A genetically encoded probe for the identification of proteins that form sulfenic acid in response to H2O2 in Saccharomyces cerevisiae.

It is widely known that reactive oxygen species (ROS), such as hydrogen peroxide, play important roles in cellular signaling and initiation of oxidative stress responses via thiol modifications. Identification of the targets of these modifications will provide a better understanding of the relationship between ROS and human diseases, such as cancer and atherosclerosis. Sulfenic acid is the principle product of a reaction between hydrogen peroxide and a reactive protein cysteine.

The role of active site residues in the oxidant specificity of the Orp1 thiol peroxidase.

In this study we investigated the role of active site residues in the peroxidase activity of Orp1 (GPx3) using three different peroxide substrates. Using a structural homology model of the reduced form of Orp1, we identified Asn126 and Phe127 as evolutionarily conserved residues that line the back of the Orp1 active site and which are likely to affect the peroxidase activity of Orp1. Additionally, we identified Phe38 as a surface residue that could influence substrate specificity as it is located adjacent to Cys36, in the same position occupied by similar hydrophobic amino acids in many Orp1 homologs.

A genetically encoded probe for cysteine sulfenic acid protein modification in vivo.

All organisms have defense mechanisms to combat the deleterious effects of oxidative damage produced by reactive oxidative species (ROS). Although it is known that ROS play a major role in oxidative damage, increasing evidence reveals that ROS have wider cellular effects through their role in many signal transduction pathways. Here we have adapted a redox-regulated domain from the Yap1 transcription factor in Saccharomyces cerevisiae to function as a general trap for proteins that form cysteine sulfenic acid (Cys-SOH) in vivo.

Molecular mechanism of oxidative stress perception by the Orp1 protein.

In this study we investigated the molecular mechanism by which the Orp1 (Gpx3) protein in Saccharomyces cerevisiae senses and reacts with hydrogen peroxide. Upon exposure to H(2)O(2) Orp1(Cys36) forms a disulfide-bonded complex with the C-terminal domain of the Yap1 protein (Yap1-cCRD). We used 4-nitrobenzo-2-oxa-1,3-diazole to identify a cysteine sulfenic acid (Cys-SOH) modification that forms on Cys(36) of Orp1(Cys36) upon exposure to H(2)O(2).

Thermosensitive TRPV channel subunits coassemble into heteromeric channels with intermediate conductance and gating properties.

Heat-sensitive transient receptor potential (TRP) channels (TRPV1-4) form the major cellular sensors for detecting temperature increases. Homomeric channels formed by thermosensitive TRPV subunits exhibit distinct temperature thresholds. While these subunits do share significant sequence similarity, whether they can coassemble into heteromeric channels has been controversial.

Metabolite of SIR2 reaction modulates TRPM2 ion channel.

The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death.

GFP-based FRET analysis in live cells.

Fluorescence resonance energy transfer (FRET) is a widely utilized optical technique for measuring small distances of 1-10 nm in live cells. In recent years, its application has been greatly popularized by the discovery of green fluorescent protein (GFP) and many improved variants which make good donor-acceptor fluorophore pairs. GFP-based proteins are structurally stable, relatively inert, and can be reliably attached to points of interest.

Accumulation of free ADP-ribose from mitochondria mediates oxidative stress-induced gating of TRPM2 cation channels.

TRPM2 is a member of the transient receptor potential melastatin-related (TRPM) family of cation channels, which possesses both ion channel and ADP-ribose hydrolase functions. TRPM2 has been shown to gate in response to oxidative and nitrosative stresses, but the mechanism through which TRPM2 gating is induced by these types of stimuli is not clear. Here we show through structure-guided mutagenesis that TRPM2 gating by ADP-ribose and both oxidative and nitrosative stresses requires an intact ADP-ribose binding cleft in the C-terminal nudix domain.

Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading.

To accommodate growth, insects must periodically replace their exoskeletons. After shedding the old cuticle, the new soft cuticle must sclerotize. Sclerotization has long been known to be controlled by the neuropeptide hormone bursicon, but its large size of 30 kDa has frustrated attempts to determine its sequence and structure.