ABSTRACT:Nanosilver particles are present in consumer and health care products. Their effects on human microsomal cytochrome P450 (P450) activities and induction in luciferase reporter-engineered Caco-2 (MDR1.C) and HepG2 (DPX2 and 1A2DRE) cells have been investigated. The LD 50 values were ϳ4 g silver/ml for HepG2 and 5 g/ml for Caco-2 cells. At silver concentrations that showed no decreased cell viability (<1 g silver/ml), the pregnane X receptor (PXR)-driven 4.5-fold induction response of MDR1.C cells to 50 M omeprazole was unaffected. In DPX2 cells, the PXR-driven 5.5-and 6.5-fold induction responses to omeprazole and 10 M rifampicin were attenuated to 4-and 3.5-fold, respectively. Nanosilver particles alone showed no induction. In 1A2DRE cells, the aryl hydrocarbon receptor-driven 5.5-fold induction response to omeprazole was attenuated to 4-fold. In 1A2DRE cells, nanosilver alone elicited slight induction at 1 g/ml. The inhibition of human P450-selective activities by nanosilver particles in vitro was proportional to the silver/microsomal protein ratio. At a fixed (0.5 mg/ml) protein concentration, P450-selective activities differed in sensitivity (IC 50 value).

Oxidative folding is a composite process that consists of both the conformational folding to the native three-dimensional structure and the regeneration of the native disulfide bonds of a protein, frequently involving over 100 disulfide intermediate species. Understanding the oxidative folding pathways of a multiple-disulfide-containing protein is a very difficult task that often requires years of devoted research due to the high complexity of the process and the very similar features of the large number of intermediates. Here we developed a method for rapidly delineating the major features of the oxidative folding pathways of a protein. The method examines the temperature dependence of the oxidative folding rate of the protein in combination with reduction pulses. Reduction pulses expose the presence of structured intermediates along the pathways. The correlation between the regeneration rate at different temperatures and the stability of the structured intermediates reveals the role that the intermediates play in determining the pathway. The method was first tested with bovine pancreatic ribonuclease A whose folding pathways were defined earlier. Then, it was explored to discern some of the major features of the folding pathways of its homologue, frog Onconase. The results suggest that the stability of the three-dimensional structure of the native protein is a major determinant of the folding rate in oxidative folding.

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ABSTRACT:When incubated with human liver microsomes, 2-diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) undergoes cytochrome P450 (P450)-dependent oxidative N-deethylation to the secondary amine metabolite 2-ethylaminoethyl-2,2-diphenylvalerate (SKF8742). P450-selective inhibitors indicated CYP3As catalyzed this reaction, and the deethylation rate correlated best with the CYP3A activity across a range of human liver microsomes. SKF525A and its metabolite and primary amine analog all inhibited CYP2B6-, CYP2C9-, CYP2C19-, CYP2D6-, and CYP3A-selective reactions to varying degrees but had little effect on CYP1A2, CYP2A6, and CYP2E1 reactions. Only the inhibition of CYP3A showed major enhancement when the inhibitors were preincubated with NADPH-fortified microsomes, and the extent of metabolic intermediate (MI) complex formation approximated typical CYP3A content. Two "lost with time" SKF525A derivatives devoid of the ethylamine moiety, 2,2-diphenylpropylethanol (SKF-Alcohol) and 2,2-diphenylpropylacetic acid (SKF-Acid) did not form an MI complex and were identified as selective inhibitors of CYP2C9. Although without detectable metabolism, their CYP2C9 inhibition fitted best with a competitive mechanism. Thus, not all the human P450s are inhibited by SKF525A and related compounds, and the mechanisms contributing to those that are inhibited vary with the isoform. P450 MI-complex formation only seems to play a role with CYP3As.

Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein. Both the cyclized and uncyclized proteins seem to follow the same oxidative folding pathways; however, cyclization altered the relative flux of the protein in these two pathways by increasing the rate of formation of a kinetically trapped intermediate. Glutaminyl cyclase (QC) catalyzed the cyclization of the unfolded, reduced protein, but had no effect on the disulfide-intact, uncyclized, folded protein. The structured intermediates of uncyclized onconase were also resistant to QC-catalysis, consistent with their having a native-like fold. These observations suggest that, in vivo, cyclization takes place during the initial stages of oxidative folding, specifically, before the formation of structured intermediates. The competition between oxidative folding and QC-mediated cyclization suggests that QC-catalyzed cyclization of the N-terminal glutamine in onconase occurs in the endoplasmic reticulum, probably co-translationally.The N-terminal glutamine residue of peptides and proteins undergoes non-enzymatic spontaneous cyclization resulting in the formation of pyroglutamic acid (1). This is a slow process requiring day(s) for completion, depending on the conditions (1). Examples of peptides and proteins with an N-terminal pyroglutamic acid include gonadotropin-releasing hormone, thyrotropin-releasing hormone, neurotensin, etc, for which biological activity depends on the presence of pyroglutamic acid at their N-termini (2,3). Loss or modification of the N-terminal pyroglutamic acid residue leads to a decrease in biological activity (4,5). This implies the existence of an enzyme that accelerates this cyclization in vivo (6)(7)(8). Indeed, enzymes with glutaminyl cyclase (QC) activity have been isolated from many sources (9-13), and the cDNA of QC was identified in many organisms (14)(15)(16) The anti-cancer activity of ONC (due to the absence of a specific intracellular inhibitor such as one that inhibits the activity of RNase A) is related to its ribonuclease activity (19,20,(26)(27)(28)(29).In this study, we examine the influence of the cyclization of N-terminal glutamine on the reductive unfolding and oxidative folding profiles (rates and pathways) in onconase. We also investigate the effect of formation of the native structure (which is coupled to disulfide-bond formation) on the QC-catalyzed cyclization of the protein.Experimental reductive unfolding (30-39) and oxidative folding studies (40-53) of proteins, in vitro and in vivo, have contributed to a better understanding of the relationship between protein structure and fol...