Molecular Basis for Mambalgin-2 Interaction with Heterotrimeric α-ENaC/ASIC1a/γ-ENaC Channels in Cancer Cells.

Cancer progression is characterized by microenvironmental acidification. Tumor cells adapt to low environmental pH by activating acid-sensing trimeric ion channels of the DEG/ENaC family. The α-ENaC/ASIC1a/γ-ENaC heterotrimeric channel is a tumor-specific acid-sensing channel, and its targeting can be considered a new strategy for cancer therapy. Mambalgin-2 from the Dendroaspis polylepis venom inhibits the α-ENaC/ASIC1a/γ-ENaC heterotrimer more effectively than the homotrimeric ASIC1a channel, initially proposed as the target of mambalgin-2. Although the molecular basis of such mambalgin selectivity remained unclear. Here, we built the models of the complexes of mambalgin-2 with the α-ENaC/ASIC1a/γ-ENaC and ASIC1a channels, performed MD and predicted the difference in the binding modes. The importance of the 'head' loop region of mambalgin-2 for the interaction with the hetero-, but not with the homotrimeric channel was confirmed by site-directed mutagenesis and electrophysiology. A new mode of allosteric regulation of the ENaC channels by linking the thumb domain of the ASIC1a subunit with the palm domain of the γ-ENaC subunit was proposed. The data obtained provide new insights into the regulation of various types of acid-sensing ion channels and the development of new strategies for cancer treatment.

Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1ß Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer.

Phα1ß (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (CaV2.2) and TRPA1. In animal models, Phα1ß administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1ß and its 15N-labeled analogue. Spatial structure and dynamics of Phα1ß were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the µs-ms time-scale fluctuations. The Phα1ß structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1ß has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1ß significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1ß as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.

Membrane-mediated interaction of non-conventional snake three-finger toxins with nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor of α7 type (α7-nAChR) presented in the nervous and immune systems and epithelium is a promising therapeutic target for cognitive disfunctions and cancer treatment. Weak toxin from Naja kaouthia venom (WTX) is a non-conventional three-finger neurotoxin, targeting α7-nAChR with weak affinity. There are no data on interaction mode of non-conventional neurotoxins with nAChRs. Using α-bungarotoxin (classical three-finger neurotoxin with high affinity to α7-nAChR), we showed applicability of cryo-EM to study complexes of α7-nAChR extracellular ligand-binding domain (α7-ECD) with toxins. Using cryo-EM structure of the α7-ECD/WTX complex, together with NMR data on membrane active site in the WTX molecule and mutagenesis data, we reconstruct the structure of α7-nAChR/WTX complex in the membrane environment. WTX interacts at the entrance to the orthosteric site located at the receptor intersubunit interface and simultaneously forms the contacts with the membrane surface. WTX interaction mode with α7-nAChR significantly differs from α-bungarotoxin's one, which does not contact the membrane. Our study reveals the important role of the membrane for interaction of non-conventional neurotoxins with the nicotinic receptors.

Mambalgin-2 Inhibits Lung Adenocarcinoma Growth and Migration by Selective Interaction With ASIC1/α-ENaC/γ-ENaC Heterotrimer.

