Evidence for modulatory sites at the lipid-protein interface of the human multidrug transporter P-glycoprotein

The human multidrug transporter P-glycoprotein (Pgp or ABCB1) sets up pharmacological barriers to many clinically important drugs, a therapeutic remedy for which has yet to be formulated. For the rational design of mechanism-based inhibitors (or modulators), it is necessary to map the potential sites for modulator interaction and understand their modes of communication with the other functional domains of Pgp. In this study, combining directed mutagenesis with homology modeling, we provide evidence of two modulator-specific sites at the lipid protein interface of Pgp. Targeting 21 variant positions in the COOH-terminal transmembrane (TM) regions, we find residues M948 (in TM11) and F983, M986, V988, and Q990 (all four in TM12) critically involved in substrate-site modulation by a thioxanthene-based allosteric modulator cis-(Z)-flupentixol. Interestingly, for ATP-site modulation by the same modulator, only two (M948 and Q990) of those four residues appear indispensable, together with two additional residues, T837 and I864 in TM9 and TM10, respectively, suggesting independent modes of communication linking the allosteric site with the substrate binding and ATPase domains. None of the seven residues identified prove to be critical for modulation of the substrate or ATP sites by Pgp modulators that are transported by the pump, such as cyclosporin A or verapamil, indicating their specificity for cis-(Z)-flupentixol. On the other hand, ATP-site modulation by verapamil proves to be highly sensitive to replacement at positions F716 (in TM7) and I765 (in TM8), and to a more moderate extent at I764 and L772 (both in TM8). Homology modeling based on the known crystal structures of the bacterial multidrug transporter SAV1866 and the mouse Pgp homologue maps the identified residues primarily at the lipid-protein interface of Pgp, in two spatially distinct modulator-specific clusters. The two modulatory sites demonstrate negative synergism in influencing ATP hydrolysis, consolidating their spatial distinctness. Because Pgp is known to recruit drug molecules directly from the lipid bilayer, identification of modulatory sites at the lipid-protein interface and at the same time outside the conventional central drug binding cavity is mechanistically revealing.     

Homology modeling, docking studies and functional analysis of various azoreductase accessory interacting proteins of Nostoc sp.PCC7120

Azo dyes have become a threat to public health because of its toxicity and carcinogenicity. Azoreductase enzyme plays a pivotal role in the degradation of azodyes released by industrial effluents and other resources. The degradation pathway has to be studied in detail for increasing the activity of azoreductase and for better degradation of azo dyes. But the data available on cyanobacterial azoreductase enzyme and its degradation pathway are still very less. Therefore the present work explored the azoreductase pathway of the cyanobacterium Nostoc sp. PCC7120 for better understanding of the degradation pathway and the other accessory interacting proteins involved. The accessory interacting proteins of azoreductase from cyanobacterium Nostoc sp. PCC7120 were obtained from STRING database. The proteins do not have a comprehensive three dimensional structure and are hypothetical. The secondary structure and functional analysis indicated that the proteins are all soluble proteins, without disulphide bonds and have alpha helices only. The structural prediction and docking study showed that alr2106, alr1063 and alr2326 have best docking result which tally with the STRING database confidence score and thus these proteins could possibly enhance the azoreductase activity and better dye degradation. These results will pave way for further increase in azoreductase activity and for better understanding of the dye degradation pathway.     

Scanning electron microscopy of the post-embryonic stages of the climbing perch,Anabas testudineus

Different developmental stages, fertilized eggs through hatchlings, of the climbing perch,Anabas testudineus, have been studied by scanning electron microscopy. The surface specialization of eggs and hatchlings reveals that while the egg surface is reticulate in appearance, the hatchlings are covered with microridges. Vitelline arteries are seen at the pharyngeal and abdominal regions. They supply nutrients directly from the yolk sac to the developing embryo. Three pairs of such arteries are distinctly seen in the pharyngeal region. Mucous glands are discernible at places over the entire body surface of the embryo before the formation of scales. The skin seems to be helpful in gaseous exchange till the gills and accessory respiratory organs develop and become functional.     

