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Characterization of a novel cell penetrating peptide derived from human Oct4.

Harreither E, Rydberg HA, Amand HL, Jadhav V, Fliedl L, Benda C, Esteban MA, Pei D, Borth N, Grillari-Voglauer R, Hommerding O, Edenhofer F, Nordén B, Grillari J - Cell Regen (Lond) (2014)

Bottom Line: 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.Oct4 is a key transcription factor in stem cell research and cellular reprogramming.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Conformational changes upon binding to LUVs. Circular dichroism spectra of 4 μM (A) FITC-labelled and (B) unlabelled Oct4-PTD (gray) and penetratin (black). Solid lines represent peptides in the presence of 0.4 mM POPG/POPC (80/20) LUVs, dotted lines show free peptide in solution. Measurements were performed in 10 mM phosphate buffer, pH 7.4. (C) LD spectra of penetratin (gray) and Oct4-PTD (black). Unlabelled peptides are represented as solid lines, FITC-labelled peptides as dashed lines.
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Fig5: Conformational changes upon binding to LUVs. Circular dichroism spectra of 4 μM (A) FITC-labelled and (B) unlabelled Oct4-PTD (gray) and penetratin (black). Solid lines represent peptides in the presence of 0.4 mM POPG/POPC (80/20) LUVs, dotted lines show free peptide in solution. Measurements were performed in 10 mM phosphate buffer, pH 7.4. (C) LD spectra of penetratin (gray) and Oct4-PTD (black). Unlabelled peptides are represented as solid lines, FITC-labelled peptides as dashed lines.

Mentions: CPPs can fold into distinct secondary structures, including α-helixes and random coils, upon interaction with lipid membranes. The resulting structures interact more or less strongly with the plasma membrane, leading to differences in uptake efficiency and cellular distribution [14]. Therefore, circular dichroism (CD) was measured to evaluate secondary structures of penetratin and Oct4-PTD, with and without fluorophore, in buffer and when bound to lipid vesicles. Both FITC-labelled peptides show a random coil structure in buffer. When bound to lipid vesicles, FITC-labelled penetratin and ditto Oct4-PTD show an evident α-helical signal, with a positive peak at 195 nm, and two negative peaks at 208 nm and 223 nm respectively (Figure 5A). If instead looking at the unlabelled peptides (Figure 5B), penetratin adopts an even more pronounced α-helix when bound to liposomes and an evident random coil when free in solution. However, the unlabeled Oct4-PTD shows an extremely weak, seemingly absent CD signal, indicating an unordered structure. The measurement was repeated with a three times higher peptide concentration, still rendering a very weak signal. The results show that a fluorescent label may induce structural changes in peptides, affecting the secondary structure of the peptide, both when free in solution or bound to lipid membranes.Figure 5


Characterization of a novel cell penetrating peptide derived from human Oct4.

Harreither E, Rydberg HA, Amand HL, Jadhav V, Fliedl L, Benda C, Esteban MA, Pei D, Borth N, Grillari-Voglauer R, Hommerding O, Edenhofer F, Nordén B, Grillari J - Cell Regen (Lond) (2014)

Conformational changes upon binding to LUVs. Circular dichroism spectra of 4 μM (A) FITC-labelled and (B) unlabelled Oct4-PTD (gray) and penetratin (black). Solid lines represent peptides in the presence of 0.4 mM POPG/POPC (80/20) LUVs, dotted lines show free peptide in solution. Measurements were performed in 10 mM phosphate buffer, pH 7.4. (C) LD spectra of penetratin (gray) and Oct4-PTD (black). Unlabelled peptides are represented as solid lines, FITC-labelled peptides as dashed lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230757&req=5

Fig5: Conformational changes upon binding to LUVs. Circular dichroism spectra of 4 μM (A) FITC-labelled and (B) unlabelled Oct4-PTD (gray) and penetratin (black). Solid lines represent peptides in the presence of 0.4 mM POPG/POPC (80/20) LUVs, dotted lines show free peptide in solution. Measurements were performed in 10 mM phosphate buffer, pH 7.4. (C) LD spectra of penetratin (gray) and Oct4-PTD (black). Unlabelled peptides are represented as solid lines, FITC-labelled peptides as dashed lines.
Mentions: CPPs can fold into distinct secondary structures, including α-helixes and random coils, upon interaction with lipid membranes. The resulting structures interact more or less strongly with the plasma membrane, leading to differences in uptake efficiency and cellular distribution [14]. Therefore, circular dichroism (CD) was measured to evaluate secondary structures of penetratin and Oct4-PTD, with and without fluorophore, in buffer and when bound to lipid vesicles. Both FITC-labelled peptides show a random coil structure in buffer. When bound to lipid vesicles, FITC-labelled penetratin and ditto Oct4-PTD show an evident α-helical signal, with a positive peak at 195 nm, and two negative peaks at 208 nm and 223 nm respectively (Figure 5A). If instead looking at the unlabelled peptides (Figure 5B), penetratin adopts an even more pronounced α-helix when bound to liposomes and an evident random coil when free in solution. However, the unlabeled Oct4-PTD shows an extremely weak, seemingly absent CD signal, indicating an unordered structure. The measurement was repeated with a three times higher peptide concentration, still rendering a very weak signal. The results show that a fluorescent label may induce structural changes in peptides, affecting the secondary structure of the peptide, both when free in solution or bound to lipid membranes.Figure 5

Bottom Line: 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.Oct4 is a key transcription factor in stem cell research and cellular reprogramming.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus