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Spontaneous membrane-translocating peptides: influence of peptide self-aggregation and cargo polarity.

Macchi S, Signore G, Boccardi C, Di Rienzo C, Beltram F, Cardarelli F - Sci Rep (2015)

Bottom Line: We unveil TM9 ability to self-aggregate in a concentration-dependent manner and demonstrate that peptide self-aggregation is a necessary--yet not sufficient--step for effective membrane translocation.These findings are discussed and compared to previous reports.The present results impose a careful rethinking of this class of sequences as direct-translocation vectors suitable for delivery purposes.

View Article: PubMed Central - PubMed

Affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12-56127 Pisa, Italy.

ABSTRACT
Peptides that translocate spontaneously across cell membranes could transform the field of drug delivery by enabling the transport of otherwise membrane-impermeant molecules into cells. In this regard, a 9-aminoacid-long motif (representative sequence: PLIYLRLLR, hereafter Translocating Motif 9, TM9) that spontaneously translocates across membranes while carrying a polar dye was recently identified by high-throughput screening. Here we investigate its transport properties by a combination of in cuvette physico-chemical assays, rational mutagenesis, live-cell confocal imaging and fluorescence correlation spectroscopy measurements. We unveil TM9 ability to self-aggregate in a concentration-dependent manner and demonstrate that peptide self-aggregation is a necessary--yet not sufficient--step for effective membrane translocation. Furthermore we show that membrane crossing can occur with apolar payloads while it is completely inhibited by polar ones. These findings are discussed and compared to previous reports. The present results impose a careful rethinking of this class of sequences as direct-translocation vectors suitable for delivery purposes.

No MeSH data available.


Related in: MedlinePlus

SMTP general structure, TP2 and derived TM9 peptide sequences, fluorophores studied here and cell uptake of dye-labeled TM9.(a) SMTP structural composition, TP2 precursor sequence as representative example and TM9 peptide sequence with ATTO 425 and TAMRA structures. (b) Confocal images of cells treated with increasing concentrations of TM9-ATTO 425 and TM9-TAMRA in free serum medium. Concerning the former, endocytic bright spots are detectable at 0.5 and 3 μM, while direct translocation is prevalent at 5 and 10 μM. Concerning the latter, only endocytosis is present at all the concentrations tested. Scale bars: 10 μm. (c) Confocal images of cells treated with 3 μM (left) and 10 μM (right) of TM9-ATTO 425 at 4 °C. While in the former case only cell plasma membranes are labelled (i.e. endocytosis is blocked), in the latter one diffuse cytoplasmic staining is still evident (i.e. direct translocation occurs).
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f1: SMTP general structure, TP2 and derived TM9 peptide sequences, fluorophores studied here and cell uptake of dye-labeled TM9.(a) SMTP structural composition, TP2 precursor sequence as representative example and TM9 peptide sequence with ATTO 425 and TAMRA structures. (b) Confocal images of cells treated with increasing concentrations of TM9-ATTO 425 and TM9-TAMRA in free serum medium. Concerning the former, endocytic bright spots are detectable at 0.5 and 3 μM, while direct translocation is prevalent at 5 and 10 μM. Concerning the latter, only endocytosis is present at all the concentrations tested. Scale bars: 10 μm. (c) Confocal images of cells treated with 3 μM (left) and 10 μM (right) of TM9-ATTO 425 at 4 °C. While in the former case only cell plasma membranes are labelled (i.e. endocytosis is blocked), in the latter one diffuse cytoplasmic staining is still evident (i.e. direct translocation occurs).