Thermodynamic Parameters of Frog Brain κ-Opioid Receptors

Abstract: The thermodynamic parameters for [³H]-ethylketocyclazocine binding in frog ( Rana esculenta ) brain membranes have been examined. Computer-based nonlinear regression analysis of the untransformed equilibrium displacement data showed that this ligand bound to two sites with different affinities and capacities in this tissue. K _A values derived from equilibrium displacement curves have been used for calculating the changes in the standard Gibbs energy, enthalpy, and entropy during the binding process. Van't Hoff plots are bipartite, with transitions occurring at 18°C for both the high- and the low-affinity sites. For the high-affinity site, the reaction appears to be associated with a decrease in enthalpy below the transition temperature and a significant gain in entropy above this temperature. The reverse appears to be true for the low-affinity site. We conclude that this profile fairly approximates the mixed agonist-antagonist nature of this ligand and surmise that thermodynamic analysis could be a very useful tool for characterization of the nature of cloned opioid receptors in vitro.     

Proline Scan of the hERG Channel S6 Helix Reveals the Location of the Intracellular Pore Gate

In Shaker-like channels, the activation gate is formed at the bundle crossing by the convergence of the inner S6 helices near a conserved proline-valine-proline motif, which introduces a kink that allows for electromechanical coupling with voltage sensor motions via the S4-S5 linker. Human ether-a-go-go-related gene (hERG) channels lack the proline-valine-proline motif and the location of the intracellular pore gate and how it is coupled to S4 movement is less clear. Here, we show that proline substitutions within the S6 of hERG perturbed pore gate closure, trapping channels in the open state. Performing a proline scan of the inner S6 helix, from Ile⁶⁵⁵ to Tyr⁶⁶⁷ revealed that gate perturbation occurred with proximal (I655P-Q664P), but not distal (R665P-Y667P) substitutions, suggesting that Gln⁶⁶⁴ marks the position of the intracellular gate in hERG channels. Using voltage-clamp fluorimetry and gating current analysis, we demonstrate that proline substitutions trap the activation gate open by disrupting the coupling between the voltage-sensing unit and the pore of the channel. We characterize voltage sensor movement in one such trapped-open mutant channel and demonstrate the kinetics of what we interpret to be intrinsic hERG voltage sensor movement.     

Role of L-thyroxine, hydrocortisone and progesterone in regulation of blood volume in a fresh-water air-breathing fish, Clarias batrachus (Linn.).

Effect of hormones on the blood volume of Clarias batrachus were ascertained and it was observed L-thyroxine and progesterone decrease, while hydrocortisone increases the total and relative blood volumes. The ratio of plasma to corpuscular volume is increased by the intraperitoneal injection of L-thyroxine, hydrocortisone and progesterone (higher dose only), thus indicating the regulation of blood volume under the complex control of homeostasis.     

Characterization and stability studies on surfactant,detergent and oxidant stable α-amylase from marine haloalkaliphilic Saccharopolyspora sp. A9

A halopalkaliphilic marine Saccharopolyspora sp. stain A9 with an ability to produce surfactants, oxidant and detergent stable α-amylase was isolated from marine sediments collected from west coast of India. The α-amylase from strain A9 was purified to homogeneity with the aid of ammonium sulfate precipitation and gel filtration chromatography by using Sephadex G-75, insoluble corn starch and sephacryl S-100 column, with a 39.01-fold increase in specific activity. SDS-PAGE and zymogram activity staining showed a single band equal to molecular mass of 66 kDa. Enzyme was found to be stable in presence of wide range of NaCl concentration with maximum activity found at 11% (w/v) of NaCl. Enzyme showed remarkable stability towards laboratory surfactants, detergents and oxidants. Glucose, maltose and maltotriose were the main end product of starch hydrolysis, indicating it is α-amylase.     

Opioid receptor labeling with the chloromethyl ketone derivative of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) II: Covalent labeling of mu opioid binding site by 3H-Tyr-D-Ala-Gly-(Me)Phe chloromethyl ketone.

Opioid receptors of rat brain membranes were prelabeled with 3H-Tyr-D-Ala2-(Phe4)-Gly-CH2Cl, a chloromethyl ketone derivative of enkephalin, and solubilized in 1% digitonin. Hydrodynamic parameters of the receptor detergent complex derived from gel filtration and sucrose density gradient ultracentrifugation were found to be 51 A and 8.7 S, respectively, and the size was estimated to be about 200 kDa. Sodium dodecyl sulfate gel electrophoresis followed by fluorography revealed specific alkylation of a major protein at 58 kDa.