9 research outputs found
Binding of cell-penetrating penetratin peptides to plasma membrane vesicles correlates directly with cellular uptake
AbstractCell-penetrating peptides (CPPs) gain access to intracellular compartments mainly via endocytosis and have capacity to deliver macromolecular cargo into cells. Although the involvement of various endocytic routes has been described it is still unclear which interactions are involved in eliciting an uptake response and to what extent affinity for particular cell surface components may determine the efficiency of a particular CPP. Previous biophysical studies of the interaction between CPPs and either lipid vesicles or soluble sugar-mimics of cell surface proteoglycans, the two most commonly suggested CPP binding targets, have not allowed quantitative correlations to be established. We here explore the use of plasma membrane vesicles (PMVs) derived from cultured mammalian cells as cell surface models in biophysical experiments. Further, we examine the relationship between affinity for PMVs and uptake into live cells using the CPP penetratin and two analogs enriched in arginines and lysines respectively. We show, using centrifugation to sediment PMVs, that the amount of peptide in the pellet fraction correlates linearly with the degree of cell internalization and that the relative efficiency of all-arginine and all-lysine variants of penetratin can be ascribed to their respective cell surface affinities. Our data show differences between arginine- and lysine-rich variants of penetratin that has not been previously accounted for in studies using lipid vesicles. Our data also indicate greater differences in binding affinity to PMVs than to heparin, a commonly used cell surface proteoglycan mimic. Taken together, this suggests that the cell surface interactions of CPPs are dependent on several cell surface moieties and their molecular organization on the plasma membrane
Effects of chirality on the intracellular localization of binuclear ruthenium(II) polypyridyl complexes
Interest in binuclear ruthenium(II) polypyridyl complexes as luminescent cellular imaging agents and for biomedical applications is increasing rapidly. We have investigated the cellular localization, uptake, and biomolecular interactions of the pure enantiomers of two structural isomers of [μ-bipb(phen)4Ru2]4+ (bipb is bis(imidazo[4,5-f]-1,10-phenanthrolin-2-yl)benzene and phen is 1,10-phenanthroline) using confocal laser scanning microscopy, emission spectroscopy, and linear dichroism. Both complexes display distinct enantiomeric differences in the staining pattern of fixed cells, which are concluded to arise from chiral discrimination in the binding to intracellular components. Uptake of complexes in live cells is efficient and nontoxic at 5 μM, and occurs through an energy-dependent mechanism. No differences in uptake are observed between the structural isomers or the enantiomers, suggesting that the interactions triggering uptake are rather insensitive to structural variations. Altogether, these findings show that the complexes investigated are promising for future applications as cellular imaging probes. In addition, linear dichroism shows that the complexes exhibit DNA-condensing properties, making them interesting as potential gene delivery vectors
Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides
Cell-penetrating peptides (CPPs) are able to traverse
cellular
membranes and deliver macromolecular cargo. Uptake occurs through
both endocytotic and nonendocytotic pathways, but the molecular requirements
for efficient internalization are not fully understood. Here we investigate
how the presence of tryptophans and their position within an oligoarginine
influence uptake mechanism and efficiency. Flow cytometry and confocal
fluorescence imaging are used to estimate uptake efficiency, intracellular
distribution and toxicity in Chinese hamster ovarian cells. Further,
membrane leakage and lipid membrane affinity are investigated. The
peptides contain eight arginine residues and one to four tryptophans,
the tryptophans positioned either at the N-terminus, in the middle,
or evenly distributed along the amino acid sequence. Our data show
that the intracellular distribution varies among peptides with different
tryptophan content and backbone spacing. Uptake efficiency is higher
for the peptides with four tryptophans in the middle, or evenly distributed
along the peptide sequence, than for the peptide with four tryptophans
at the N-terminus. All peptides display low cytotoxicity except for
the one with four tryptophans at the N-terminus, which was moderately
toxic. This finding is consistent with their inability to induce efficient
leakage of dye from lipid vesicles. All peptides have comparable affinities
for lipid vesicles, showing that lipid binding is not a decisive parameter
for uptake. Our results indicate that tryptophan content and backbone
spacing can affect both the CPP uptake efficiency and the CPP uptake
mechanism. The low cytotoxicity of these peptides and the possibilities
of tuning their uptake mechanism are interesting from a therapeutic
point of view
Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides
Cell-penetrating peptides (CPPs) are able to traverse
cellular
membranes and deliver macromolecular cargo. Uptake occurs through
both endocytotic and nonendocytotic pathways, but the molecular requirements
for efficient internalization are not fully understood. Here we investigate
how the presence of tryptophans and their position within an oligoarginine
influence uptake mechanism and efficiency. Flow cytometry and confocal
fluorescence imaging are used to estimate uptake efficiency, intracellular
distribution and toxicity in Chinese hamster ovarian cells. Further,
membrane leakage and lipid membrane affinity are investigated. The
peptides contain eight arginine residues and one to four tryptophans,
the tryptophans positioned either at the N-terminus, in the middle,
or evenly distributed along the amino acid sequence. Our data show
that the intracellular distribution varies among peptides with different
tryptophan content and backbone spacing. Uptake efficiency is higher