Lung cancer is one of the most common cancer types in the world. Despite existing treatment strategies, overall patient survival remains low and new targeted therapies are required. Acidification of the tumor microenvironment drives the growth and metastasis of many cancers. Acid sensors such as acid-sensing ion channels (ASICs) may become promising targets for lung cancer therapy. Previously, we showed that inhibition of the ASIC1 channels by a recombinant analogue of mambalgin-2 from Dendroaspis polylepis controls oncogenic processes in leukemia, glioma, and melanoma cells. Here, we studied the effects and molecular targets of mambalgin-2 in lung adenocarcinoma A549 and Lewis cells, lung transformed WI-38 fibroblasts, and lung normal HLF fibroblasts. We found that mambalgin-2 inhibits the growth and migration of A549, metastatic Lewis P29 cells, and WI-38 cells, but not of normal fibroblasts. A549, Lewis, and WI-38 cells expressed different ASIC and ENaC subunits, while normal fibroblasts did not at all. Mambalgin-2 induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma cells. In line, acidification-evoked inward currents were observed only in A549 and WI-38 cells. Gene knockdown showed that the anti-proliferative and anti-migratory activity of mambalgin-2 is dependent on the expression of ASIC1a, α-ENaC, and γ-ENaC. Using affinity extraction and immunoprecipitation, mambalgin-2 targeting of ASIC1a/α-ENaC/γ-ENaC heteromeric channels in A549 cells was shown. Electrophysiology studies in Xenopus oocytes revealed that mambalgin-2 inhibits the ASIC1a/α-ENaC/γ-ENaC channels with higher efficacy than the ASIC1a channels, pointing on the heteromeric channels as a primary target of the toxin in cancer cells. Finally, bioinformatics analysis showed that the increased expression of ASIC1 and γ-ENaC correlates with a worse survival prognosis for patients with lung adenocarcinoma. Thus, the ASIC1a/α-ENaC/γ-ENaC heterotrimer can be considered a marker of cell oncogenicity and its targeting is promising for the design of new selective cancer therapeutics.

Extracellular Vesicles Derived from Acidified Metastatic Melanoma Cells Stimulate Growth, Migration, and Stemness of Normal Keratinocytes.

Metastatic melanoma is a highly malignant tumor. Melanoma cells release extracellular vesicles (EVs), which contribute to the growth, metastasis, and malignancy of neighboring cells by transfer of tumor-promoting miRNAs, mRNA, and proteins. Melanoma microenvironment acidification promotes tumor progression and determines EVs' properties. We studied the influence of EVs derived from metastatic melanoma cells cultivated at acidic (6.5) and normal (7.4) pH on the morphology and homeostasis of normal keratinocytes. Acidification of metastatic melanoma environment made EVs more prooncogenic with increased expression of prooncogenic mi221 RNA, stemless factor CD133, and pro-migration factor SNAI1, as well as with downregulated antitumor mir7 RNA. Incubation with EVs stimulated growth and migration both of metastatic melanoma cells and keratinocytes and changed the morphology of keratinocytes to stem-like phenotype, which was confirmed by increased expression of the stemness factors KLF and CD133. Activation of the AKT/mTOR and ERK signaling pathways and increased expression of epidermal growth factor receptor EGFR and SNAI1 were detected in keratinocytes upon incubation with EVs. Moreover, EVs reduced the production of different cytokines (IL6, IL10, and IL12) and adhesion factors (sICAM-1, sICAM-3, sPecam-1, and sCD40L) usually secreted by keratinocytes to control melanoma progression. Bioinformatic analysis revealed the correlation between decreased expression of these secreted factors and worse survival prognosis for patients with metastatic melanoma. Altogether, our data mean that metastatic melanoma EVs are important players in the transformation of normal keratinocytes.

SLURP-1 Controls Growth and Migration of Lung Adenocarcinoma Cells, Forming a Complex With α7-nAChR and PDGFR/EGFR Heterodimer.

Secreted Ly6/uPAR-related protein 1 (SLURP-1) is a secreted Ly6/uPAR protein that negatively modulates the nicotinic acetylcholine receptor of α7 type (α7-nAChR), participating in control of cancer cell growth. Previously we showed, that a recombinant analogue of human SLURP-1 (rSLURP-1) diminishes the lung adenocarcinoma A549 cell proliferation and abolishes the nicotine-induced growth stimulation. Here, using multiplex immunoassay, we demonstrated a decrease in PTEN and mammalian target of rapamycin (mTOR) kinase phosphorylation in A549 cells upon the rSLURP-1 treatment pointing on down-regulation of the PI3K/AKT/mTOR signaling pathway. Decreased phosphorylation of the platelet-derived growth factor receptor type ß (PDGFRß) and arrest of the A549 cell cycle in the S and G2/M phases without apoptosis induction was also observed. Using a scratch migration assay, inhibition of A549 cell migration under the rSLURP-1 treatment was found. Affinity extraction demonstrated that rSLURP-1 in A549 cells forms a complex not only with α7-nAChR, but also with PDGFRα and epidermal growth factor receptor (EGFR), which are known to be involved in regulation of cancer cell growth and migration and are able to form a heterodimer. Knock-down of the genes encoding α7-nAChR, PDGFRα, and EGFR confirmed the involvement of these receptors in the anti-migration effect of SLURP-1. Thus, SLURP-1 can target the α7-nAChR complexes with PDGFRα and EGFR in the membrane of epithelial cells. Using chimeric proteins with grafted SLURP-1 loops we demonstrated that loop I is the principal active site responsible for the SLURP-1 interaction with α7-nAChR and its antiproliferative effect. Synthetic peptide mimicking the loop I cyclized by a disulfide bond inhibited ACh-evoked current at α7-nAChR, as well as A549 cell proliferation and migration. This synthetic peptide represents a promising prototype of new antitumor drug with the properties close to that of the native SLURP-1 protein.

