Search

TRPV1, a member of the transient receptor potential (TRP) channels family, has been found to be involved in redox sensing. The crystal structure of the human TRPV1 ankyrin-repeat domain (TRPV1-ARD) was determined at 4.5 Å resolution under nonreducing conditions. This is the first report of the crystal structure of a ligand-free form of TRPV1-ARD and in particular of the human homologue. The structure showed a unique conformation in finger loop 3 near Cys258, which is most likely to be involved in inter-subunit disulfide-bond formation. Also, in human TRPV1-ARD it was possible for solvent to access Cys258. This structural feature might be related to the high sensitivity of human TRPV1 to oxidants. ESI-MS revealed that Cys258 did not form an S–OH functionality even under nonreducing conditions.

Cellular redox status is maintained by the balance between series of antioxidant systems and production of reactive oxygen/nitrogenous species. Cells utilize this redox balance to mediate diverse physiological functions. Transient receptor potential (TRP) channels are non-selective cation channels that act as biosensors for environmental and noxious stimuli, such as capsaicin and allicin, as well as changes in temperature and conditions inside the cell. TRP channels also have an emerging role as essential players in detecting cellular redox status to regulate cellular signals mediating physiological phenomena. Reactive species activate TRP channels either directly through oxidative amino acid modifications or indirectly through second messengers. For instance, TRPA1, TRPV1 and TRPC5 channels are directly activated by oxidizing agents through cysteine modification; whereas, TRPM2 channel is indirectly activated by production of ADP-ribose. One intriguing property of several TRP channels is susceptibility to both oxidizing and reducing stimuli, suggesting TRP channels could potentially act as a bidirectional sensor for detecting deviations in redox status. In this review, we discuss the unique chemical physiologies of redox sensitive TRP channels and their physiological significance in Ca(2+) signaling.

The effects of dietary soybean β-conglycinin on lipid metabolism and energy consumption were studied in Wistar adult rats. Rats were fed, a diet containing casein (control group) or β-conglycinin (β-conglycinin group), for 4 weeks. Carbohydrate consumption was higher and fat consumption was lower in the β-conglycinin group than in the control group, whereas the total energy consumption was the same between the two groups. Serum adiponectin was higher in the β-conglycinin group than in the control group. Serum triacylglycerol levels in the β-conglycinin group were significantly lower than those in the control group. The secretion rate of triacylglycerols from the liver after the administration of tyloxapol, an inhibitor of lipolysis, was significantly lower in the β-conglycinin group than in the control group. These results suggest the possibility that β-conglycinin exerts hypolipidemic effects through an acceleration in carbohydrate consumption associated with an increase in adiponectin in rats.

Environmental and endogenous reactive species such as reactive oxygen species (ROS), reactive nitrogen species (RNS), and other electrophiles are not only known to exert toxic effects on organisms, but are also emerging as molecules that mediate cell signaling responses. However, the mechanisms underlying this cellular redox signaling by reactive species remains largely uncharacterized.Ca2+-permeable cation channels encoded by the transient receptor potential (trp) gene superfamily are characterized by a wide variety of activation triggers that act from outside and inside the cell. Recent studies have revealed that multiple TRP channels sense reactive species and induce diverse physiological and pathological responses, such as cell death, chemokine production, and pain transduction. TRP channels sense reactive species either indirectly through second messengers or directly via oxidative modification of cysteine residues. In this review, we describe the activation mechanisms and biological roles of redox-sensitive TRP channels, including TRPM2, TRPM7, TRPC5, TRPV1, and TRPA1.The sensitivity of TRP channels to reactive species in vitro has been well characterized using molecular and pharmacological approaches. However, the precise activation mechanism(s) and in vivo function(s) of ROS/RNS-sensitive TRP channels remain elusive.Redox sensitivity of TRP channels has been shown to mediate previously unexplained biological phenomena and is involved in various pathologies. Understanding the physiological significance and activation mechanisms of TRP channel regulation by reactive species may lead to TRP channels becoming viable pharmacological targets, and modulators of these channels may offer therapeutic options for previously untreatable diseases.

An ability to adapt to changes in oxygen availability is essential for survival in both prokaryotic and eukaryotic organisms. Recently, cation channels encoded by the transient receptor potential (trp) gene superfamily have been recognized as multimodal sensors of a wide variety of factors inside the cells and in the extracellular environment and also as transducers of electrical and chemical signals mediated by ions such as Ca(2+). The functional features of TRP channels enable the body to react and adapt to different forms of environmental changes, including oxygen levels. A subclass of TRP channels regulates various cellular processes in response to fluctuations in oxygen. In this article, we describe the physiological and pathological significance of the oxygen-sensitive TRP channels, which are heterogeneous in the cellular responses to acute changes in oxygen, by contrasting their oxygen monitoring function with that of other ion channels, transporters, and enzymes. We also discuss the physiological relevance of oxygen-sensitive TRP channels as a novel class of target proteins for pharmaceutical therapeutics.

