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By admin, on April 23rd, 2012
PLoS Pathog. 2012 Apr; 8(4): e1002639
Vijayapalani P, Maeshima M, Nagasaki-Takekuchi N, Miller WA
A small open reading frame (ORF), pipo, overlaps with the P3 coding region of the potyviral polyprotein ORF. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. Here, we provide immunoblotting evidence that the protein PIPO is expressed as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO (P3N-PIPO). P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. Using a yeast two-hybrid screen, co-immunoprecipitation assays, and bimolecular fluorescence complementation (BiFC) assays, we found that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. BiFC revealed that it is the PIPO domain of P3N-PIPO that binds PCaP1 and that myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. In PCaP1 knockout mutants (pcap1) of Arabidopsis, accumulation of TuMV harboring a GFP gene (TuMV-GFP) was drastically reduced relative to the virus level in wild-type plants, only small localized spots of GFP were visible, and the plants showed few symptoms. In contrast, TuMV-GFP infection in wild-type Arabidopsis yielded large green fluorescent patches, and caused severe stunting. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. In contrast to TuMV, the tobamovirus Oilseed rape mosaic virus did not require PCaP1 to infect Arabidopsis plants. We conclude that potyviral P3N-PIPO interacts specifically with the host plasma membrane protein PCaP1 to participate in cell-to-cell movement. We speculate that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement. The PCaP1 knockout may contribute to a new strategy for recessive resistance to potyviruses.
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Interaction of the Trans-Frame Potyvirus Protein P3N-PIPO with Host Protein PCaP1 Facilitates Potyvirus Movement.
By admin, on April 20th, 2012
PLoS One . 2012; 7(4): e33073 Smani Y, McConnell MJ, Pachón J Adhesion to host cells is an initial and important step in Acinetobacter baumannii pathogenesis. However, there is relatively little information on the mechanisms by which A.
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Role of Fibronectin in the Adhesion of Acinetobacter baumannii to Host Cells.
By admin, on April 17th, 2012
PLoS One. 2012; 7(4): e34173
Hoa N, Tsui S, Afifiyan NF, Sinha Hikim A, Li B, Douglas RS, Smith TJ
Insulin-like growth factor-1 receptor (IGF-1R) comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rβ. IGF-1R is over-expressed by orbital fibroblasts in the autoimmune syndrome, Graves' disease (GD). When activated by IGF-1 or GD-derived IgG (GD-IgG), these fibroblasts produce RANTES and IL-16, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD fibroblasts where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rβ mAbs. Nuclear redistribution of IGF-1R is absent in control orbital fibroblasts. In GD fibroblasts, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with (125)I IGF-1, (125)I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD fibroblasts. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD fibroblasts. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived fibroblasts. Nuclear IGF-1R may play a role in disease pathogenesis.
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Nuclear Targeting of IGF-1 Receptor in Orbital Fibroblasts from Graves’ Disease: Apparent Role of ADAM17.
By admin, on April 17th, 2012
PLoS One. 2012; 7(4): e34173
Hoa N, Tsui S, Afifiyan NF, Sinha Hikim A, Li B, Douglas RS, Smith TJ
Insulin-like growth factor-1 receptor (IGF-1R) comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rβ. IGF-1R is over-expressed by orbital fibroblasts in the autoimmune syndrome, Graves' disease (GD). When activated by IGF-1 or GD-derived IgG (GD-IgG), these fibroblasts produce RANTES and IL-16, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD fibroblasts where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rβ mAbs. Nuclear redistribution of IGF-1R is absent in control orbital fibroblasts. In GD fibroblasts, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with (125)I IGF-1, (125)I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD fibroblasts. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD fibroblasts. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived fibroblasts. Nuclear IGF-1R may play a role in disease pathogenesis.
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Nuclear Targeting of IGF-1 Receptor in Orbital Fibroblasts from Graves’ Disease: Apparent Role of ADAM17.
