Monitoring the action of redox-directed cancer therapeutics using a human peroxiredoxin-2-based probe.

Redox cancer therapeutics target the increased reliance on intracellular antioxidant systems and enhanced susceptibility to oxidant-induced stress of some cancer cells compared to normal cells. Many of these therapeutics are thought to perturb intracellular levels of the oxidant hydrogen peroxide (HO), a signaling molecule that modulates a number of different processes in human cells. However, fluorescent probes for this species remain limited in their ability to detect the small perturbations induced during successful treatments.

A reaction-diffusion model of cytosolic hydrogen peroxide.

As a signaling molecule in mammalian cells, hydrogen peroxide (H2O2) determines the thiol/disulfide oxidation state of several key proteins in the cytosol. Localization is a key concept in redox signaling; the concentrations of signaling molecules within the cell are expected to vary in time and in space in manner that is essential for function. However, as a simplification, all theoretical studies of intracellular hydrogen peroxide and many experimental studies to date have treated the cytosol as a well-mixed compartment.

Insights into electron leakage in the reaction cycle of cytochrome P450 BM3 revealed by kinetic modeling and mutagenesis.

As a single polypeptide, cytochrome P450 BM3 fuses oxidase and reductase domains and couples each domain's function to perform catalysis with exceptional activity upon binding of substrate for hydroxylation. Mutations introduced into the enzyme to change its substrate specificity often decrease coupling efficiency between the two domains, resulting in unproductive consumption of cofactors and formation of water and/or reactive species. This phenomenon can correlate with leakage, in which P450 BM3 uses electrons from NADPH to reduce oxygen to water and/or reactive species even without bound substrate.

Analysis of the lifetime and spatial localization of hydrogen peroxide generated in the cytosol using a reduced kinetic model.

Hydrogen peroxide (H2O2) acts as a signaling molecule via its reactions with particular cysteine residues of certain proteins. Determining the roles of direct oxidation by H2O2 versus disulfide exchange reactions (i.e.

Use of a genetically encoded hydrogen peroxide sensor for whole cell screening of enzyme activity.

We report the use of HyPer, a genetically encoded, fluorescent sensor that reacts with hydrogen peroxide (H2O2), in a novel screen to engineer enzymes for enhanced production of H2O2. We co-expressed HyPer with cytochrome P450 BM3 variants and, using HyPer's ratiometric signal, found variants that produce greater amounts of H2O2 than the wild-type enzyme through the leakage reaction. The screen avoids lysis procedures and the addition of reagents to assay intracellular contents.

In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress.

Genetically encoded, fluorescent biosensors have been developed to probe the activities of various signaling molecules inside cells ranging from changes in intracellular ion concentrations to dynamics of lipid second messengers. HyPer is a member of this class of biosensors and is the first to dynamically respond to hydrogen peroxide (H2O2), a reactive oxygen species that functions as a signaling molecule. However, detailed characterization of HyPer's signal is not currently available within the context of bacteria exposed to external oxidative stress, which occurs in the immunological response of higher organisms against invasive pathogenic bacteria.

CHARMM36 united atom chain model for lipids and surfactants.

Molecular simulations of lipids and surfactants require accurate parameters to reproduce and predict experimental properties. Previously, a united atom (UA) chain model was developed for the CHARMM27/27r lipids (Hénin, J., et al.

Influence of ester-modified lipids on bilayer structure.

Lipid membranes function as barriers for cells to prevent unwanted chemicals from entering the cell and wanted chemicals from leaving. Because of their hydrophobic interior, membranes do not allow water to penetrate beyond the headgroup region. We performed molecular simulations to examine the effects of ester-modified lipids, which contain ester groups along their hydrocarbon chains, on bilayer structure.

Update of the cholesterol force field parameters in CHARMM.

A modification of the CHARMM36 lipid force field (C36) for cholesterol, henceforth, called C36c, is reported. A fused ring compound, decalin, was used to model the steroid section of cholesterol. For decalin, C36 inaccurately predicts the heat of vaporization (~10 kJ/mol) and molar volume (~10 cc/mol), but C36c resulted in near perfect comparison with experiment.

Sterol binding and membrane lipid attachment to the Osh4 protein of yeast.

Osh4 is an oxysterol-binding protein homologue found in yeast that is essential for the intracellular transport of sterols and cell life. In this study, molecular dynamics simulations were used to investigate the binding of ergosterol, 25-hydroxycholesterol, and lipid moieties to Osh4. The binding energies between both sterols and Osh4 were dominated by van der Waals interactions with residues within the sterol binding pocket, and were further stabilized by water-mediated interactions with polar residues at the bottom of the binding pocket (W46, Q96, Y97, N165, Q181).

Cholesterol flip-flop: insights from free energy simulation studies.

The mechanism of lipid flip-flop motion is important for maintaining the asymmetric distribution of lipids in a biological membrane. To explore the energetics and mechanism of passive cholesterol flip-flop and its dependence on chain saturation, we performed two-dimensional umbrella sampling simulations in DPPC, POPC, and DAPC (dipalmitoyl-, palmitoyloleoyl-, and diarachidonylphosphatidylcholine) and used the string method to identify the most probable flip-flop paths based on the two-dimensional free energy maps. The resulting paths indicate that cholesterol prefers to tilt first and then move to the bilayer center where the free energy barrier exists.

Lipid chain branching at the iso- and anteiso-positions in complex Chlamydia membranes: a molecular dynamics study.

Membranes in the intracellular eubacterial parasite Chlamydia trachomatis consist of the elementary body (EB) and reticular body (RB), and contain methyl branches at the iso- and anteiso-positions for some phospholipid chains. Acyl chain branching is the focus of this study. Molecular dynamics simulations were used to study bilayers of 1-13-methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (13-MpPPC), 1-14-methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (14-MpPPC), and diphytanoylphosphatidylcholine (DPhPC).

CHARMM-GUI Membrane Builder for mixed bilayers and its application to yeast membranes.

The CHARMM-GUI Membrane Builder (http://www.charmm-gui.org/input/membrane), an intuitive, straightforward, web-based graphical user interface, was expanded to automate the building process of heterogeneous lipid bilayers, with or without a protein and with support for up to 32 different lipid types.