Electrophile-Induced Conformational Switch of the Human TRPA1 Ion Channel Detected by Mass Spectrometry.
ABSTRACT: The human Transient Receptor Potential A1 (hTRPA1) ion channel, also known as the wasabi receptor, acts as a biosensor of various potentially harmful stimuli. It is activated by a wide range of chemicals, including the electrophilic compound N-methylmaleimide (NMM), but the mechanism of activation is not fully understood. Here, we used mass spectrometry to map and quantify the covalent labeling in hTRPA1 at three different concentrations of NMM. A functional truncated version of hTRPA1 (?1-688 hTRPA1), lacking the large N-terminal ankyrin repeat domain (ARD), was also assessed in the same way. In the full length hTRPA1, the labeling of different cysteines ranged from nil up to 95% already at the lowest concentration of NMM, suggesting large differences in reactivity of the thiols. Most important, the labeling of some cysteine residues increased while others decreased with the concentration of NMM, both in the full length and the truncated protein. These findings indicate a conformational switch of the proteins, possibly associated with activation or desensitization of the ion channel. In addition, several lysines in the transmembrane domain and the proximal N-terminal region were labeled by NMM, raising the possibility that lysines are also key targets for electrophilic activation of hTRPA1.
Project description:TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 ?M versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.
Project description:We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (?1-688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid ?(9)-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and ?1-688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca(2+), or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease).
Project description:BACKGROUND AND PURPOSE: Thymol, a major component of thyme and oregano, has medical uses in oral care products as an astringent and antibiotic. Its distinctive sharp odour and pungent flavour are considered aversive properties. The molecular basis of these aversive properties is not well understood. EXPERIMENTAL APPROACH: The ability of thymol to activate human transient receptor potential channel A1 (hTRPA1) expressed in stably transfected human embryonic kidney 293 (HEK293) cells was measured by membrane potential and calcium-sensitive dyes in a fluorescence-imaging plate reader (FLIPR) assay. Direct activation of hTRPA1 currents was measured by whole-cell voltage clamp recording. Intracellular calcium changes were measured using fura-2 dye. The FLIPR assay was also used to measure membrane potential changes elicited by thymol after pretreatment with camphor, a known TRPA1 inhibitor. The ability of related alkyl phenols to activate hTRPA1 was also determined. KEY RESULTS: Thymol potently activated a membrane potential response and intracellular calcium increase in hTRPA1-expressing HEK293 cells in a concentration-dependent manner. Activation by thymol desensitized hTRPA1 to further exposure to thymol or the known ligand allyl isothiocyanate (AITC). The related phenols 2-tert-butyl-5-methylphenol, 2,6-diisopropylphenol (propofol) and carvacrol also activated hTRPA1. Phenols with less bulky carbon substitutions and lower logP values were less potent in general. The response to thymol was blocked by camphor. CONCLUSIONS AND IMPLICATIONS: These results suggest a role for hTRPA1 activation in the reported pungent and aversive properties of some of these pharmaceutically important phenols.
Project description:[Structure: see text] A strategically novel approach to the formation of syn-1,3-diol mono- and diethers through electrophilic activation of homoallylic alkoxymethyl ethers has been developed. The resulting polyketide-like synthetic fragments are generated in good yield and with excellent stereocontrol. A chairlike transition state is proposed to account for the high stereoselectivity. Varying the conditions of the reaction workup results in the efficient generation of mono- and diether containing structural units common to polyketide natural products.
Project description:We show that the physiological activity of solid aerosolized benzylidenemalononitriles (BMNs) including 'tear gas' (CS) in historic human volunteer trials correlates with activation of the human transient receptor potential ankyrin 1 ion channel (hTRPA1). This suggests that the irritation caused by the most potent of these compounds results from activation of this channel. We prepared 50 BMNs and measured their hTRPA1 agonist potencies. A mechanism of action consistent with their physiological activity, involving their dissolution in water on contaminated body surfaces, cell membrane penetration and reversible thiolation by a cysteine residue of hTRPA1, supported by data from nuclear magnetic resonance experiments with a model thiol, explains the structure-activity relationships. The correlation provides evidence that hTRPA1 is a receptor for irritants on nociceptive neurons involved in pain perception; thus, its activation in the eye, nose, mouth and skin would explain the symptoms of lachrymation, sneezing, coughing and stinging, respectively. The structure-activity results and the use of the BMNs as pharmacological tools in future by other researchers may contribute to a better understanding of the TRPA1 channel in humans (and other animals) and help facilitate the discovery of treatments for human diseases involving this receptor.
