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By admin, on February 21st, 2012
Plant Physiol. 2012 Feb 13;
Qi D, Deyoung BJ, Innes RW
The Arabidopsis RPS5 disease resistance protein mediates recognition of the Pseudomonas syringae effector protein AvrPphB. RPS5 belongs to the coiled-coil-nucleotide binding site-leucine-rich repeat (CC-NBS-LRR) family and is activated by AvrPphB-mediated cleavage of the protein kinase PBS1. Here we present a structure-function analysis of the CC and LRR domains of RPS5 using transient expression assays in Nicotiana benthamiana. We found that substituting the CC domain of RPS2 for the RPS5 CC domain did not alter RPS5 specificity and only moderately reduced its ability to activate programmed cell death, suggesting that the CC domain does not play a direct role in the recognition of PBS1 cleavage. Analysis of an RPS5-super Yellow Fluorescent Protein (sYFP) fusion revealed that RPS5 localizes to the plasma membrane (PM). Alanine substitutions of predicted myristoylation (glycine 2) and palmitoylation residues (cysteine 4) affected RPS5 PM localization, protein stability, and function in an additive manner, indicating that PM localization is essential to RPS5 function. The first 20 amino acids of RPS5 were sufficient for directing sYFP to the PM. C-terminal truncations of RPS5 revealed that the first 4 LRR repeats are sufficient for inhibiting RPS5 autoactivation; however, the complete LRR domain was required for recognition of PBS1 cleavage. Substitution of the RPS2 LRR domain resulted in autoactivation of RPS5, indicating that the LRR domain must co-evolve with the NBS domain. We conclude that the RPS5 LRR domain functions to suppress RPS5 activation in the absence of PBS1 cleavage, and promotes RPS5 activation in its presence.
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Structure-Function Analysis of the Coiled-Coil and Leucine-Rich Repeat Domains of the RPS5 Disease Resistance Protein.
By admin, on February 16th, 2012
Biochem Cell Biol . 2012 Feb 14; Arutyunova E, Brooks CL, Beddek A, Mak MW, Schryvers AB, Lemieux MJ Lactoferrin (Lf) is a bi-lobed, iron-binding protein found on mucosal surfaces and at sites of inflammation. Gram-negative pathogens from the Neisseriaceae and Moraxellaceae families are capable of using Lf as a source of iron for growth through a process mediated by a bacterial surface receptor that directly binds host Lf.
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Crystal structure of the N-lobe of lactoferrin binding protein B from Moraxella bovis (1) () This paper is an invited article as a result of a presentation at the International Lactoferrin Conference held in Mazatlan, Mexico (May 2011), and has undergone the Journal’s usual peer review process.
By admin, on February 12th, 2012
Microbiology . 2012 Feb 9; Shiba Y, Miyagawa H, Nagahama H, Matsumoto K, Kondo D, Matsuoka S, Matsumoto K, Hara H The Rcs phosphorelay signal transduction system controls genes for capsule production and many other envelope-related functions and is implicated in biofilm formation. We investigated the activation of the Rcs system in a pgsA null mutant of Escherichia coli, which completely lacks the major acidic phospholipids, phosphatidylglycerol and cardiolipin.
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Exploring the relationship between lipoprotein mislocalisation and activation of the Rcs signal transduction system in Escherichia coli.
By admin, on February 12th, 2012
Microbiology . 2012 Feb 9; Shiba Y, Miyagawa H, Nagahama H, Matsumoto K, Kondo D, Matsuoka S, Matsumoto K, Hara H The Rcs phosphorelay signal transduction system controls genes for capsule production and many other envelope-related functions and is implicated in biofilm formation. We investigated the activation of the Rcs system in a pgsA null mutant of Escherichia coli, which completely lacks the major acidic phospholipids, phosphatidylglycerol and cardiolipin.
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Exploring the relationship between lipoprotein mislocalisation and activation of the Rcs signal transduction system in Escherichia coli.
