Browsing by Subject "Adenosine Diphosphate"

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    A kinesin motor in a force-producing conformation.
    (BMC Struct Biol, 2010-07-05) Heuston, Elisabeth; Bronner, C Eric; Kull, F Jon; Endow, Sharyn A
    BACKGROUND: Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states. RESULTS: Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central beta-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1. CONCLUSIONS: Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor.
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    Comparing Inverse Probability of Treatment Weighting and Instrumental Variable Methods for the Evaluation of Adenosine Diphosphate Receptor Inhibitors After Percutaneous Coronary Intervention.
    (JAMA cardiology, 2016-09) Federspiel, Jerome J; Anstrom, Kevin J; Xian, Ying; McCoy, Lisa A; Effron, Mark B; Faries, Douglas E; Zettler, Marjorie; Mauri, Laura; Yeh, Robert W; Peterson, Eric D; Wang, Tracy Y; Treatment With Adenosine Diphosphate Receptor Inhibitors–Longitudinal Assessment of Treatment Patterns and Events After Acute Coronary Syndrome (TRANSLATE-ACS) Investigators
    IMPORTANCE:There is increasing interest in performing comparative effectiveness analyses in large observational databases, yet these analyses must adjust for treatment selection issues. OBJECTIVES:To conduct comparative safety and efficacy analyses of prasugrel vs clopidogrel bisulfate after percutaneous coronary intervention and to evaluate inverse probability of treatment weighting (a propensity score method) and instrumental variable methods. DESIGN, SETTING, AND PARTICIPANTS:This study used data from the Treatment With Adenosine Diphosphate Receptor Inhibitors-Longitudinal Assessment of Treatment Patterns and Events After Acute Coronary Syndrome (TRANSLATE-ACS) study. Included in the study were patients undergoing percutaneous coronary intervention for myocardial infarction, 26.0% of whom received prasugrel. The study dates were April 4, 2010, to October 31, 2012. EXPOSURES:Choice of initial antiplatelet agent (prasugrel or clopidogrel). MAIN OUTCOMES AND MEASURES:Safety and efficacy outcomes included 1-year composite major adverse cardiovascular events, moderate to severe bleeding, and stent thrombosis. Hospitalizations for pneumonia, bone fractures, and planned percutaneous coronary intervention were used as the falsification end points. RESULTS:The study cohort comprised 11 784 participants (mean [SD] age, 60.0 [11.6] years, and 28.0% were female). Using inverse probability of treatment weighting adjustment, prasugrel and clopidogrel had similar major adverse cardiovascular events (hazard ratio [HR], 0.98; 95% CI, 0.83-1.16) and bleeding outcomes (1.18; 0.77-1.80), but prasugrel had a lower rate of stent thrombosis (0.51; 0.31-0.85). Using instrumental variable methods, prasugrel use was associated with a lower rate of the major adverse cardiovascular event end point (HR, 0.68; 95% CI, 0.47-1.00) but nonsignificant differences in the rates of bleeding (0.95; 0.41-2.08) and stent thrombosis (0.67; 0.16-2.00). There was no significant treatment difference noted in any of the falsification end-point rates when analyses were performed using inverse probability of treatment weighting, although the bone fracture end point approached statistical significance. Nevertheless, a lower rate of pneumonia-related hospitalizations was noted in the prasugrel-treated patients when analyses were performed using instrumental variable methods. CONCLUSIONS AND RELEVANCE:Conclusions regarding the safety and efficacy of antiplatelet therapy varied depending on analytic technique, and none were concordant with the results from randomized trials. In addition, both statistical strategies demonstrated concerning associations when tested in the falsification analyses. A high level of scrutiny and careful attention to assumptions and validity are required when interpreting complex analyses of observational data.
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    Regulation of Integrin α6 Recycling by Calcium-independent Phospholipase A2 (iPLA2) to Promote Microglia Chemotaxis on Laminin.