Project description:Over the last 50 years, the posttranslational modification (PTM) of proteins has emerged as a central mechanism for cells to regulate metabolism, growth, differentiation, cell-cell interactions, and immune responses. By influencing protein structure and function, PTM leads to a multiplication of proteome diversity. Redox-dependent PTMs, mediated by environmental and endogenously generated reactive species, induce cell signaling responses and can have toxic effects in organisms. PTMs induced by the electrophilic by-products of redox reactions most frequently occur at protein thiols; other nucleophilic amino acids serve as less favorable targets. Advances in mass spectrometry and affinity-chemistry strategies have improved the detection of electrophile-induced protein modifications both in vitro and in vivo and have revealed a high degree of amino acid and protein selectivity of electrophilic PTM. The identification of biological targets of electrophiles has motivated further study of the functional impact of various PTM reactions on specific signaling pathways and how this might affect organisms.
Project description:Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.
Project description:An emerging aspect of redox signaling is the pathway mediated by electrophilic byproducts, such as nitrated cyclic nucleotide (for example, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP)) and nitro or keto derivatives of unsaturated fatty acids, generated via reactions of inflammation-related enzymes, reactive oxygen species, nitric oxide and secondary products. Here we report that enzymatically generated hydrogen sulfide anion (HS(-)) regulates the metabolism and signaling actions of various electrophiles. HS(-) reacts with electrophiles, best represented by 8-nitro-cGMP, via direct sulfhydration and modulates cellular redox signaling. The relevance of this reaction is reinforced by the significant 8-nitro-cGMP formation in mouse cardiac tissue after myocardial infarction that is modulated by alterations in HS(-) biosynthesis. Cardiac HS(-), in turn, suppresses electrophile-mediated H-Ras activation and cardiac cell senescence, contributing to the beneficial effects of HS(-) on myocardial infarction-associated heart failure. Thus, this study reveals HS(-)-induced electrophile sulfhydration as a unique mechanism for regulating electrophile-mediated redox signaling.
Project description:Pain is a harmful sensation that arises from noxious stimuli. Transient receptor potential ankyrin 1 (TRPA1) is one target for studying pain mechanisms. TRPA1 is activated by various stimuli such as noxious cold, pungent natural products and environmental irritants. Since TRPA1 is an attractive target for pain therapy, a few TRPA1 antagonists have been developed and some function as analgesic agents. The responses of TRPA1 to agonists and antagonists vary among species and these species differences have been utilized to identify the structural basis of activation and inhibition mechanisms. The TRPA1 antagonist HC-030031 (HC) failed to inhibit frog TRPA1 (fTRPA1) and zebrafish TRPA1 activity induced by cinnamaldehyde (CA), but did inhibit human TRPA1 (hTRPA1) in a heterologous expression system. Chimeric studies between fTRPA1 and hTRPA1, as well as analyses using point mutants, revealed that a single amino acid residue (N855 in hTRPA1) significantly contributes to the inhibitory action of HC. Moreover, the N855 residue and the C-terminus region exhibited synergistic effects on the inhibition by HC. Molecular dynamics simulation suggested that HC stably binds to hTRPA1-N855. These findings provide novel insights into the structure-function relationship of TRPA1 and could lead to the development of more effective analgesics targeted to TRPA1.
Project description:A novel gas-phase electrophilic cyclization, initiated by the protonation of a nitro group, occurs for 2-nitrophenyl phenyl ether and for the analogous sulfide and amine, leading to heterocyclic intermediates in each case. Subsequently, the cyclic intermediates dissociate via two pathways: (1) unusual step-wise eliminations of two OH radicals to afford heterocyclic cations, [phenoxazine - H](+), [phenothiazine - H](+), and [phenazine + H](+), and (2) expulsion of H(2)O, to yield a heterocyclic ketone, followed by loss of CO. The proposed structures of the gas-phase product ions and reaction mechanisms are supported by chemical substitution, deuterium labeling, accurate mass measurements at high mass resolving power, product-ion mass spectra obtained by tandem mass spectrometry, mass spectra of reference compounds, and molecular orbital calculations. Using a mass spectrometer as a reaction vessel, we demonstrate that, upon protonation, a nitro group becomes an electrophile and participates in cyclization reactions in the gas phase.