By admin, on February 12th, 2012
Mol Biol Cell. 2012 Feb 9;
Morén B, Shah C, Howes MT, Schieber NL, McMahon HT, Parton RG, Daumke O, Lundmark R
EHD2 belongs to the Eps15 homology (EH) domain containing protein family of dynamin-related ATPases involved in membrane remodeling in the endosomal system. EHD2 dimers oligomerize into rings on highly curved membranes resulting in stimulation of the intrinsic ATPase activity. Here, we report that EHD2 is specifically and stably associated with caveolae at the plasma membrane and not involved in clathrin-mediated endocytosis or endosomal recycling, as previously suggested. EHD2 interacts with pacsin2 and cavin1 and ordered membrane assembly of EHD2 is dependent on cavin1 and caveolae integrity. While the EH domain of EHD2 is dispensable for targeting, we identified a loop in the nucleotide binding domain that together with ATP-binding is required for caveola localization. EHD2 was not essential for the formation or shaping of caveolae but high levels of EHD2 caused distortion and loss of endogenous caveolae. Assembly of EHD2 stabilized and constrained caveolae to the plasma membrane to control turnover, and depletion of EHD2 resulted in endocytic and more dynamic and short-lived caveolae. Thus, following the identification of caveolin and cavins, EHD2 constitutes a third structural component of caveolae involved in controlling the stability and turnover of this organelle.
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EHD2 regulates caveola dynamics via ATP-driven targeting and oligomerization.
By admin, on February 8th, 2012
J Lipid Res. 2012 Feb 3;
Irino Y, Tokuda E, Hasegawa J, Itoh T, Takenawa T
Phosphoinositides (PIs) play important regulatory roles in cell physiology. Localization and quantitation of PIs within the cell is necessary to understand their precise function. Currently, ectopic expression of green fluorescent protein (GFP)-fused PI-binding domains is used to visualize PIs localized to the cell membrane. However, ectopically expressed PI-binding domains may compete with endogenous binding proteins, thus altering the physiological functions of the PIs. Here, we establish a novel method for quantification and visualization of PIs in cells and tissue samples, using PI-binding domains labeled with quantum dots (Qdots) as specific probes. This method allowed us to simultaneously quantify 3 distinct PIs; phosphatidylinositol 3,4,5-triphosphatase (PtdIns(3,4,5)P3), PtdIns(3,4)P2, and PtdIns(4,5)P2, in crude acidic lipids extracted from insulin-stimulated cells. In addition, the method allows the PIs to be visualized within fixed cells and tissues. Sequential and spatial changes in PI production and distribution were detected in platelet-derived growth factor (PDGF)-stimulated NRK49F cells. We also observed accumulation of PtdIns(3,4)P2 at the dorsal ruffle in PDGF-stimulated NIH3T3 cells. Finally, we found PtdIns(3,4,5)P3 was enriched in lung cancer tissues, which also showed high levels of phosphorylated Akt. Our new method to quantify and visualize PIs is expected to provide further insight into the role of lipid signaling in a wide range of cellular events.
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Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes.
By admin, on February 8th, 2012
J Lipid Res. 2012 Feb 3;
Irino Y, Tokuda E, Hasegawa J, Itoh T, Takenawa T
Phosphoinositides (PIs) play important regulatory roles in cell physiology. Localization and quantitation of PIs within the cell is necessary to understand their precise function. Currently, ectopic expression of green fluorescent protein (GFP)-fused PI-binding domains is used to visualize PIs localized to the cell membrane. However, ectopically expressed PI-binding domains may compete with endogenous binding proteins, thus altering the physiological functions of the PIs. Here, we establish a novel method for quantification and visualization of PIs in cells and tissue samples, using PI-binding domains labeled with quantum dots (Qdots) as specific probes. This method allowed us to simultaneously quantify 3 distinct PIs; phosphatidylinositol 3,4,5-triphosphatase (PtdIns(3,4,5)P3), PtdIns(3,4)P2, and PtdIns(4,5)P2, in crude acidic lipids extracted from insulin-stimulated cells. In addition, the method allows the PIs to be visualized within fixed cells and tissues. Sequential and spatial changes in PI production and distribution were detected in platelet-derived growth factor (PDGF)-stimulated NRK49F cells. We also observed accumulation of PtdIns(3,4)P2 at the dorsal ruffle in PDGF-stimulated NIH3T3 cells. Finally, we found PtdIns(3,4,5)P3 was enriched in lung cancer tissues, which also showed high levels of phosphorylated Akt. Our new method to quantify and visualize PIs is expected to provide further insight into the role of lipid signaling in a wide range of cellular events.
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Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes.