Mambalgin-2 Inhibits Growth, Migration, and Invasion of Metastatic Melanoma Cells by Targeting the Channels Containing an ASIC1a Subunit Whose Up-Regulation Correlates with Poor Survival Prognosis.

Melanoma is an aggressive cancer characterized by the acidification of the extracellular environment. Here, we showed for the first time that extracellular media acidification increases proliferation, migration, and invasion of patient-derived metastatic melanoma cells and up-regulates cell-surface expression of acid-sensitive channels containing the ASIC1a, α-ENaC, and γ-ENaC subunits. No influence of media acidification on these processes was found in normal keratinocytes. To control metastatic melanoma progression associated with the ASIC1a up-regulation, we proposed the ASIC1a inhibitor, -mambalgin-2 from Dendpoaspis polylepis venom. Recombinant analog of mambalgin-2 cancelled acidification-induced proliferation, migration, and invasion of metastatic melanoma cells, promoted apoptosis, and down-regulated cell-surface expression of prooncogenic factors CD44 and Frizzled 4 and phosphorylation of transcription factor SNAI. Confocal microscopy and affinity purification revealed that mambalgin-2 interacts with heterotrimeric ASIC1a/α-ENaC/γ-ENaC channels on the surface of metastatic melanoma cells. Using the mutant variant of mambalgin-2 with reduced activity toward ASIC1a, we confirmed that the principal molecular target of mambalgin-2 in melanoma cells is the ASIC1a subunit. Bioinformatic analysis confirmed up-regulation of the ASIC1 expression as a marker of poor survival prognosis for patients with metastatic melanoma. Thus, targeting ASIC1a by drugs such as mambalgin-2 could be a promising strategy for metastatic melanoma treatment.

Biochemical Basis of Skin Disease Mal de Meleda: SLURP-1 Mutants Differently Affect Keratinocyte Proliferation and Apoptosis.

Mal de Meleda is an autosomal recessive palmoplantar keratoderma associated with mutations in a gene encoding SLURP-1. SLURP-1 controls growth, differentiation, and apoptosis of keratinocytes by interaction with α7-type nicotinic acetylcholine receptors. SLURP-1 has a three-finger structure with a ß-structural core (head) and three prolonged loops (fingers). To determine the role of SLURP-1 mutations, we produced 22 mutant variants of the protein, including those involved in Mal de Meleda pathogenesis. All mutants except R71H, R71P, T52A, R96P, and L98P were produced in the folded form. SLURP-1 reduces the growth of Het-1A keratinocytes; thus, we studied the influence of the mutations on its antiproliferative activity. Mutations in loops I and III led to the protein inactivation, whereas most mutations in loop II increased SLURP-1 antiproliferative activity. Alanine substitutions of R96 and L98 residues located in the protein head resulted in the appearance of additional pro-apoptotic activity. Our results agree with the diversity of Mal de Meleda phenotypes. Using obtained functional data, the SLURP-1/α7 type nicotinic acetylcholine receptor complex was modeled in silico. Our study provides functional and structural information about the role of the SLURP-1 mutations in Mal de Meleda pathogenesis and predicts SLURP-1 variants, which could drive the disease.

Human secreted protein SLURP-1 abolishes nicotine-induced proliferation, PTEN down-regulation and α7-nAChR expression up-regulation in lung cancer cells.