Mentions: Marks and co-workers recently introduced a straightforward combinatorial peptide chemistry and orthogonal high-throughput screening to select peptides that spontaneously translocate across synthetic lipid bilayer membranes without permeabilization effects, while carrying a small polar cargo (TAMRA)7. A family of Spontaneous Membrane Translocating Peptide (SMTP) sequences with the general structure reported in Fig. 1a was identified. In particular, the first 9 residues represent the screened segment, with putative translocating properties (example: 1PLIYLRLLR9, as in Fig. 1a). A Glycine in position 10 constitutes a flexible linker, while Glutamate in position 11 and Phenylalanine in position 12 are functional to the in vitro test of membrane permeability used7. A C-terminal Cysteine residue was also added for labeling purposes. Following this general strategy, and in order to better evaluate the properties of the putative 9-aa-long translocating motif, we removed the Glutamate and Phenylalanine residues, while preserving the Glycine as a flexible linker and Cysteine to chemically attach a cargo moiety (overall sequence: PLIYLRLLR-G-C, hereafter named Translocating Motif 9, TM9). The purified peptide was labeled on the Cys residue with either ATTO 425 (as model of apolar cargo) or TAMRA (as a model of polar cargo, in analogy with previous studies71314) (Fig. 1a). Labeled peptides were tested on live CHO-K1 cells by confocal fluorescence microscopy. As a first test, we performed experiments on cells exposed to increasing concentrations of either TM9-ATTO 425 or TM9-TAMRA. As detailed in the Experimental Procedures section, purified dye-labeled peptides were pre-dissolved in water solution, sonicated, quantified by UV-Vis absorbance, and added to the cell medium at the desired concentration (in both cases, fluorescent peptides are incubated with cells for 20 minutes before washing). As reported in Fig. 1b, we observed that for TM9-ATTO 425 concentrations up to 3 μM only fluorescent vesicles (bright spots in Fig. 1b-0.5 and 3 μM) are detected, indicating that endocytosis is the dominant mechanism responsible of peptide uptake. On the contrary, an intense and diffuse fluorescence signal is present at higher peptide concentrations (e.g. 5 and 10 μM), suggesting that a vesicle-independent direct translocation process across the plasma membrane becomes dominant in this regime (Fig. 1b-5 and 10 μM). The observed fluorescence staining (labeled cytoplasm and apparently dark nucleus) is consistent with the peculiar spectral properties of ATTO 425 (Fig. S1): just like many coumarin derivatives18, in fact, this fluorophore markedly increases its fluorescence quantum yield in apolar, lipophilic environments of the cell (cytoplasm) compared to aqueous environments (nucleus and surrounding medium). By contrast, under the same experimental conditions, TM9-TAMRA clearly shows punctuate fluorescence throughout the complete range of tested concentrations, with no apparent direct translocation into the intracellular medium (Fig. 1b). To reinforce our conclusion that the massive cytoplasmic staining observed for TM9-ATTO 425 at high concentrations depends on direct plasma membrane translocation we performed an uptake experiment at 4 °C, a well-known strategy to block active endocytosis while preserving direct permeation5. As can be seen in Fig. 1c, endocytosis of TM9-ATTO 425 at 3 μM is almost completely inhibited (i.e. only accumulation of the peptide at the level of the plasma membrane can be detected, Fig. 1c, left panels). On the contrary, under the same experimental conditions, TM9-ATTO 425 at 10 μM still shows diffuse cytoplasmic staining, thus corroborating the hypothesis of a temperature-independent direct translocation process across the membrane. To exclude any possible toxic effect of the labeled peptides on cell viability, we incubated cells for 2 hours in presence of varying peptide concentrations and performed the standard WST-8 assay 24 hours after removal of the peptide solution (Fig. S2).


Spontaneous membrane-translocating peptides: influence of peptide self-aggregation and cargo polarity.

Macchi S, Signore G, Boccardi C, Di Rienzo C, Beltram F, Cardarelli F - Sci Rep (2015)

SMTP general structure, TP2 and derived TM9 peptide sequences, fluorophores studied here and cell uptake of dye-labeled TM9.(a) SMTP structural composition, TP2 precursor sequence as representative example and TM9 peptide sequence with ATTO 425 and TAMRA structures. (b) Confocal images of cells treated with increasing concentrations of TM9-ATTO 425 and TM9-TAMRA in free serum medium. Concerning the former, endocytic bright spots are detectable at 0.5 and 3 μM, while direct translocation is prevalent at 5 and 10 μM. Concerning the latter, only endocytosis is present at all the concentrations tested. Scale bars: 10 μm. (c) Confocal images of cells treated with 3 μM (left) and 10 μM (right) of TM9-ATTO 425 at 4 °C. While in the former case only cell plasma membranes are labelled (i.e. endocytosis is blocked), in the latter one diffuse cytoplasmic staining is still evident (i.e. direct translocation occurs).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4645181&req=5