By admin, on January 21st, 2012
By admin, on January 17th, 2012
J Biochem. 2012 Jan 13;
Tokuda N, Kawai K, Lee YH, Ikegami T, Yamaguchi S, Yagisawa H, Fukui Y, Tuzi S
Differences in the conformation of the pleckstrin homology (PH) domain of switch-associated protein-70 (SWAP-70) in solution and at the lipid bilayer membrane surface were examined using CD, fluorescence, and NMR spectroscopy. Intracellular relocalization of SWAP-70 from the cytoplasm to the plasma membrane and then to the nucleus is associated with its cellular functions. The PH domain of SWAP-70 contains a phosphoinositide-binding site and a nuclear localization signal (NLS) which localize SWAP-70 to the plasma membrane and nucleus, respectively. CD and fluorescence spectra showed that a significant conformational alteration involving formation of disordered structure occurs when the PH domain binds to D-myo-phosphatidylinositol 3,4,5-trisphosphate or D-myo-phosphatidylinositol 4,5-bisphosphate embedded in lipid bilayer vesicles. NMR spectra indicate that Ala and Trp residues located in the C-terminal α-helix of the PH domain undergo conformational alterations to form a disordered structure at the vesicle surface. These conformational alterations were not induced by association with inositol 1,3,4,5-tetrakisphosphate in solution or coexistence of phosphatidylcholine vesicles. Interaction with the plane of the lipid bilayer via association with the phosphoinositides is required for the unfolding of the C-terminal α-helix of the PH domain. The unwinding of the C-terminal α-helix could regulate the functions of SWAP-70 at the plasma membrane surface.
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Membrane-induced alteration of the secondary structure in the SWAP-70 pleckstrin homology domain.
By admin, on January 17th, 2012
J Biochem. 2012 Jan 13;
Tokuda N, Kawai K, Lee YH, Ikegami T, Yamaguchi S, Yagisawa H, Fukui Y, Tuzi S
Differences in the conformation of the pleckstrin homology (PH) domain of switch-associated protein-70 (SWAP-70) in solution and at the lipid bilayer membrane surface were examined using CD, fluorescence, and NMR spectroscopy. Intracellular relocalization of SWAP-70 from the cytoplasm to the plasma membrane and then to the nucleus is associated with its cellular functions. The PH domain of SWAP-70 contains a phosphoinositide-binding site and a nuclear localization signal (NLS) which localize SWAP-70 to the plasma membrane and nucleus, respectively. CD and fluorescence spectra showed that a significant conformational alteration involving formation of disordered structure occurs when the PH domain binds to D-myo-phosphatidylinositol 3,4,5-trisphosphate or D-myo-phosphatidylinositol 4,5-bisphosphate embedded in lipid bilayer vesicles. NMR spectra indicate that Ala and Trp residues located in the C-terminal α-helix of the PH domain undergo conformational alterations to form a disordered structure at the vesicle surface. These conformational alterations were not induced by association with inositol 1,3,4,5-tetrakisphosphate in solution or coexistence of phosphatidylcholine vesicles. Interaction with the plane of the lipid bilayer via association with the phosphoinositides is required for the unfolding of the C-terminal α-helix of the PH domain. The unwinding of the C-terminal α-helix could regulate the functions of SWAP-70 at the plasma membrane surface.
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Membrane-induced alteration of the secondary structure in the SWAP-70 pleckstrin homology domain.
By admin, on January 13th, 2012
PLoS One. 2012; 7(1): e29520
Guo X, Li J, Wang T, Liu Z, Chen X, Li Y, Gu Z, Mao X, Guan W, Li Y
Multidrug resistance protein Pdr5p is a yeast ATP-binding cassette (ABC) transporter in the plasma membrane. It confers multidrug resistance by active efflux of intracellular drugs. However, the highly polymorphic Pdr5p from clinical strain YJM789 loses its ability to expel azole and cyclohexmide. To investigate the role of amino acid changes in this functional change, PDR5 chimeras were constructed by segmental replacement of homologous BY4741 PDR5 fragments. Functions of PDR5 chimeras were evaluated by fluconazole and cycloheximide resistance assays. Their expression, ATPase activity, and efflux efficiency for other substrates were also analyzed. Using multiple lines of evidence, we show that an alanine-to-methionine mutation at position 1352 located in the predicted short intracellular loop 4 significantly contributes to the observed transport deficiency. The degree of impairment is likely correlated to the size of the mutant residue.
