Functional and Structural Divergence in Human TRPV1 Channel Subunits by Oxidative Cysteine Modification.

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.

Soybean ß-conglycinin improves carbohydrate and lipid metabolism in Wistar rats.

The effects of dietary soybean ß-conglycinin on lipid metabolism and energy consumption were studied in Wistar adult rats. Rats were fed, a diet containing casein (control group) or ß-conglycinin (ß-conglycinin group), for 4 weeks. Carbohydrate consumption was higher and fat consumption was lower in the ß-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 ß-conglycinin group than in the control group. Serum triacylglycerol levels in the ß-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 ß-conglycinin group than in the control group. These results suggest the possibility that ß-conglycinin exerts hypolipidemic effects through an acceleration in carbohydrate consumption associated with an increase in adiponectin in rats.

TRP channels as sensors of oxygen availability.

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.

Impact of nutritional guidance on various clinical parameters in patients with moderate obesity: A retrospective study.

Context: This study aims to investigate whether there is adequate provision of nutritional guidance through interventions by registered dietitians, especially for patients with moderate obesity. This is particularly important as such interventions may prove to be more effective for Japanese patients. Methods: In Japan, since there is a system of nutritional guidance with a registered dietitian for patients with a BMI over 30 kg/m2, we recruited 636 patients with obesity who had a BMI over 30 kg/m2 admitted to the Kawasaki Medical School General Medical Center between April 2018 and March 2020 through a review of their medical records. Second, we recruited 153 patients who underwent a blood examination before receiving nutritional guidance and at least one time every 3 to 6 months thereafter after receiving it. We aimed to evaluate whether continued nutritional guidance and follow-up interventions for patients with obesity were effective. We compared the BMI and metabolic markers of the patients who received nutritional guidance from a registered dietitian against those who did not. Results: A total of 636 patients with obesity who have a BMI over 30 kg/m2 were included in this study. A total of 164 patients with obesity received nutritional guidance from a registered dietitian at least one time, but 472 patients did not. Most interventions on nutritional guidance conducted by a registered dietitian were ordered from internal medicine (81.1%). However, internal medicine was the most common department that did not perform these interventions; however, less than half of the (49.2%) received them. In the second analysis, we compared two groups of patients with obesity. The first group (n = 70) who underwent blood examinations received nutritional guidance from a registered dietitian, while the second group (n = 54) did not receive such guidance. We found that there was no significant difference in body weight and BMI between the two groups of patients. We observed a significant decrease in dyslipidemia-associated metabolic markers among the patients who received nutritional guidance compared to those who did not [total cholesterol, -9.7 ± 29.3 vs. 2.3 ± 22.0 mg/dL (p = 0.0208); low-density lipoprotein cholesterol, -10.4 ± 30.5 vs. -2.0 ± 51.0 mg/dL (p = 0.0147), respectively]. Other metabolic markers also tended to decrease, although they did not reach statistical significance. Conclusion: It is rare for patients with only obesity to receive nutritional guidance. However, when nutritional guidance from a registered dietitian is provided, improvements in BMI and metabolic parameters can be expected.

Structure determination of the human TRPV1 ankyrin-repeat domain under nonreducing conditions.

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 Šresolution 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.

Sensing of redox status by TRP channels.

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.

Redox regulation of transient receptor potential channels.

SIGNIFICANCE: 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. RECENT ADVANCES: 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. CRITICAL ISSUES: 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. FUTURE DIRECTIONS: 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.

Resolving double disulfide bond patterns in SNAP25B using liquid chromatography-ion trap mass spectrometry.

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.