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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

Oct4-PTD is able to mediate uptake of protein cargo. (A) Oct4-PTD-Cre fusion proteins are taken up by CV1-5B Cre reporter cells as determined by microscopy of cells stained positive for b-Gal activity. Increasing protein concentrations of recombinant wild type Oct4-PTD-Cre fusion protein (WT) and mutated versions of the Oct4-PTD (R16A as wells as K13A/R16A) in fusion with Cre, respectively, were subjected to a Cre recombinase assay where the intracellular activity of transduced protein is assessed by b-galactosidase activity [45]. TAT-modified Cre protein [47] and unmodified Cre protein carrying no transduction peptide served as controls (first two rows). Quantification of Cre-reporter assay outlined in (B) (n = 5). All values relative to TAT-Cre 1 μM. Error bars and n represent standard deviations and image sections quantified for b-Gal activity, respectively. Two-tailed t-test was used for statistical analysis. *p <0.05, **p <0.01, ***p < 0.001. (C-F) Detection of recombinant human Oct4 protein in mammalian cells after extracellular exposure. CV1-5B (C) and human BJ foreskin fibroblasts (E) were incubated with 100 nM recombinant human Oct4 protein for 3 hours, washed and subsequently stained for Oct4 and DAPI. (D, F) Untreated CV1-5B cells (D) and BJ fibroblasts (F) stained with first (anti-Oct3/4) and secondary antibody (Alexa 488) served as controls. Scale bar: 25 μm.
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Fig6: Oct4-PTD is able to mediate uptake of protein cargo. (A) Oct4-PTD-Cre fusion proteins are taken up by CV1-5B Cre reporter cells as determined by microscopy of cells stained positive for b-Gal activity. Increasing protein concentrations of recombinant wild type Oct4-PTD-Cre fusion protein (WT) and mutated versions of the Oct4-PTD (R16A as wells as K13A/R16A) in fusion with Cre, respectively, were subjected to a Cre recombinase assay where the intracellular activity of transduced protein is assessed by b-galactosidase activity [45]. TAT-modified Cre protein [47] and unmodified Cre protein carrying no transduction peptide served as controls (first two rows). Quantification of Cre-reporter assay outlined in (B) (n = 5). All values relative to TAT-Cre 1 μM. Error bars and n represent standard deviations and image sections quantified for b-Gal activity, respectively. Two-tailed t-test was used for statistical analysis. *p <0.05, **p <0.01, ***p < 0.001. (C-F) Detection of recombinant human Oct4 protein in mammalian cells after extracellular exposure. CV1-5B (C) and human BJ foreskin fibroblasts (E) were incubated with 100 nM recombinant human Oct4 protein for 3 hours, washed and subsequently stained for Oct4 and DAPI. (D, F) Untreated CV1-5B cells (D) and BJ fibroblasts (F) stained with first (anti-Oct3/4) and secondary antibody (Alexa 488) served as controls. Scale bar: 25 μm.

Mentions: In order to test if Oct4-PTD is able to translocate other cargo than fluorescent labels into a cell and determine their intracellular activity after transduction, we used a Cre/loxP reporter system [45]. We generated a recombinant Oct4-PTD-Cre fusion protein as well as mutations in the Oct4-PTD as controls. The design of the mutants was based on a structure-activity relationship study of penetratin that led us to perform a R16A and a K13A/R16A exchange [46]. TAT-Cre, comprising the highly potent TAT transduction peptide in fusion with Cre served as a positive control [47]. After application of purified fusion proteins to CVI-5B reporter cells we observed a dose dependent activation of the loxP-modified reporter gene indicating Cre uptake via the Oct4-PTD (Figure 6A, B). Quantification of b-gal activity showed that the two Oct4-PTD mutatnts inverstigated did not result in b-gal activity above untagged Cre as observed by light microscopy (Figure 6B). However, our Oct4-PTD-Cre constructs were less efficient than the TAT-tagged Cre control.Figure 6


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)