Complex disulfide bond patterns in synaptosomal-associated protein of 25 kD B (SNAP25B) are thought to regulate neurotransmitter release in response to oxidative stress. However, the steric feasibility of each possible disulfide pattern in SNAP25B has not been assessed. To assess the steric feasibility of hypothesized closely spaced complex disulfide patterning in SNAP25B and also the feasibility of identifying complex disulfide bond patterns with MS, we have developed a novel probabilistic analysis to unambiguously resolve complex double disulfide bond patterns by using an ion trap mass spectrometer. We analyzed fragmentation patterns of singly linked peptides to determine likely fragmentation events in an ion trap mass spectrometer and observed double and single backbone cleavage along with heterolytic cleavage of the disulfide bond. We modeled these same events in the doubly disulfide linked SNAP25B peptide and used a cumulative hypergeometric distribution with top–down scoring to both identify and differentiate these bonding patterns. Because of the presence of unique MS/MS peaks, two of the bonding patterns were directly identified. The third was assigned on the basis of full chromatographic separation and confirmed by modeling triple breakage fragments. In total, this work demonstrates the feasibility – and also limitations – of identification of complex intradisulfide patterns by using ion trap-based collision-induced dissociation-based fragmentation methods. Copyright © 2013 John Wiley & Sons, Ltd.

Transient receptor potential vanilloid 1 (TRPV1) channel is a tetrameric protein that acts as a sensor for noxious stimuli such as heat and for diverse inflammatory mediators such as oxidative stress to mediate nociception in a subset of sensory neurons. In TRPV1 oxidation sensing, cysteine (Cys) oxidation has been considered as the principle mechanism; however, its biochemical basis remains elusive. Here, we characterize the oxidative status of Cys residues in differential redox environments and propose a model of TRPV1 activation by oxidation. Through employing a combination of non-reducing SDS-PAGE, electrophysiology, and mass spectrometry we have identified the formation of subunit dimers carrying a stable intersubunit disulfide bond between Cys-258 and Cys-742 of human TRPV1 (hTRPV1). C258S and C742S hTRPV1 mutants have a decreased protein half-life, reflecting the role of the intersubunit disulfide bond in supporting channel stability. Interestingly, the C258S hTRPV1 mutant shows an abolished response to oxidants. Mass spectrometric analysis of Cys residues of hTRPV1 treated with hydrogen peroxide shows that Cys-258 is highly sensitive to oxidation. Our results suggest that Cys-258 residues are heterogeneously modified in the hTRPV1 tetrameric complex and comprise Cys-258 with free thiol for oxidation sensing and Cys-258, which is involved in the disulfide bond for assisting subunit dimerization. Thus, the hTRPV1 channel has a heterogeneous subunit composition in terms of both redox status and function.

SNAP-25 contains two of the four helixes that form the SNARE complex, critical for neuronal exocytosis. The helixes are linked together with a cysteine-rich domain, which is the site of various post-translational modifications such as palmitoylation and possibly oxidation. SNAP-25's linker contains 4 cysteines and thus could be both palmitoylated and oxidized. Palmitoylation anchors SNAP-25 to the membrane, while the level of oxidation may regulate transmitter release.Using standard MS/MS collision-induced dissociation (CID) in an Ion Trap mass spectrometer we analyzed peptide fragmentation patterns to determine fragmentation events, and observed single and double backbone cleavage along with heterolytic cleavage of disulfide bonds. We modeled these same events in the doubly disulfide linked SNAP25B peptide and used a cumulative hypergeometric distribution with top-down scoring to first identify, and then to differentiate all three bonding patterns. This is the first study to assign all double disulfide bonding patterns in a protein, and uses methodology fully compatible with standard large scale shotgun proteomics.In addition, the structural stability of oxidized and reduced SNAP-25 was determined with Circular Dichroism (CD). We observe that oxidized protein is less stable (lower melting temperature) than reduced protein. Additional experiments focus on the stability of the SNARE complex with oxidized samples and on the extent of SNAP-25 oxidation in brain tissue, with the ultimate goal to elucidate the effects of palmitoylation and oxidative stress on SNARE complex formation and vesicle fusion.