    (The Journal of biological chemistry, 2016-11) Lee, Sang-Hyun; Sud, Neetu; Lee, Narae; Subramaniyam, Selvaraj; Chung, Chang Y
    Microglia are the immune effector cells that are activated in response to pathological changes in the central nervous system. Microglial activation is accompanied by the alteration of integrin expression on the microglia surface. However, changes of integrin expression upon chemoattractant (ADP) stimulation still remain unknown. In this study, we investigated whether ADP induces the alteration of integrin species on the cell surface, leading to changes in chemotactic ability on different extracellular matrix proteins. Flow cytometry scans and on-cell Western assays showed that ADP stimulation induced a significant increase of α6 integrin-GFP, but not α5, on the surface of microglia cells. Microglia also showed a greater motility increase on laminin than fibronectin after ADP stimulation. Time lapse microscopy and integrin endocytosis assay revealed the essential role of calcium-independent phospholipase A2 activity for the recycling of α6 integrin-GFP from the endosomal recycling complex to the plasma membrane. Lack of calcium-independent phospholipase A2 activity caused a reduced rate of focal adhesion formation on laminin at the leading edge. Our results suggest that the alteration of integrin-mediated adhesion may regulate the extent of microglial infiltration into the site of damage by controlling their chemotactic ability.
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    Role of iPLA(2) in the regulation of Src trafficking and microglia chemotaxis.
    (Traffic (Copenhagen, Denmark), 2011-07) Lee, Sang-Hyun; Schneider, Claus; Higdon, Ashlee N; Darley-Usmar, Victor M; Chung, Chang Y
    Microglia are immune effector cells in the central nervous system (CNS) and their activation, migration and proliferation play crucial roles in brain injuries and diseases. We examined the role of intracellular Ca(2+) -independent phospholipase A(2) (iPLA(2)) in the regulation of microglia chemotaxis toward ADP. Inhibition of iPLA(2) by 4-bromoenol lactone (BEL) or iPLA(2) knockdown exerted a significant inhibition on phosphatidylinositol-3-kinase (PI3K) activation and chemotaxis. Further examination revealed that iPLA(2) knockdown abrogated Src activation, which is required for PI3K activation and chemotaxis. Colocalization studies showed that cSrc-GFP was retained in the endosomal recycling compartment (ERC) in iPLA(2) knockdown cells, but the addition of arachidonic acid (AA) could restore cSrc trafficking to the plasma membrane by allowing the formation/release of recycling endosomes associated with cSrc-GFP. Using BODIPY-AA, we showed that AA is selectively enriched in recycling endosomes. These results suggest that AA is required for the cSrc trafficking to the plasma membrane by controlling the formation/release of recycling endosomes from the ERC.
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    β-arrestin 2-dependent activation of ERK1/2 is required for ADP-induced paxillin phosphorylation at Ser(83) and microglia chemotaxis.
    (Glia, 2012-09) Lee, Sang-Hyun; Hollingsworth, Ryan; Kwon, Hyeok-Yil; Lee, Narae; Chung, Chang Y
    Microglia play crucial roles in increased inflammation in the central nervous system upon brain injuries and diseases. Extracellular ADP has been reported to induce microglia chemotaxis and membrane ruffle formation through P2Y(12) receptor. In this study, we examined the role of ERK1/2 activation in ADP-induced microglia chemotaxis. ADP stimulation increases the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and paxillin phosphorylation at Tyr(31) and Ser(83) . Inhibition of ERK1/2 significantly inhibited paxillin phosphorylation at Ser(83) and the retraction of membrane ruffles, causing inefficient chemotaxis. Close examination of dynamics of focal adhesion (FA) formation with green fluorescent protein-paxillin revealed that the disassembly of FAs in U0126-treated cells was significantly impaired. Depletion of β-Arrestin 2 (β-Arr2) with short hairpin RNA markedly reduced the phosphorylation of ERK1/2 and Pax/Ser(83) , indicating that β-Arr2 is required for ERK1/2 activation upon ADP stimulation. A large fraction of phosphorylated ERK1/2 and β-Arr2 were translocated and co-localized at focal contacts in the newly forming lamellipodia. Examination of kinetics and rate constant of paxillin formation and disassembly revealed that the phosphorylation of paxillin at Tyr(31) by c-Src appears to be involved in adhesion formation upon ADP stimulation while Ser(83) required for adhesion disassembly.