By admin, on April 11th, 2012
J Struct Biol . 2012 Mar 29; Efremov RG, Sazanov LA Outer membrane protein F, a major component of the Escherichia coli outer membrane, was crystallized for the first time in lipidic mesophase of monoolein in novel space groups, P1 and H32. Due to ease of its purification and crystallization OmpF can be used as a benchmark protein for establishing membrane protein crystallization in meso, as a “membrane lyzozyme”.
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Structure of Escherichia coli OmpF porin from lipidic mesophase.
By admin, on April 8th, 2012
Commun Integr Biol. 2012 Jan 1; 5(1): 68-70
Bernabò N, Palestini P, Chiarini M, Maccarrone M, Mattioli M, Barboni B
Mammalian spermatozoa reach the ability to fertilize only after they complete a complex series of physical-chemical modification, the capacitation. Recently, the endocannabinoid-binding type-1cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 (TRPV1) channel have been proposed to play a key role in the control of capacitation. In particular CB1, acting via a Gi protein/cAMP/PKA pathway, maintains low cAMP levels in early stages of post ejaculatory life of male gametes. By this way it promotes the maintenance of membrane stability, thus avoiding the premature fusion of plasma membrane (PM) and outer acrosome membrane (OAM), which is mandatory for the exocytosis of acrosome content. TRPV1, on the contrary, becomes active during the latest stages of capacitation, and allows the rapid increase in intracellular calcium concentration that leads to the removal of the F-actin network interposed between PM and OAM, leading to their fusion and, ultimately, to the acrosome reaction.
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Endocannabinoid-binding CB1 and TRPV1 receptors as modulators of sperm capacitation.
By admin, on April 8th, 2012
Biochim Biophys Acta. 2012 Mar 28;
Pangon L, Van Kralingen C, Abas M, Daly RJ, Musgrove EA, Kohonen-Corish MR
In this study, we describe a new post-translational modification at position -1 of the PDZ-binding motif in the mutated in colorectal cancer (MCC) protein and its role in lamellipodia formation. Serine 828 at position -1 of this motif is phosphorylated, which is predicted to increase MCC binding affinity with the polarity protein Scrib. We show that endogenous MCC localizes at the active migratory edge of cells, where it interacts with Scrib and the non-muscle motor protein Myosin-IIB. Expression of MCC harboring a phosphomimetic mutation MCC-S828D strongly impaired lamellipodia formation and resulted in accumulation of Myosin-IIB in the membrane cortex fraction. We propose that MCC regulates lamellipodia formation by binding to Scrib and its downstream partner Myosin-IIB in a multiprotein complex. Importantly, we propose that the function of this complex is under the regulation of a newly described phosphorylation of the PDZ-binding motif at position -1.
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The PDZ-binding motif of MCC is phosphorylated at position -1 and controls lamellipodia formation in colon epithelial cells.
By admin, on April 7th, 2012
PLoS One. 2012; 7(3): e33640
Chen HC, Ziemba BP, Landgraf KE, Corbin JA, Falke JJ
The second messenger lipid PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) is generated by the lipid kinase PI3K (phosphoinositide-3-kinase) in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3)-specific pleckstrin homology (PH) domains to the membrane surface. Despite the broad importance of PIP(3)-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3) lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3). The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i) PIP(3) target lipid that provides specificity and affinity, and (ii) PS facilitator lipid that enhances the PIP(3) on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3) headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3) headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3) headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral diffusion observed for PIP(3)-bound GRP1 PH domain on supported lipid bilayers.
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Membrane Docking Geometry of GRP1 PH Domain Bound to a Target Lipid Bilayer: An EPR Site-Directed Spin-Labeling and Relaxation Study.