SNAP-25 plays a critical role in neuronal exocytosis. The SNAP-25 linker region contains a cysteine-rich domain, which is the site of various post-translational modifications such as palmitoylation and possibly oxidation. Palmitoylation anchors SNAP-25 to the membrane, while the level of oxidation may regulate the extent of transmitter release. Using a biotinylation/chemiluminescence assay, within a reconstituted system, and mass spectrometry (LTQ Orbitrap) we characterize the modification of the four cysteine residues in the SNAP-25B linker. We show that the palmitoyl transferase DHHC17 increases the extent of SNAP-25B palmitoylation in vitro. We verified that oxidation of SNAP-25B leads to disulfide bond formation within the cysteine-rich domain, the predominant form containing two disulfide bonds. As expected, oxidation of cysteines prevented palmitoylation. Through additional experimentation we hope to elucidate the effects of palmitoylation and oxidative stress on SNARE complex formation and vesicle fusion.

Ever since Pfluger, it has been always a major issue of physiology to identify the core principles underlying our body’s ability to sense and adapt to the changes in the availability of ambient molecular oxygen (O2) [3, 5]. The milestone discovery of an O2 sensor was first achieved at the organ level by Heymans, who demonstrated that inspired hypoxia triggers increased breathing through the stimulation of the carotid bodies (CBs) [2]. With regard to the cellular and molecular mechanisms underlying the acute hypoxia-sensing in the CBs, it is generally accepted that hypoxia inhibits K+ channels to depolarize the chemoreceptor glomus cells, leading to the activation of voltage-dependent Ca2+ channels and consequent release of neurotransmitters that relays information to regulate the respiration [10]. Another milestone is the discovery of a soluble factor erythropoietin (Epo) that is essential for the chronic phases of O2 adaptation. At the end of nineteenth century, it was recognized by Viault that exposure to high altitude hypoxia elicits a robust burst of erythropoiesis in humans [1]. Almost a half century later, Epo was demonstrated to mediate hypoxia-induced erythropoiesis. While the initial characterizations of the expression of Epo suggested its production in the fetal liver and adult kidney, it was only recently that the localization of the renal Epo-producing (REP) cell was found to be in the peritubular interstitial space located in the deep renal outer medulla region [4]. In addition, hypoxia-inducible factor (HIF) [6] is recognized as the transcription factor central to chronic cellular responses to hypoxia including erythropoiesis via Epo production. Thus, CBs, Epo, and HIF provide solid bases for understanding the body’s ability to sense and adapt to the ambient O2. Even after the above discoveries of the core principles, the physiology of O2 sensing and adaptation never loses its glory. This is highlighted by recent findings concerning the diversity and ubiquity of O2-sensing mechanisms. For example, in addition to multiple K+ channel subtypes and mediators previously reported responsible for hypoxia sensing, transient receptor potential (TRP) cation channels have emerged as O2–regulated ion channels critical for O2 sensing in non-CB chemoreceptors and tissues [7, 11]. Importantly, HIF, which binds to the hypoxia-responsive element present in the 3′ enhancer region of the Epo gene [6], is expressed widely throughout the body and is intriguingly found in lower organisms that produce neither Epo nor red blood cells. In addition to erythropoiesis, chronic hypoxia induces the expression of numerous genes whose products facilitate non-oxidative synthesis of ATP and multiply the number of vessels supplying the hypoxic area of tissues [8] through the action of HIFs. These lines of evidence suggest the ubiquitous presence of O2-sensing cells and organs in the animal body. In this context, it is interesting to note that two HIF isoforms HIF1 and HIF2 play distinctive roles in the regulation of hypoxic responses of CBs by differentially promoting cellular redox status [12]. Moreover, the physiological importance of hypoxia has been demonstrated in maintaining the hematopoietic stem cell niche for cell cycle quiescence through the action of HIF in the bone marrow [9]. In this special issue, reviews of these recent developments are included together with several important original research articles, which indeed enrich our knowledge in O2 physiology. We believe that there is a great prospect that O2 physiology will further enhance its current status as a major issue in physiology. A future direction of O2 physiology is systems physiology, in which regulation of O2 sensing and adaptation through interplays among different chemoreceptors and molecules are studied. Another possible direction is to understand O2 sensing and adaptation in the context of redox biology, where alterations in redox homeostasis (via reactive chemical species and antioxidant systems) in response to changes in the O2 availability are integrated with other types of stress such as metabolic perturbation, heat, and proinflammatory mediators. Adding these future developments to O2 physiology should lead us to understand our body’s ability as a total system to adapt to changes in ambient O2 availability.