passamonti S
Flavonoids are dietary components involved in decreasing oxidative stress in the vascular endothelium, and thus the risk of endothelial dysfunction. However, their very low concentrations in plasma place this role in doubt. Thus, a relationship between the effective intracellular concentration of flavonoids and their bioactivity needs to be assessed. This study examined the uptake of physiological concentrations of cyanidin 3-glucoside, a widespread dietary flavonoid, into the human vascular endothelial cells. Furthermore, the involvement of the membrane transporter bilitranslocase (TC #2.A.65.1.1) as the key underlying molecular mechanism for membrane transport was investigated by using purified anti-sequence antibodies binding at the extracellular domain of the protein. The experimental observations were carried out in isolated plasma membrane vesicles and intact endothelial cells from human endothelial cells (EA.hy926), and on the ischemia-reperfusion model in the isolated rat hearts. Cyanidin 3-glucoside was transported via bilitranslocase into endothelial cells, where it acted as a powerful intracellular antioxidant and a cardioprotective agent in the reperfusion phase after ischemia. These findings suggest that dietary flavonoids, in spite of their limited oral bioavailability and very low post-absorption plasma concentrations, may provide protection against oxidative stress-based cardiovascular diseases. Bilitranslocase, by mediating the cellular uptake of some flavonoids, is thus a key factor for their protective activity on the endothelial function.
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Transport and Bioactivity of Cyanidin 3-Glucoside into the Vascular Endothelium.
By admin, on March 5th, 2012
Gene Expr Patterns. 2012 Feb 15;
Ihara KI, Nishimura T, Fukuda T, Ookura T, Nishimori K
The membrane-associated guanylate kinase inverted 2 (MAGI-2) protein, which is known to localize at the tight junction of epithelial cells, contains multiple copies of the PDZ and WW domains in its structure. Although the expression pattern of Magi2 mRNA in representative organs has been previously published, its detailed cellular distribution at the histological level remains unknown. Such detailed information would be useful to clarify the biological function of MAGI-2. Here, we report the generation of Venus reporter knock-in mice for Magi2 in which exon 6 of the gene was substituted by the Venus-encoding sequence. We detected the expression of the Venus reporter protein in kidney podocytes from these knock-in mice. We also detected Venus reporter protein expression in spermatids within the testes and within neurons in various regions of the brain. Detection of the reporter protein from these diverse locations indicated the endogenous expression of MAGI-2 in these tissues. Our data suggested a potential function of MAGI-2 in the glomerular filtration process and sperm cell maturation. These data indicate that the Venus reporter knock-in mouse for Magi2 is a useful model for the further study of Magi2 gene function.
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Generation of Venus reporter knock-in mice revealed MAGI-2 expression patterns in adult mice.
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