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A Mutation in Intracellular Loop 4 Affects the Drug-Efflux Activity of the Yeast Multidrug Resistance ABC Transporter Pdr5p.
By admin, on January 13th, 2012
Biophys J . 2012 Jan 4; 102(1): 39-47 Mahendran KR, Romero-Ruiz M, Schlösinger A, Winterhalter M, Nussberger S Mitochondrial proteins are almost exclusively imported into mitochondria from the cytosol in an unfolded or partially folded conformation.
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Protein Translocation through Tom40: Kinetics of Peptide Release.
By admin, on January 7th, 2012
By admin, on January 7th, 2012
By admin, on January 6th, 2012
J Neurosci. 2012 Jan 4; 32(1): 282-96
Marsick BM, San Miguel-Ruiz JE, Letourneau PC
The development of a functioning neural network relies on responses of axonal growth cones to molecular guidance cues that are encountered en route to their target tissue. Nerve growth factor (NGF) and neurotrophin-3 serve as attractive cues for chick embryo sensory growth cones in vitro and in vivo, but little is known about the actin-binding proteins necessary to mediate this response. The evolutionarily conserved ezrin/radixin/moesin (ERM) family of proteins can tether actin filaments to the cell membrane when phosphorylated at a conserved threonine residue. Here we show that acute neurotrophin stimulation rapidly increases active phospho-ERM levels in chick sensory neuron growth cone filopodia, coincident with an increase in filopodial L1 and β-integrin. Disrupting ERM function with a dominant-negative construct (DN-ERM) results in smaller and less motile growth cones with disorganized actin filaments. Previously, we found that NGF treatment increases actin-depolymerizing factor (ADF)/cofilin activity and growth cone F-actin (Marsick et al., 2010). Here, we show this F-actin increase, as well as attractive turning to NGF, is blocked when ERM function is disrupted despite normal activation of ADF/cofilin. We further show that DN-ERM expression disrupts leading edge localization of active ADF/cofilin and free F-actin barbed ends. Moreover, filopodial phospho-ERM levels are increased by incorporation of active ADF/cofilin and reduced by knockdown of L1CAM.Together, these data suggest that ERM proteins organize actin filaments in sensory neuron growth cones and are crucial for neurotrophin-induced remodeling of F-actin and redistribution of adhesion receptors.
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Activation of Ezrin/Radixin/Moesin Mediates Attractive Growth Cone Guidance through Regulation of Growth Cone Actin and Adhesion Receptors.
By admin, on January 6th, 2012
J Neurosci. 2012 Jan 4; 32(1): 282-96
Marsick BM, San Miguel-Ruiz JE, Letourneau PC
The development of a functioning neural network relies on responses of axonal growth cones to molecular guidance cues that are encountered en route to their target tissue. Nerve growth factor (NGF) and neurotrophin-3 serve as attractive cues for chick embryo sensory growth cones in vitro and in vivo, but little is known about the actin-binding proteins necessary to mediate this response. The evolutionarily conserved ezrin/radixin/moesin (ERM) family of proteins can tether actin filaments to the cell membrane when phosphorylated at a conserved threonine residue. Here we show that acute neurotrophin stimulation rapidly increases active phospho-ERM levels in chick sensory neuron growth cone filopodia, coincident with an increase in filopodial L1 and β-integrin. Disrupting ERM function with a dominant-negative construct (DN-ERM) results in smaller and less motile growth cones with disorganized actin filaments. Previously, we found that NGF treatment increases actin-depolymerizing factor (ADF)/cofilin activity and growth cone F-actin (Marsick et al., 2010). Here, we show this F-actin increase, as well as attractive turning to NGF, is blocked when ERM function is disrupted despite normal activation of ADF/cofilin. We further show that DN-ERM expression disrupts leading edge localization of active ADF/cofilin and free F-actin barbed ends. Moreover, filopodial phospho-ERM levels are increased by incorporation of active ADF/cofilin and reduced by knockdown of L1CAM.Together, these data suggest that ERM proteins organize actin filaments in sensory neuron growth cones and are crucial for neurotrophin-induced remodeling of F-actin and redistribution of adhesion receptors.
Link:
Activation of Ezrin/Radixin/Moesin Mediates Attractive Growth Cone Guidance through Regulation of Growth Cone Actin and Adhesion Receptors.
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