The Prototypic Cyclotide Kalata B1 Has a Unique Mechanism of Entering Cells

Henriques, Sónia Troeira; Huang, Yen‐Hua; Chaousis, Stephanie; Sani, Marc‐Antoine; Poth, Aaron G.; Separovic, Frances; Craik, David J.

doi:10.1016/j.chembiol.2015.07.012

Cited by 73 publications

(112 citation statements)

References 54 publications

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“…SUVs made with POPC, POPE, and NBD-dioleoylglycerophosphoethanolamine (NBD-DOPE) (POPC/POPE/NBD-DOPE (3:1:1 molar ratio)) were dispersed in PSS and prepared by extrusion as described above. NBDlabeled phospholipids were incorporated into cells by incubating POPC/POPE/NBD-DOPE SUVs (final NBD-DOPE concentration was 150 M) for 30 min at 37°C (72). Phospholipids with NBD label in the headgroup cannot undergo membrane flip-flop (73).…”

Section: Membrane Dipolar Potential Changes Induced In Luvs-mentioning

confidence: 99%

Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes Is a Prerequisite for Its Inhibition of Human Voltage-gated Sodium Channel NaV1.7

Henriques

Deplazes

Lawrence

et al. 2016

Journal of Biological Chemistry

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132

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ProTx-II is a disulfide-rich peptide toxin from tarantula venom able to inhibit the human voltage-gated sodium channel 1.7 (hNa V 1.7), a channel reported to be involved in nociception, and thus it might have potential as a pain therapeutic. ProTx-II acts by binding to the membrane-embedded voltage sensor domain of hNa V 1.7, but the precise peptide channel-binding site and the importance of membrane binding on the inhibitory activity of ProTx-II remain unknown. In this study, we examined the structure and membrane-binding properties of ProTx-II and several analogues using NMR spectroscopy, surface plasmon resonance, fluorescence spectroscopy, and molecular dynamics simulations. Our results show a direct correlation between ProTx-II membrane binding affinity and its potency as an hNa V 1.7 channel inhibitor. The data support a model whereby a hydrophobic patch on the ProTx-II surface anchors the molecule at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. This is the first study to demonstrate that binding of ProTx-II to the lipid membrane is directly linked to its potency as an hNa V 1.7 channel inhibitor.Voltage-gated ion channels (VGICs) 4 are transmembrane proteins responsible for voltage-dependent movement of ions across cell membranes. They are involved in a wide range of physiological processes, including action potential generation in excitable cells, muscle and nerve relaxation, regulation of blood pressure, and sensory transduction. Many disorders are associated with VGIC abnormalities, and hence VGICs are actively pursued as drug targets for the treatment of a range of neuromuscular, neurological, or inflammatory disorders (1-3). For instance, the human voltage-gated sodium channel subtype 1.7 (hNa V 1.7) is involved in pain sensation, and molecules that selectively inhibit this channel might therefore be useful as leads for the development of novel analgesics (4). However, high sequence identity and structural homology exist between the nine subtypes of voltage-gated sodium channels, and given the critical role of Na V channels in the normal electrical activity of neurons, skeletal muscles, and cardiomyocytes, subtype selectivity is crucial but often difficult to achieve with small molecule inhibitors (2).Disulfide-rich peptide toxins isolated from animal venoms (e.g. spiders, snakes, and cone snails) have attracted much attention as potential analgesics because of their well defined three-dimensional structure and ability to inhibit voltage-gated sodium (Na V ), potassium (K V ), and calcium (Ca V ) ion channels with high potency and selectivity (5-9). Because of their stability, selectivity, and potency, these disulfide-rich peptides have been extensively characterized and are vigorously being pursued as drug leads as well as pharmacological tools to characterize VGICs (5).In general, peptide toxins that inhibit VGICs can be divided into two main groups based on their inhibitory strategy as fol-

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Section: Membrane Dipolar Potential Changes Induced In Luvs-mentioning

confidence: 99%

Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes Is a Prerequisite for Its Inhibition of Human Voltage-gated Sodium Channel NaV1.7

Henriques

Deplazes

Lawrence

et al. 2016

Journal of Biological Chemistry

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132

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“…After the treatment with fluorescent peptides, the percentage of the fluorescent cells is similar before and after the addition of trypan blue, with virtually all the cells having peptide internalized (TAT‐Alexa and kB1‐T20K‐Alexa examples in Figure A). This suggests that the cells are viable and the fluorescent peptides were internalized by cells . The internalization rate of kB1‐T20K‐Alexa and kL4‐N29K‐Alexa is comparable to that of the gold standard TAT‐Alexa and ∼5‐fold higher than that of TAT‐G‐Alexa.…”

Section: Resultsmentioning

confidence: 87%

Structural and functional characterization of chimeric cyclotides from the Möbius and trypsin inhibitor subfamilies

et al. 2017

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Cyclotides are plant-derived host defense peptides displaying exceptional stability due to their cyclic cystine knot comprising three intertwined disulfide bonds and a cyclic backbone. Their six conserved cysteine residues are separated by backbone loops with diverse sequences. Prototypical cyclotides from the Möbius (kalata B1) and trypsin inhibitor (MCoTI-II) subfamilies lack sequence homology with one another, but both are able to penetrate cells, apparently via different mechanisms. To delineate the influence of the sequences of the loops on the structure and cell internalization of these two cyclotide subfamilies, a series of Möbius/trypsin inhibitor loop-chimeras of kalata B1 and MCoTI-II were synthesized, and structurally and functionally characterized. NMR analysis showed that the structural fold of the majority of chimeric peptides was minimally affected by the loop substitutions. Substituting loops 3, 5, or 6 of MCoTI-II into the corresponding loops of kalata B1 attenuated its hemolytic and cytotoxic activities, and greatly reduced its cell-penetrating properties. On the other hand, replacing loops of MCoTI-II with the corresponding loops of kalata B1 did not introduce cytotoxicity into the chimeras. Loops 2, 3, and 4 of MCoTI-II were found to contribute little to cell-penetrating properties. Overall, this study provides valuable insights into the structural basis for the hemolytic, cytotoxic, and cell-penetrating properties of kalata B1 and MCoTI-II, which could be useful for future engineering of cyclotides to carry bioactive epitopes to intracellular targets.

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“…In this pH range the conformational change in the structure of kB1 is minimal, 27 and earlier work has indicated that pH has little effect on the mass loading of kB1 to PE within supported membranes in the range of pH 5.5 -7.4. 28 However, a recent study has shown the membrane organisation is strongly dependent on pH over the same range. 20 At lower pH the membrane is more ordered with a significantly reduced Qm suggesting an increased inter-molecular attraction of the lipids at lower pH is modifying the interaction of kB1 with the PE population of the lipid bilayer.…”

Section: Resultsmentioning

confidence: 99%

Kalata B1 and Kalata B2 Have a Surfactant-Like Activity in Phosphatidylethanolomine-Containing Lipid Membranes

et al. 2017

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Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2) on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores, but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behaviour is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore, but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.

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The Prototypic Cyclotide Kalata B1 Has a Unique Mechanism of Entering Cells

Cited by 73 publications

References 54 publications

Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes Is a Prerequisite for Its Inhibition of Human Voltage-gated Sodium Channel NaV1.7

Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes Is a Prerequisite for Its Inhibition of Human Voltage-gated Sodium Channel NaV1.7

Structural and functional characterization of chimeric cyclotides from the Möbius and trypsin inhibitor subfamilies

Kalata B1 and Kalata B2 Have a Surfactant-Like Activity in Phosphatidylethanolomine-Containing Lipid Membranes

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