Lynx1 and Aβ1–42 bind competitively to multiple nicotinic acetylcholine receptor subtypes

AbstractLynx1 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, β2, and β4 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 α4β2 nAChRs, suggesting that Lynx1 can affect the function of native non-α7, non-α4β2 nAChR subtypes. We further show that Lynx1 and oligomeric β-amyloid1–42 compete for binding to several nAChR subunits, that Ws-Lynx1 prevents β-amyloid1–42–induced cytotoxicity in cortical neurons, and that cortical Lynx1 levels are decreased in a transgenic mouse model with concomitant β-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

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

Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider <i>Phoneutria nigriventer</i>

Phα1β (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, Phα1β administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1β and its 15N-labeled analogue. Spatial structure and dynamics of Phα1β 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 Phα1β 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. Phα1β 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 Phα1β 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 Phα1β as a gating modifier toxin, probably interacting with S1–S4 gating domains from a membrane-bound state

Structural Diversity and Dynamics of Human Three-Finger Proteins Acting on Nicotinic Acetylcholine Receptors

Ly-6/uPAR or three-finger proteins (TFPs) contain a disulfide-stabilized &beta;-structural core and three protruding loops (fingers). In mammals, TFPs have been found in epithelium and the nervous, endocrine, reproductive, and immune systems. Here, using heteronuclear NMR, we determined the three-dimensional (3D) structure and backbone dynamics of the epithelial secreted protein SLURP-1 and soluble domains of GPI-anchored TFPs from the brain (Lynx2, Lypd6, Lypd6b) acting on nicotinic acetylcholine receptors (nAChRs). Results were compared with the data about human TFPs Lynx1 and SLURP-2 and snake &alpha;-neurotoxins WTX and NTII. Two different topologies of the &beta;-structure were revealed: one large antiparallel &beta;-sheet in Lypd6 and Lypd6b, and two &beta;-sheets in other proteins. &alpha;-Helical segments were found in the loops I/III of Lynx2, Lypd6, and Lypd6b. Differences in the surface distribution of charged and hydrophobic groups indicated significant differences in a mode of TFPs/nAChR interactions. TFPs showed significant conformational plasticity: the loops were highly mobile at picosecond-nanosecond timescale, while the &beta;-structural regions demonstrated microsecond-millisecond motions. SLURP-1 had the largest plasticity and characterized by the unordered loops II/III and cis-trans isomerization of the Tyr39-Pro40 bond. In conclusion, plasticity could be an important feature of TFPs adapting their structures for optimal interaction with the different conformational states of nAChRs

New Three-Finger Protein from Starfish Asteria rubens Shares Structure and Pharmacology with Human Brain Neuromodulator Lynx2

Three-finger proteins (TFPs) are small proteins with characteristic three-finger &beta;-structural fold stabilized by the system of conserved disulfide bonds. These proteins have been found in organisms from different taxonomic groups and perform various important regulatory functions or act as components of snake venoms. Recently, four TFPs (Lystars 1&ndash;4) with unknown function were identified in the coelomic fluid proteome of starfish A. rubens. Here we analyzed the genomes of A. rubens and A. planci starfishes and predicted additional five and six proteins containing three-finger domains, respectively. One of them, named Lystar5, is expressed in A. rubens coelomocytes and has sequence homology to the human brain neuromodulator Lynx2. The three-finger structure of Lystar5 close to the structure of Lynx2 was confirmed by NMR. Similar to Lynx2, Lystar5 negatively modulated &alpha;4&beta;2 nicotinic acetylcholine receptors (nAChRs) expressed in X. laevis oocytes. Incubation with Lystar5 decreased the expression of acetylcholine esterase and &alpha;4 and &alpha;7 nAChR subunits in the hippocampal neurons. In summary, for the first time we reported modulator of the cholinergic system in starfish

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

Effect of rSLURP-1 on the growth of Het-1A cells.

<p>(A). Amino acid sequence alignment of human SLURP-1, SLURP-2, ws-Lynx1, and non-conventional toxin WTX from <i>N</i>. <i>kaouthia</i>. Cysteine residues are labeled in gray, and the disulfide linkages are shown; additional <i>N</i>-terminal Met residues are underlined. (B). Influence of rSLURP-1 (diamonds) and ws-Lynx1 (squares) on cell growth. Each point is mean ± S.E. of six independent experiments. The Hill equation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149733#pone.0149733.e001" target="_blank">Eq 1</a>) was fitted to rSLURP-1 data (% of control) for each of the six experiments independently. After averaging the following values for EC₅₀, nH and A1 were obtained 4.3 ± 0.6 nM, 1.4 ± 0.2 and 59.5 ± 1.3% (mean ± S.E., n = 6). (C). Effects of rSLURP-1 (1 μM), atropine (1 μM), Mec (10 μM), α-Bgtx (1 μM), polyclonal antibodies against α7 (IgG-α7, 1 μg per 50 μl), and their co-application. Each bar is mean ± S.E. of 4–6 independent experiments. ** and *** indicate significant (p<0.01 and p<0.001, t-test) differences.</p