for the peptides with four tryptophans in the middle, or evenly distributed
along the peptide sequence, than for the peptide with four tryptophans
at the N-terminus. All peptides display low cytotoxicity except for
the one with four tryptophans at the N-terminus, which was moderately
toxic. This finding is consistent with their inability to induce efficient
leakage of dye from lipid vesicles. All peptides have comparable affinities
for lipid vesicles, showing that lipid binding is not a decisive parameter
for uptake. Our results indicate that tryptophan content and backbone
spacing can affect both the CPP uptake efficiency and the CPP uptake
mechanism. The low cytotoxicity of these peptides and the possibilities
of tuning their uptake mechanism are interesting from a therapeutic
point of view
Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides
Cell-penetrating peptides (CPPs) are able to traverse
cellular
membranes and deliver macromolecular cargo. Uptake occurs through
both endocytotic and nonendocytotic pathways, but the molecular requirements
for efficient internalization are not fully understood. Here we investigate
how the presence of tryptophans and their position within an oligoarginine
influence uptake mechanism and efficiency. Flow cytometry and confocal
fluorescence imaging are used to estimate uptake efficiency, intracellular
distribution and toxicity in Chinese hamster ovarian cells. Further,
membrane leakage and lipid membrane affinity are investigated. The
peptides contain eight arginine residues and one to four tryptophans,
the tryptophans positioned either at the N-terminus, in the middle,
or evenly distributed along the amino acid sequence. Our data show
that the intracellular distribution varies among peptides with different
tryptophan content and backbone spacing. Uptake efficiency is higher
for the peptides with four tryptophans in the middle, or evenly distributed
along the peptide sequence, than for the peptide with four tryptophans
at the N-terminus. All peptides display low cytotoxicity except for
the one with four tryptophans at the N-terminus, which was moderately
toxic. This finding is consistent with their inability to induce efficient
leakage of dye from lipid vesicles. All peptides have comparable affinities
for lipid vesicles, showing that lipid binding is not a decisive parameter
for uptake. Our results indicate that tryptophan content and backbone
spacing can affect both the CPP uptake efficiency and the CPP uptake
mechanism. The low cytotoxicity of these peptides and the possibilities
of tuning their uptake mechanism are interesting from a therapeutic
point of view
Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides
Cell-penetrating peptides (CPPs) are able to traverse
cellular
membranes and deliver macromolecular cargo. Uptake occurs through
both endocytotic and nonendocytotic pathways, but the molecular requirements
for efficient internalization are not fully understood. Here we investigate
how the presence of tryptophans and their position within an oligoarginine
influence uptake mechanism and efficiency. Flow cytometry and confocal
fluorescence imaging are used to estimate uptake efficiency, intracellular
distribution and toxicity in Chinese hamster ovarian cells. Further,
membrane leakage and lipid membrane affinity are investigated. The
peptides contain eight arginine residues and one to four tryptophans,
the tryptophans positioned either at the N-terminus, in the middle,
or evenly distributed along the amino acid sequence. Our data show
that the intracellular distribution varies among peptides with different
tryptophan content and backbone spacing. Uptake efficiency is higher
for the peptides with four tryptophans in the middle, or evenly distributed
along the peptide sequence, than for the peptide with four tryptophans
at the N-terminus. All peptides display low cytotoxicity except for
the one with four tryptophans at the N-terminus, which was moderately
toxic. This finding is consistent with their inability to induce efficient
leakage of dye from lipid vesicles. All peptides have comparable affinities
for lipid vesicles, showing that lipid binding is not a decisive parameter
for uptake. Our results indicate that tryptophan content and backbone
spacing can affect both the CPP uptake efficiency and the CPP uptake
mechanism. The low cytotoxicity of these peptides and the possibilities
of tuning their uptake mechanism are interesting from a therapeutic
point of view
Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans
Characterization of a novel cell penetrating peptide derived from human Oct4
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.
Results:
A 16 amino acid peptide representing the third helix of the human Oct4 homeodomain, referred to as Oct4 protein transduction domain (Oct4-PTD), can internalize in mammalian cells upon conjugation to a fluorescence moiety thereby acting as a cell penetrating peptide (CPP). The cellular distribution of Oct4-PTD shows diffuse cytosolic and nuclear staining, whereas penetratin is strictly localized to a punctuate pattern in the cytoplasm. By using a Cre/loxP-based reporter system, we show that this peptide also drives translocation of a functionally active Oct4-PTD-Cre-fusion protein. We further provide evidence for translocation of full length Oct4 into human and mouse cell lines without the addition of any kind of cationic fusion tag. Finally, physico-chemical properties of the novel CPP are characterized, showing that in contrast to penetratin a helical structure of Oct4-PTD is only observed if the FITC label is present on the N-terminus of the peptide.
Conclusions:
Oct4 is a key transcription factor in stem cell research and cellular reprogramming. Since it has been shown that recombinant Oct4 fused to a cationic fusion tag can drive generation of iPSCs, our finding might contribute to further development of protein-based methods to generate iPSCs.
Moreover, our data support the idea that transcription factors might be part of an alternative paracrine signalling pathway, where the proteins are transferred to neighbouring cells thereby actively changing the behaviour of the recipient cell.(VLID)90690