182 research outputs found
Carba-Cyclophellitols are Neutral Retaining Glucosidase Inhibitors
The
conformational analysis of glycosidases affords a route to
their specific inhibition through transition-state mimicry. Inspired
by the rapid reaction rates of cyclophellitol and cyclophellitol aziridineboth
covalent retaining β-glucosidase inhibitorswe postulated
that the corresponding carba “cyclopropyl” analogue
would be a potent retaining β-glucosidase inhibitor for those
enzymes reacting through the <sup>4</sup>H<sub>3</sub> transition-state
conformation. <i>Ab initio</i> metadynamics simulations
of the conformational free energy landscape for the cyclopropyl inhibitors
show a strong bias for the <sup>4</sup>H<sub>3</sub> conformation,
and carba-cyclophellitol, with an <i>N</i>-(4-azidobutyl)carboxamide
moiety, proved to be a potent inhibitor (<i>K</i><sub>i</sub> = 8.2 nM) of the <i>Thermotoga maritima</i> <i>Tm</i>GH1 β-glucosidase. 3-D structural analysis and comparison with
unreacted epoxides show that this compound indeed binds in the <sup>4</sup>H<sub>3</sub> conformation, suggesting that conformational
strain induced through a cyclopropyl unit may add to the armory of
tight-binding inhibitor designs
Inside-Out Regulation of ICAM-1 Dynamics in TNF-α-Activated Endothelium
Background: During transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions. Principal Findings: Detailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions. Conclusions: Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesio
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