Characterization of a novel cell penetrating peptide derived from human Oct4

Abstract

BackgroundOct4 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.ResultsA 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.ConclusionsOct4 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.Electronic supplementary materialThe online version of this article (doi: 10.1186/2045-9769-3-2) contains supplementary material, which is available to authorized users.

Cell-Penetrating Function of the Poly(ADP-Ribose) (PAR)-Binding Motif Derived from the PAR-Dependent E3 Ubiquitin Ligase Iduna

“…Most CPPs, including the ones mentioned here, are derived from non-human sources, such as viruses or drosophila, or are synthesized artificially. Due to possible immunogenicity or toxicity of those CPPs, human-originated CPPs have been identified such as Hph-1 [ 13 , 14 ], Sim-2 [ 15 ], ECP 32–41 [ 16 ] and Oct4 [ 17 ]. We also have previously reported novel human protein-derived CPPs including LPIN [ 18 ], 2pIL-1aNLS [ 19 ], dNP2 [ 20 ], and AP [ 21 ].…”

Design and Characterization of a Cell-Penetrating Peptide Derived from the SOX2 Transcription Factor

“…We synthesized a parental and mutant SOX2-iPep N-terminally conjugated with TAMRA, respectively, which facilitated the intracellular detection of the peptide by IF (Figure 4A). A similar approach has been used for the characterization of an OCT4 peptide with a protein transduction domain capable of translocating into human and mouse cells [25]. We found that the SOX2-iPep was internalized very rapidly and effectively in 4.9% of T11 cells within two hours and co-localized with Hoechst staining into the nucleus of the breast cancer cells (Supplementary Figure S1).…”

“…When bound to lipid membranes, both FITClabelled OCT4 peptide and penetratin were found to adopt α-helical conformations, and the unlabeled OCT4 peptide exhibited a disordered structure when free in solution or bound to lipid membranes. The unlabeled and labeled OCT4 and penetratin not only internalized but were shown to translocate entire proteins such as Cre and its natural cargo [25,39]. Owing to the role of SOX2 in triple-negative breast cancer and its inherent cell-penetrating sequence, this SOX2 iPep when physically linked with small molecules such as doxorubicin or cisplatin (cargo) might help to localize them specifically in the nucleus of cancer cells overexpressing SOX2.…”

Cupid, a cell permeable peptide derived from amoeba, capable of delivering GFP into a diverse range of species

“…Although natural cell permeable peptides (CPPs) are present in diverse species, the potential for sequences to act as CPPs is unpredictable 40 and none have yet been shown in Dictyostelium amoeba. This suggests either the amoebae have no requirement for a protein transduction mechanism via a CPP, or it might achieve this end with a cryptic CPP sequence.…”