A spin label study of horseradish peroxidase.

The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid (N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)-p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe3+ or Mn3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant Ka approximately 4.7 . 10(5) M-1. Since the interaction of ligands (e.g. F-, CN-) and H2O2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did not indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the pK value of the acid-alkali transition of horseradish peroxidase was found to be 10.5. The 2Tm value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0 degree C and 46.5 G at 52 degrees C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label bound to horseradish peroxidase. From this finding, the minimum distance between the iron atom and nitroxide group and hence a lower limit to the depth of the heme pocket of horseradish peroxidase was estimated to be 22 A.     

Tethering of the platelet-derived growth factor beta receptor to G-protein-coupled receptors. A novel platform for integrative signaling by these receptor classes in mammalian cells

Here we provide evidence to show that the platelet-derived growth factor beta receptor is tethered to endogenous G-protein-coupled receptor(s) in human embryonic kidney 293 cells. The tethered receptor complex provides a platform on which receptor tyrosine kinase and G-protein-coupled receptor signals can be integrated to produce more efficient stimulation of the p42/p44 mitogen-activated protein kinase pathway. This was based on several lines of evidence. First, we have shown that pertussis toxin (which uncouples G-protein-coupled receptors from inhibitory G-proteins) reduced the platelet-derived growth factor stimulation of p42/p44 mitogen-activated protein kinase. Second, transfection of cells with inhibitory G-protein alpha subunit increased the activation of p42/p44 mitogen-activated protein kinase by platelet-derived growth factor. Third, platelet-derived growth factor stimulated the tyrosine phosphorylation of the inhibitory G-protein alpha subunit, which was blocked by the platelet-derived growth factor kinase inhibitor, tyrphostin AG 1296. We have also shown that the platelet-derived growth factor beta receptor forms a tethered complex with Myc-tagged endothelial differentiation gene 1 (a G-protein-coupled receptor whose agonist is sphingosine 1-phosphate) in cells co-transfected with these receptors. This facilitates platelet-derived growth factor-stimulated tyrosine phosphorylation of the inhibitory G-protein alpha subunit and increases p42/p44 mitogen-activated protein kinase activation. In addition, we found that G-protein-coupled receptor kinase 2 and beta-arrestin I can associate with the platelet-derived growth factor beta receptor. These proteins play an important role in regulating endocytosis of G-protein-coupled receptor signal complexes, which is required for activation of p42/p44 mitogen-activated protein kinase. Thus, platelet-derived growth factor beta receptor signaling may be initiated by G-protein-coupled receptor kinase 2/beta-arrestin I that has been recruited to the platelet-derived growth factor beta receptor by its tethering to a G-protein-coupled receptor(s). These results provide a model that may account for the co-mitogenic effect of certain G-protein-coupled receptor agonists with platelet-derived growth factor on DNA synthesis.     

In vitro activity of novel dual action MDR anthranilamide modulators with inhibitory activity on CYP-450 (Part 2)

Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency on the P-glycoprotein (P-gp) transporter. Previous studies showed that the replacement of the aromatic spacer group between nitrogen atoms (N(1) and N(2)) in the P-gp inhibitor XR9576 with ethyl or propyl chain is optimal for P-gp inhibition potency. To confirm that observation, the ethyl or the propyl linker arm was replaced with a pyrrolidine or an alicyclic group such as cyclohexyl. In addition, an arylpiperazinyl group and two methoxyl groups onto the anthranilic part were introduced to assess their effect on the anti P-gp activity. Five molecules were prepared and evaluated on CEM/VLB500. All new anthranilamides were more potent than verapamil, most of them exhibited a lower cytotoxicity than XR9576. Compound 5 was the most potent and its inhibition activity was similar to XR9576. Interestingly, in vitro biotransformation studies of compounds 4 and 5 using human CYP-450 isoforms revealed, that conversely to XR9576, compounds 4 and 5 inhibited CYP3A4, an enzyme that colocalizes with P-gp in the intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of numerous drugs, notably paclitaxel. In that context, 5 might be suitable for further drug development.     