Human Ly-6/uPAR-related protein-1 (SLURP-1) is an allosteric negative modulator of the α7-type nicotinic acetylcholine receptor (α7-nAChR), one of the key receptors promoting nicotine-induced proliferation of lung cancer cells. Incubation of lung adenocarcinoma A549 cells with recombinant SLURP-1 (rSLURP-1) at concentrations >10 nM resulted in the significant decrease of the cell growth (~70%), while treatment of normal lung-derived WI-38 fibroblasts with rSLURP-1 did not influence the cell proliferation up to 1 µM of the protein. rSLURP-1 fully abolished the nicotine-induced increase of the cell proliferation, down-regulation of the expression of PTEN (the negative regulator of the AKT pathway, controlling the growth, survival, and proliferation of cancer cells), and up-regulation of the α7-nAChR expression in the A549 cells. Using the siRNA against α7-nAChR and inhibitors of different cell-surface receptors, we showed that rSLURP-1 antiproliferative effect in A549 cells is connected with α7-nAChR, epidermal growth factor receptors, and ß-adrenergic receptors. Moreover, we found that downstream effectors of rSLURP-1 are IP3 receptors and the STAT3 transcription factor. Implication of the IP3 receptors and PTEN in the rSLURP-1 antiproliferative activity points on the AKT-mediated signaling pathway. Co-application of rSLURP-1 with gefitinib and bortezomib (currently used anticancer drugs) resulted in an additive suppression of the A549 cells proliferation up to ~44% and 35%, respectively. Thus, rSLURP-1 could be considered a promising prototype of drugs to prevent nicotine-induced pathologies and cancer treatment.

Lynx1 and Aß1-42 bind competitively to multiple nicotinic acetylcholine receptor subtypes.

Lynx1 regulates synaptic plasticity in the brain by regulating nicotinic acetylcholine receptors (nAChRs). It is not known to which extent Lynx1 can bind to endogenous nAChR subunits in the brain or how this interaction is affected by Alzheimer's disease pathology. We apply affinity purification to demonstrate that a water-soluble variant of human Lynx1 (Ws-Lynx1) isolates α3, α4, α5, α6, α7, ß2, and ß4 nAChR subunits from human and rat cortical extracts, and rat midbrain and olfactory bulb extracts, suggesting that Lynx1 forms complexes with multiple nAChR subtypes in the human and rodent brain. Incubation with Ws-Lynx1 decreases nicotine-mediated extracellular signal-regulated kinase phosphorylation in PC12 cells and striatal neurons, indicating that binding of Ws-Lynx1 is sufficient to inhibit signaling downstream of nAChRs. The effect of nicotine in PC12 cells is independent of α7 or α4ß2 nAChRs, suggesting that Lynx1 can affect the function of native non-α7, non-α4ß2 nAChR subtypes. We further show that Lynx1 and oligomeric ß-amyloid1-42 compete for binding to several nAChR subunits, that Ws-Lynx1 prevents ß-amyloid1-42-induced cytotoxicity in cortical neurons, and that cortical Lynx1 levels are decreased in a transgenic mouse model with concomitant ß-amyloid and tau pathology. Our data suggest that Lynx1 binds to multiple nAChR subtypes in the brain and that this interaction might have functional and pathophysiological implications in relation to Alzheimer's disease.

Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor.

SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 µM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

Structural investigation of influenza virus hemagglutinin membrane-anchoring peptide.

Hemagglutinin (HA), the trimeric spike of influenza virus, catalyzes fusion of viral and cellular membranes. We have synthesized the anchoring peptide including the linker, transmembrane region and cytoplasmic tail (HA-TMR-CT) in a cell-free system. Furthermore, to mimic the palmitoylation of three conserved cysteines within the CT, we chemically alkylated HA-TMR-CT using hexadecyl-methanethiosulfonate. While the nuclear magnetic resonance spectroscopy showed pure and refolded peptides, the formation of multiple oligomers of higher order impeded further structural analysis. Circular dichroism spectroscopy of both alkylated and non-alkylated HA-TMR-CT revealed an α-helical secondary structure. No major impact of the fatty acids on the secondary structure was detected.