Oct4-PTD is able to mediate uptake of protein cargo. (A) Oct4-PTD-Cre fusion proteins are taken up by CV1-5B Cre reporter cells as determined by microscopy of cells stained positive for b-Gal activity. Increasing protein concentrations of recombinant wild type Oct4-PTD-Cre fusion protein (WT) and mutated versions of the Oct4-PTD (R16A as wells as K13A/R16A) in fusion with Cre, respectively, were subjected to a Cre recombinase assay where the intracellular activity of transduced protein is assessed by b-galactosidase activity [45]. TAT-modified Cre protein [47] and unmodified Cre protein carrying no transduction peptide served as controls (first two rows). Quantification of Cre-reporter assay outlined in (B) (n = 5). All values relative to TAT-Cre 1 μM. Error bars and n represent standard deviations and image sections quantified for b-Gal activity, respectively. Two-tailed t-test was used for statistical analysis. *p <0.05, **p <0.01, ***p < 0.001. (C-F) Detection of recombinant human Oct4 protein in mammalian cells after extracellular exposure. CV1-5B (C) and human BJ foreskin fibroblasts (E) were incubated with 100 nM recombinant human Oct4 protein for 3 hours, washed and subsequently stained for Oct4 and DAPI. (D, F) Untreated CV1-5B cells (D) and BJ fibroblasts (F) stained with first (anti-Oct3/4) and secondary antibody (Alexa 488) served as controls. Scale bar: 25 μm.
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Related In: Results  -  Collection

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Fig6: Oct4-PTD is able to mediate uptake of protein cargo. (A) Oct4-PTD-Cre fusion proteins are taken up by CV1-5B Cre reporter cells as determined by microscopy of cells stained positive for b-Gal activity. Increasing protein concentrations of recombinant wild type Oct4-PTD-Cre fusion protein (WT) and mutated versions of the Oct4-PTD (R16A as wells as K13A/R16A) in fusion with Cre, respectively, were subjected to a Cre recombinase assay where the intracellular activity of transduced protein is assessed by b-galactosidase activity [45]. TAT-modified Cre protein [47] and unmodified Cre protein carrying no transduction peptide served as controls (first two rows). Quantification of Cre-reporter assay outlined in (B) (n = 5). All values relative to TAT-Cre 1 μM. Error bars and n represent standard deviations and image sections quantified for b-Gal activity, respectively. Two-tailed t-test was used for statistical analysis. *p <0.05, **p <0.01, ***p < 0.001. (C-F) Detection of recombinant human Oct4 protein in mammalian cells after extracellular exposure. CV1-5B (C) and human BJ foreskin fibroblasts (E) were incubated with 100 nM recombinant human Oct4 protein for 3 hours, washed and subsequently stained for Oct4 and DAPI. (D, F) Untreated CV1-5B cells (D) and BJ fibroblasts (F) stained with first (anti-Oct3/4) and secondary antibody (Alexa 488) served as controls. Scale bar: 25 μm.
Mentions: In order to test if Oct4-PTD is able to translocate other cargo than fluorescent labels into a cell and determine their intracellular activity after transduction, we used a Cre/loxP reporter system [45]. We generated a recombinant Oct4-PTD-Cre fusion protein as well as mutations in the Oct4-PTD as controls. The design of the mutants was based on a structure-activity relationship study of penetratin that led us to perform a R16A and a K13A/R16A exchange [46]. TAT-Cre, comprising the highly potent TAT transduction peptide in fusion with Cre served as a positive control [47]. After application of purified fusion proteins to CVI-5B reporter cells we observed a dose dependent activation of the loxP-modified reporter gene indicating Cre uptake via the Oct4-PTD (Figure 6A, B). Quantification of b-gal activity showed that the two Oct4-PTD mutatnts inverstigated did not result in b-gal activity above untagged Cre as observed by light microscopy (Figure 6B). However, our Oct4-PTD-Cre constructs were less efficient than the TAT-tagged Cre control.Figure 6

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