Chaperone-mediated inhibition of tubulin self-assembly

Molecular chaperones are known to play an important role in facilitating the proper folding of many newly synthesized proteins. Here, we have shown that chaperone proteins exhibit another unique property to inhibit tubulin self-assembly efficiently. Chaperones tested include alpha-crystallin from bovine eye lenses, HSP16.3, HSP70 from Mycobacterium tuberculosis and alpha (s)-casein from milk. All of them inhibit polymerization in a dose-dependent manner independent of assembly inducers used. The critical concentration of MTP polymerization increases with increasing concentration of HSP16.3. Increase in chaperone concentration lowers the extent of polymerization and increases the lag time of self-assembly reaction. Although the addition of a chaperone at the early stage of elongation phase shows no effect on polymerization, the same concentration of chaperone inhibits polymerization completely when added before the initiation of polymerization. Bindings of HSP16.3 and alpha (s)-casein to tubulin have been confirmed using isothermal titration calorimetry. Affinity constants of tubulin are 5.3 xx 10(4) and 9.8 xx 10(5) M(-1) for HSP16.3 and alpha (s)-casein, respectively. Thermodynamic parameters indicate favourable entropy and enthalpy changes for both chaperones-tubulin interactions. Positive entropy change suggests that the interaction is hydrophobic in nature and desolvation occurring during formation of tubulin-chaperone complex. On the basis of thermodynamic data and observations made upon addition of chaperone at early elongation phase or before the initiation of polymerization, we hypothesize that chaperones bind tubulin at the protein-protein interaction site involved in the nucleation phase of self-assembly.     

1,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

A series of 1,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase. A variety of flexible and restricted basic amine side chain substitutions was explored at the 1-position of the indole ring, while keeping the amidine group fixed at the 5-position. Compounds having N-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)- (12, (R)-12, (S)-12 and 13) and N-(1-(1-methylazepan-4-yl)- side chains (14, 15, (-)-15 and (+)-15) showed increased inhibitory activity for the human nNOS isoform and selectivity over eNOS and iNOS isoforms. The most potent compound of the series for human nNOS (IC(50)=0.02 μM) (S)-12 showed very good selectivity over the eNOS (eNOS/nNOS=96-fold) and iNOS (iNOS/nNOS=850-fold) isoforms.     

Monomeric Cu,Zn-superoxide dismutase is a common misfolding intermediate in the oxidation models of sporadic and familial amyotrophic lateral sclerosis

Proteinacious intracellular aggregates in motor neurons are a key feature of both sporadic and familial amyotrophic lateral sclerosis (ALS). These inclusion bodies are often immunoreactive for Cu,Zn-superoxide dismutase (SOD1) and are implicated in the pathology of ALS. On the basis of this and a similar clinical presentation of symptoms in the familial (fALS) and sporadic forms of ALS, we sought to investigate the possibility that there exists a common disease-related aggregation pathway for fALS-associated mutant SODs and wild type SOD1. We have previously shown that oxidation of fALS-associated mutant SODs produces aggregates that have the same morphological, structural, and tinctorial features as those found in SOD1 inclusion bodies in ALS. Here, we show that oxidative damage of wild type SOD at physiological concentrations ( approximately 40 microm) results in destabilization and aggregation in vitro. Oxidation of either mutant or wild type SOD1 causes the enzyme to dissociate to monomers prior to aggregation. Only small changes in secondary and tertiary structure are associated with monomer formation. These results indicate a common aggregation prone monomeric intermediate for wild type and fALS-associated mutant SODs and provides a link between sporadic and familial ALS.     

Expression and Purification of Soluble, Active Heterodimeric Guanylyl Cyclase from Baculovirus

A method for expression and purification of active cytosolic heterodimeric histidine (His)-tagged guanylyl cyclase of the α1/β1 isoform has been developed using recombinant baculovirus-transfected insect cells. Confirmation of expression of active cyclase was obtained by both Western analysis and enzymatic activity. A His tag on the COOH-terminus of the α1 and β1 subunits allowed rapid purification of the heterodimeric form of guanylyl cyclase in a single affinity step using a nickel column. A second gel-filtration step was applied to reconstitute into the complex heme, a required cofactor. This was confirmed spectroscopically by absorbance in the Soret region. Like enzyme purified from tissue, the activity of recombinant guanylyl cyclase was increased by protoporphyrin IX and inhibited by both Zn- and Sn-protoporphyrin. The method described here should provide a general approach for the expression and purification of alternate forms of cytosolic guanylyl cyclase and facilitate mechanistic and structural studies of this important family of enzymes. Furthermore, the procedure demonstrates the utility of the His-tag system to purify multimeric proteins.