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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

Overview of the investigated peptides. (A) Oct4-PTD derives from the third helix of the homeodomain (orange) of the transcription factor Oct4 (POU5F1), which possesses a POU-specific and a homeodomain [42], pdb: 3L1P. (B) The sequence alignment of Oct4-PTD and Penetratin shows great conservation, almost half of the residues are identical (*), more than two thirds of the peptide share at least same charge (* or :) (C, D) Helical wheel projection of Oct4-PTD or penetratin.
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Fig1: Overview of the investigated peptides. (A) Oct4-PTD derives from the third helix of the homeodomain (orange) of the transcription factor Oct4 (POU5F1), which possesses a POU-specific and a homeodomain [42], pdb: 3L1P. (B) The sequence alignment of Oct4-PTD and Penetratin shows great conservation, almost half of the residues are identical (*), more than two thirds of the peptide share at least same charge (* or :) (C, D) Helical wheel projection of Oct4-PTD or penetratin.

Mentions: The cell penetrating peptide penetratin is derived from a DNA binding domain of a homeobox transcription factor. Oct4 belongs to the same class of transcription factors and also contains a bi-partite DNA-binding domain, the POU-specific domain (POUs), which recognises the octamer motif as well as the homeodomain (POUhd) (Figure 1A). Therefore, we aligned Oct4 and penetratin sequences and found a high degree of homology in the third helix of the respective homeodomains. The homology between the 16 aa of penetratin and the respective region of Oct4 is 7 out of 16 (43%) identities and 11 of 16 (68%) similarities (Figure 1B). Due to this similarity we termed this putative CPP Oct4-protein transduction domain (Oct4-PTD). The Oct4-PTD differs from penetratin mainly by having less lysines but more arginines – a feature that might be of advantage as far as uptake is concerned [41]. These differences are visualized by a helical wheel projection showing the differing distribution of the charges over the peptide suggesting diverging structural features of Oct4-PTD compared to Penetratin. Still, we considered the similarities to be sufficiently high to test Oct4-PTD for a putative activity as CPP.Figure 1


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)

Overview of the investigated peptides. (A) Oct4-PTD derives from the third helix of the homeodomain (orange) of the transcription factor Oct4 (POU5F1), which possesses a POU-specific and a homeodomain [42], pdb: 3L1P. (B) The sequence alignment of Oct4-PTD and Penetratin shows great conservation, almost half of the residues are identical (*), more than two thirds of the peptide share at least same charge (* or :) (C, D) Helical wheel projection of Oct4-PTD or penetratin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Overview of the investigated peptides. (A) Oct4-PTD derives from the third helix of the homeodomain (orange) of the transcription factor Oct4 (POU5F1), which possesses a POU-specific and a homeodomain [42], pdb: 3L1P. (B) The sequence alignment of Oct4-PTD and Penetratin shows great conservation, almost half of the residues are identical (*), more than two thirds of the peptide share at least same charge (* or :) (C, D) Helical wheel projection of Oct4-PTD or penetratin.
Mentions: The cell penetrating peptide penetratin is derived from a DNA binding domain of a homeobox transcription factor. Oct4 belongs to the same class of transcription factors and also contains a bi-partite DNA-binding domain, the POU-specific domain (POUs), which recognises the octamer motif as well as the homeodomain (POUhd) (Figure 1A). Therefore, we aligned Oct4 and penetratin sequences and found a high degree of homology in the third helix of the respective homeodomains. The homology between the 16 aa of penetratin and the respective region of Oct4 is 7 out of 16 (43%) identities and 11 of 16 (68%) similarities (Figure 1B). Due to this similarity we termed this putative CPP Oct4-protein transduction domain (Oct4-PTD). The Oct4-PTD differs from penetratin mainly by having less lysines but more arginines – a feature that might be of advantage as far as uptake is concerned [41]. These differences are visualized by a helical wheel projection showing the differing distribution of the charges over the peptide suggesting diverging structural features of Oct4-PTD compared to Penetratin. Still, we considered the similarities to be sufficiently high to test Oct4-PTD for a putative activity as CPP.Figure 1

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