The human TRPA1 intrinsic cold and heat sensitivity involves separate channel structures beyond the N-ARD domain

TRP channels sense temperatures ranging from noxious cold to noxious heat. Whether specialized TRP thermosensor modules exist and how they control channel pore gating is unknown. We studied purified human TRPA1 (hTRPA1) truncated proteins to gain insight into the temperature gating of hTRPA1. In patch-clamp bilayer recordings, ∆1–688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas ∆1–854 hTRPA1, also lacking the S1–S4 voltage sensing-like domain (VSLD), gained sensitivity to cold but lost its heat sensitivity. In hTRPA1 intrinsic tryptophan fluorescence studies, cold and heat evoked rearrangement of VSLD and the C-terminus domain distal to the transmembrane pore domain S5–S6 (CTD). In whole-cell electrophysiology experiments, replacement of the CTD located cysteines 1021 and 1025 with alanine modulated hTRPA1 cold responses. It is proposed that hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5–S6 pore region and the VSLD, respectively

Electrophile-Induced Conformational Switch of the Human TRPA1 Ion Channel Detected by Mass Spectrometry

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 (Δ1-688 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

Thermo- and chemosensitive properties of Transient Receptor Potential Ankyrin 1 ion channels

The ability to sense and accommodate to an ever-changing environment is crucial for the survival of living organisms. Transient Receptor Potential (TRP) ion channels comprise a large superfamily of cation conducting membrane proteins that function as molecular sensors in diverse sensory processes including perception of light, taste, smell, sound, touch and temperature. The TRP Ankyrin 1 (TRPA1) ion channel is a unique member of the mammalian TRP superfamily, containing a large N-terminal ankyrin repeat domain (ARD) which constitutes half of the entire protein. TRPA1 responds to a variety of unrelated noxious stimuli such as chemicals, temperature and mechanical stress. It seems convincing that TRPA1 acts as a noxious chemical sensor and also plays a role in the detection of warm temperatures in non-mammalian species. The role of mammalian TRPA1 as a cold sensor is, however, controversial. The mammalian TRPA1 has been implicated in acute and inflammatory pain conditions and has been proposed as an important target for analgesics. If TRPA1 ion channels are intrinsically chemo-, thermo- and mechanosensitive proteins, regardless of species, remains to be shown. The overall of aim of this thesis was to investigate possible inherent thermo- and chemosensitive properties of human TRPA1 (hTRPA1) and the malaria mosquito Anopheles gambiae TRPA1 (AgTRPA1), and the role of the N-terminal ARD, containing suggested key temperature elements as well as cysteines targeted by thiol reactive oxidants and electrophiles known to activate TRPA1. Difficulties to express and purify large amounts of proteins have hampered structural and functional studies of TRPs but were here overcome by heterologous expression in the yeast Pichia pastoris. Single-channel electrophysiological recordings of purified hTRPA1 reconstituted into artificial lipid bilayers indicated that cold- and chemosensitivity are inherent channel properties recognized by structures outside the N-terminal ARD. Surprisingly, hTRPA1 is also intrinsically sensitive to warm temperatures, and thus displays U-shaped thermosensitivity. Notably, redox state and ligands showed modulatory effects on the mammalian TRPA1 thermosensitivity. The purified AgTRPA1 was activated by heat and the electrophile allyl isothiocyanate (a major pungent ingredient in wasabi and mustard) independently of the N-terminal ARD. The TRPA1 proteins displayed fluorescence quenching upon exposure to temperature and ligands, suggesting that conformational changes occur in a membrane-independent manner and thus that TRPA1 is an intrinsic chemo- and thermosensitive protein. Mass spectrometry was used to map hTRPA1 binding sites of the frequently used electrophilic TRPA1 activator N-methyl maleimide. Our results indicate that thermal and chemical sensitivities of mammalian and non-mammalian TRPA1 are intrinsic channel properties and that the N-terminal ARD is not key but plays a modulatory role in TRPA1 chemo- and thermosensation. Our findings provide a better understanding of the function of hTRPA1, which can help to develop novel treatments for pain and illnesses/symptoms caused by sensory hypersensitivity

Wnt signaling in intestinal inflammation

Chronic inflammatory bowel diseases, including Crohns disease and ulcerative colitis, are a major health burden worldwide. Numerous conserved signaling pathways control tissue injury and repair during colitis, but owing to the complexity of the inflammatory process, their individual contribution remains poorly understood. A key regulatory pathway in the intestinal mucosa is Wnt/beta-catenin signaling, which acts as the central organizer of epithelial stem cell identity and maintenance. Apart from this core function, there is mounting evidence that the Wnt pathway is highly interconnected with numerous other signaling cascades, and that combinatorial signaling events shape epithelial homeostasis and tissue regeneration. Here we provide an updated view of how Wnt signaling intersects with major inflammatory pathways, with a particular focus on intestinal inflammation. Elucidating the reciprocal actions of Wnt ligands and cytokines has the potential to reveal new treatment options for chronic colitis and other inflammatory disorders.Funding Agencies|Knut and Alice Wallenberg Foundation (KAW); European Crohns and Colitis Organisation (ECCO), Austria; Ferring Pharmaceuticals, Switzerland</p