By admin, on April 7th, 2012
PLoS One. 2012; 7(3): e33640
Chen HC, Ziemba BP, Landgraf KE, Corbin JA, Falke JJ
The second messenger lipid PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) is generated by the lipid kinase PI3K (phosphoinositide-3-kinase) in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3)-specific pleckstrin homology (PH) domains to the membrane surface. Despite the broad importance of PIP(3)-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3) lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3). The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i) PIP(3) target lipid that provides specificity and affinity, and (ii) PS facilitator lipid that enhances the PIP(3) on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3) headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3) headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3) headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral diffusion observed for PIP(3)-bound GRP1 PH domain on supported lipid bilayers.
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Membrane Docking Geometry of GRP1 PH Domain Bound to a Target Lipid Bilayer: An EPR Site-Directed Spin-Labeling and Relaxation Study.
By admin, on April 6th, 2012
Biochemistry. 2012 Apr 4;
Joung MJ, Mohan SK, Yu C
Synaptotagmin I (Syt I) is a synaptic vesicle membrane protein that serves as a multifunctional regulator during the exocytosis of neurotransmitter release. It contains C2A and C2B domains. The binding of Ca2+ to the C2A domain activates the exocytosis of secretory vesicles, while the binding of inositol polyphosphates (IP4, IP5 and IP6) to the C2B domain inhibits this process. To understand the IP6-induced inhibition of exocytosis of secretory vesicles, we solved the 3D structure of the C2B-IP6 complex by NMR. In this study, we have determined the binding constant by isothermal titration calorimetry. The CD measurements demonstrated that IP6 can stabilize the C2B molecule. We identified the binding site using 1H-15N HSQC titration data and solved the C2B-IP6 complex structure using multi-dimensional NMR studies. This information will aid in the design of better pharmacological treatments for neurological disorders.
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Molecular Level Interaction of Inositol Hexaphosphate with C2B domain of the Human Synaptotagmin I.
By admin, on April 6th, 2012
Biochemistry. 2012 Apr 4;
Joung MJ, Mohan SK, Yu C
Synaptotagmin I (Syt I) is a synaptic vesicle membrane protein that serves as a multifunctional regulator during the exocytosis of neurotransmitter release. It contains C2A and C2B domains. The binding of Ca2+ to the C2A domain activates the exocytosis of secretory vesicles, while the binding of inositol polyphosphates (IP4, IP5 and IP6) to the C2B domain inhibits this process. To understand the IP6-induced inhibition of exocytosis of secretory vesicles, we solved the 3D structure of the C2B-IP6 complex by NMR. In this study, we have determined the binding constant by isothermal titration calorimetry. The CD measurements demonstrated that IP6 can stabilize the C2B molecule. We identified the binding site using 1H-15N HSQC titration data and solved the C2B-IP6 complex structure using multi-dimensional NMR studies. This information will aid in the design of better pharmacological treatments for neurological disorders.
Original post:
Molecular Level Interaction of Inositol Hexaphosphate with C2B domain of the Human Synaptotagmin I.
By admin, on April 6th, 2012
Biochemistry. 2012 Apr 4;
Joung MJ, Mohan SK, Yu C
Synaptotagmin I (Syt I) is a synaptic vesicle membrane protein that serves as a multifunctional regulator during the exocytosis of neurotransmitter release. It contains C2A and C2B domains. The binding of Ca2+ to the C2A domain activates the exocytosis of secretory vesicles, while the binding of inositol polyphosphates (IP4, IP5 and IP6) to the C2B domain inhibits this process. To understand the IP6-induced inhibition of exocytosis of secretory vesicles, we solved the 3D structure of the C2B-IP6 complex by NMR. In this study, we have determined the binding constant by isothermal titration calorimetry. The CD measurements demonstrated that IP6 can stabilize the C2B molecule. We identified the binding site using 1H-15N HSQC titration data and solved the C2B-IP6 complex structure using multi-dimensional NMR studies. This information will aid in the design of better pharmacological treatments for neurological disorders.
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Molecular Level Interaction of Inositol Hexaphosphate with C2B domain of the Human Synaptotagmin I.
By admin, on April 4th, 2012
By admin, on April 4th, 2012
By admin, on April 4th, 2012
By admin, on April 4th, 2012
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