Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans

Åmand, Helene L.; Rydberg, Hanna; Fornander, Louise; Lincoln, Per; Nordén, Bengt; Esbjörner, Elin K.

doi:10.1016/j.bbamem.2012.06.006

Cited by 120 publications

(112 citation statements)

References 47 publications

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“…heparan sulfate) and other negatively charged/Hbond donor molecules rather than to the lipid membrane (45). Arg-containing peptides have been shown to possess stronger affinity to glycosaminoglycans, compared with Lys counterparts (46,47) …”

Section: Lipidmentioning

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

135

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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: Lipidmentioning

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

135

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“…The cell uptake of the (K/W)9-ASPNA variant, in which all arginine where mutated in lysines, was decreased six fold compared to (R/W)9-ASPNA in PPT/HeLa cells and in four other cell lines, indicating that the arginine residues confer a cell uptake advantage to (R/W)9 conjugates. It has been reported also that a penetratin variant in which all lysine residues were substituted with arginines (PenArg) is internalized more efficiently than penetratin itself [40], [41]. We show that the uptake advantage of (R/W)9-ASPNA conjugate is correlated with reduction in cytoplasmic mRNA target expression.…”

Section: Discussionmentioning

confidence: 53%

“…Moreover, the strong decrease in (R/W)9-ASPNA and (K/W)9-ASPNA cell uptake into CHO-745 cells, in which proteoglycan synthesis is defective, shows that glycosaminoglycans are necessary for cell surface binding and internalization of both conjugates. It has been shown that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts electrostatically with sugars [41]. It is noteworthy that the free uptake studies of CPPs indicate that tryptophan content and backbone spacing within basic peptide also affect uptake efficiency [42], [43].…”

Section: Discussionmentioning

confidence: 99%

Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9

et al. 2014

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Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.

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“…Especially, the guanidinium head group of arginine has been shown to confer more efficient uptake of arginine, possibly caused by formation of bidentite hydrogen bonds with oxo-anions from e.g. sulfated sugars present in the extracellular matrix [50,51]. Predominant cyotosolic and nuclear staining has been observed before with other CPPs or CPP-containing proteins in different cell lines [52][53][54].…”

Section: Discussionmentioning

confidence: 96%

Characterization of a novel cell penetrating peptide derived from human Oct4

et al. 2014

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Background: Oct4 is a transcription factor that plays a major role for the preservation of the pluripotent state in embryonic stem cells as well as for efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) or other progenitors. Protein-based reprogramming methods mainly rely on the addition of a fused cell penetrating peptide. This study describes that Oct4 inherently carries a protein transduction domain, which can translocate into human and mouse cells.

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Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans

Cited by 120 publications

References 47 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

Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9

Characterization of a novel cell penetrating peptide derived from human Oct4

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