A uniform expression library for the exploration of FOX transcription factor biology

Forkhead box (FOX) family transcription factors play essential roles in development, tissue homeostasis, and disease. Although the biology of several FOX proteins has been studied in depth, it is unclear to what extent these findings apply to even closely related family members, which frequently exert overlapping but non-redundant functions. To help address this question, we have generated a uniform, ready-to-use expression library of all 44 human FOX transcription factors with a convenient peptide tag for parallel screening assays. In addition, we have generated multiple universal forkhead box reporter plasmids, which can be used to monitor the transcriptional activity of most FOX proteins with high fidelity. As a proof-of-principle, we use our plasmid library to identify the DNA repair protein XRCC6/Ku70 as a selective FOX interaction partner and regulator of FOX transcriptional activity. We believe that these tools, which we make available via the Addgene plasmid repository, will considerably expedite the investigation of FOX protein biology.Funding agencies: Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation; Rotary Club Borgholm; Lions Foundation</p

FOX transcription factors are common regulators of Wnt/beta- catenin-dependent gene transcription

The Wnt/fl-catenin pathway is a critical regulator of devel-opment and stem cell maintenance. Mounting evidence suggests that the outcome of Wnt signaling is determined by the collab-orative action of multiple transcription factors, including members of the highly conserved forkhead box (FOX) protein family. However, the contribution of FOX transcription factors to Wnt signaling has not been investigated in a systematic manner. Here, we performed complementary screens of all 44 human FOX proteins to identify new Wnt pathway regulators. By combining fl-catenin reporter assays with Wnt pathway-focused qPCR arrays and proximity proteomics of selected candidates, we determine that most FOX proteins are involved in the regu-lation of Wnt pathway activity. As proof-of-principle, we addi-tionally characterize class D and I FOX transcription factors as physiologically relevant regulators of Wnt/fl-catenin signaling. We conclude that FOX proteins are common regulators of Wnt/ fl-catenin-dependent gene transcription that may control Wnt pathway activity in a tissue-specific manner.Funding Agencies|Knut and Alice Wallenberg Foundation; Swedish Research Council [2020-01084]; Swedish Cancer Society (Cancerfonden) [20 0737 Pj 01 H]</p

Calcium activates purified human TRPA1 with and without its N-terminal ankyrin repeat domain in the absence of calmodulin

Extracellular influx of calcium or release of calcium from intracellular stores have been shown to activate mammalian TRPA1 as well as to sensitize and desensitize TRPA1 electrophilic activation. Calcium binding sites on both intracellular N- and C-termini have been proposed. Here, we demonstrate based on Forster resonance energy transfer (FRET) and bilayer patch-clamp studies, a direct calmodulin-independent action of calcium on the purified human TRPA1 (hTRPA1), causing structural changes and activation without immediate subsequent desensitization of hTRPA1 with and without its N-terminal ankyrin repeat domain (N-ARD). Thus, calcium alone activates hTRPA1 by a direct interaction with binding sites outside the N-ARD.Funding Agencies|Swedish Research CouncilSwedish Research Council [2014-3801]; Medical Faculty of Lund University - ALF [ALFSKANE-451751]; Agence Nationale de la Recherche (ANR)French National Research Agency (ANR) [ANR-17-CE09-0026-01]; European Research Council (ERC) under the European Commissions Seventh Framework Programme [278242]</p

Human TRPA1 is an inherently mechanosensitive bilayer-gated ion channel

The role of mammalian Transient Receptor Potential Ankyrin 1 (TRPA1) as a mechanosensor is controversial. Here, we report that purified human TRPA1 (hTRPA1) with and without its N-terminal ankyrin repeat domain responded with pressure-dependent single-channel current activity when reconstituted into artificial lipid bilayers. The hTRPA1 activity was abolished by the thiol reducing agent TCEP. Thus, depending on its redox state, hTRPA1 is an inherent mechanosensitive ion channel gated by force-from-lipids

hTRPA1: Structural dynamics of pain sensor by FCS

International audienc

Wnt activator FOXB2 drives the neuroendocrine differentiation of prostate cancer

The Wnt signaling pathway is of paramount importance for development and disease. However, the tissue-specific regulation of Wnt pathway activity remains incompletely understood. Here we identify FOXB2, an uncharacterized forkhead box family transcription factor, as a potent activator of Wnt signaling in normal and cancer cells. Mechanistically, FOXB2 induces multiple Wnt ligands, including WNT7B, which increases TCF/LEF-dependent transcription without activating Wnt coreceptor LRP6 or beta-catenin. Proximity ligation and functional complementation assays identified several transcription regulators, including YY1, JUN, and DDX5, as cofactors required for FOXB2-dependent pathway activation. Although FOXB2 expression is limited in adults, it is induced in select cancers, particularly advanced prostate cancer. RNA-seq data analysis suggests that FOXB2/WNT7B expression in prostate cancer is associated with a transcriptional program that favors neuronal differentiation and decreases recurrence-free survival. Consistently, FOXB2 controls Wnt signaling and neuroendocrine differentiation of prostate cancer cell lines. Our results suggest that FOXB2 is a tissue-specific Wnt activator that promotes the malignant transformation of prostate cancer.Funding Agencies|Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation</p