f1: SMTP general structure, TP2 and derived TM9 peptide sequences, fluorophores studied here and cell uptake of dye-labeled TM9.(a) SMTP structural composition, TP2 precursor sequence as representative example and TM9 peptide sequence with ATTO 425 and TAMRA structures. (b) Confocal images of cells treated with increasing concentrations of TM9-ATTO 425 and TM9-TAMRA in free serum medium. Concerning the former, endocytic bright spots are detectable at 0.5 and 3 μM, while direct translocation is prevalent at 5 and 10 μM. Concerning the latter, only endocytosis is present at all the concentrations tested. Scale bars: 10 μm. (c) Confocal images of cells treated with 3 μM (left) and 10 μM (right) of TM9-ATTO 425 at 4 °C. While in the former case only cell plasma membranes are labelled (i.e. endocytosis is blocked), in the latter one diffuse cytoplasmic staining is still evident (i.e. direct translocation occurs).
Mentions: Marks and co-workers recently introduced a straightforward combinatorial peptide chemistry and orthogonal high-throughput screening to select peptides that spontaneously translocate across synthetic lipid bilayer membranes without permeabilization effects, while carrying a small polar cargo (TAMRA)7. A family of Spontaneous Membrane Translocating Peptide (SMTP) sequences with the general structure reported in Fig. 1a was identified. In particular, the first 9 residues represent the screened segment, with putative translocating properties (example: 1PLIYLRLLR9, as in Fig. 1a). A Glycine in position 10 constitutes a flexible linker, while Glutamate in position 11 and Phenylalanine in position 12 are functional to the in vitro test of membrane permeability used7. A C-terminal Cysteine residue was also added for labeling purposes. Following this general strategy, and in order to better evaluate the properties of the putative 9-aa-long translocating motif, we removed the Glutamate and Phenylalanine residues, while preserving the Glycine as a flexible linker and Cysteine to chemically attach a cargo moiety (overall sequence: PLIYLRLLR-G-C, hereafter named Translocating Motif 9, TM9). The purified peptide was labeled on the Cys residue with either ATTO 425 (as model of apolar cargo) or TAMRA (as a model of polar cargo, in analogy with previous studies71314) (Fig. 1a). Labeled peptides were tested on live CHO-K1 cells by confocal fluorescence microscopy. As a first test, we performed experiments on cells exposed to increasing concentrations of either TM9-ATTO 425 or TM9-TAMRA. As detailed in the Experimental Procedures section, purified dye-labeled peptides were pre-dissolved in water solution, sonicated, quantified by UV-Vis absorbance, and added to the cell medium at the desired concentration (in both cases, fluorescent peptides are incubated with cells for 20 minutes before washing). As reported in Fig. 1b, we observed that for TM9-ATTO 425 concentrations up to 3 μM only fluorescent vesicles (bright spots in Fig. 1b-0.5 and 3 μM) are detected, indicating that endocytosis is the dominant mechanism responsible of peptide uptake. On the contrary, an intense and diffuse fluorescence signal is present at higher peptide concentrations (e.g. 5 and 10 μM), suggesting that a vesicle-independent direct translocation process across the plasma membrane becomes dominant in this regime (Fig. 1b-5 and 10 μM). The observed fluorescence staining (labeled cytoplasm and apparently dark nucleus) is consistent with the peculiar spectral properties of ATTO 425 (Fig. S1): just like many coumarin derivatives18, in fact, this fluorophore markedly increases its fluorescence quantum yield in apolar, lipophilic environments of the cell (cytoplasm) compared to aqueous environments (nucleus and surrounding medium). By contrast, under the same experimental conditions, TM9-TAMRA clearly shows punctuate fluorescence throughout the complete range of tested concentrations, with no apparent direct translocation into the intracellular medium (Fig. 1b). To reinforce our conclusion that the massive cytoplasmic staining observed for TM9-ATTO 425 at high concentrations depends on direct plasma membrane translocation we performed an uptake experiment at 4 °C, a well-known strategy to block active endocytosis while preserving direct permeation5. As can be seen in Fig. 1c, endocytosis of TM9-ATTO 425 at 3 μM is almost completely inhibited (i.e. only accumulation of the peptide at the level of the plasma membrane can be detected, Fig. 1c, left panels). On the contrary, under the same experimental conditions, TM9-ATTO 425 at 10 μM still shows diffuse cytoplasmic staining, thus corroborating the hypothesis of a temperature-independent direct translocation process across the membrane. To exclude any possible toxic effect of the labeled peptides on cell viability, we incubated cells for 2 hours in presence of varying peptide concentrations and performed the standard WST-8 assay 24 hours after removal of the peptide solution (Fig. S2).

Bottom Line: We unveil TM9 ability to self-aggregate in a concentration-dependent manner and demonstrate that peptide self-aggregation is a necessary--yet not sufficient--step for effective membrane translocation.These findings are discussed and compared to previous reports.The present results impose a careful rethinking of this class of sequences as direct-translocation vectors suitable for delivery purposes.

View Article: PubMed Central - PubMed

Affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12-56127 Pisa, Italy.

ABSTRACT
Peptides that translocate spontaneously across cell membranes could transform the field of drug delivery by enabling the transport of otherwise membrane-impermeant molecules into cells. In this regard, a 9-aminoacid-long motif (representative sequence: PLIYLRLLR, hereafter Translocating Motif 9, TM9) that spontaneously translocates across membranes while carrying a polar dye was recently identified by high-throughput screening. Here we investigate its transport properties by a combination of in cuvette physico-chemical assays, rational mutagenesis, live-cell confocal imaging and fluorescence correlation spectroscopy measurements. We unveil TM9 ability to self-aggregate in a concentration-dependent manner and demonstrate that peptide self-aggregation is a necessary--yet not sufficient--step for effective membrane translocation. Furthermore we show that membrane crossing can occur with apolar payloads while it is completely inhibited by polar ones. These findings are discussed and compared to previous reports. The present results impose a careful rethinking of this class of sequences as direct-translocation vectors suitable for delivery purposes.

No MeSH data available.


Related in: MedlinePlus