Cell Penetrating Peptide Delivers Proteins Into Cells
By Klaus D. Linse
Cell-penetrating peptides, or CPPs, are short peptides that can facilitate cellular uptake of a variety of molecular cargo. CPPs (sometimes also called protein transduction domains, or PTDs, are a group of short, highly basic peptides. These peptides can penetrate cell membranes either alone or along with cargo molecules. This “cargo” can range from nano-sized particles to small chemical molecules, peptides, proteins and large fragments of DNA as well as RNA molecules such as siRNA duplexes. Often the "cargo" is co-inserted into target cells together with the CPPs either via covalent attachment or through non-covalent interactions. The function of the CPP is to help deliver the cargo into cells.
In a paper published online on June 2014 in the Journal Nature Methods, a research group at Texas A & M (Erazo-Oliveras et al. 2014) reported the development of a protein transduction approach using a cell-penetrating peptide (CPP) to deliver proteins as cargo across the cell membrane into living cells. The TAT sequence derived from the HIV-1 trans-activator gene product was used as the template for the design of a dimeric delivery molecule or vehicle. The peptide was modified with a lysine and fluorophore tetramethylrhodamine (TMR) to allow for fluorescence imaging as well as a cysteine, both added at the N-terminal end of the TAT peptide to permit dimerization by disulfide bond formation. The researchers used this design since it is known that disulfide bonds are relatively stable inside endosomes but are cleaved following endosomal escape and upon entry into the reducing cytosol which releases the monomeric molecule. It was found that the CPP dfTAT can penetrate live cells by escaping from endosomes with high efficiency. The scientists demonstrated that this peptide allows the delivery of proteins, and potentially other cargo molecules, into the cells with the help of a mechanism called endosomal leakage. Furthermore, the cytosolic delivery of proteins as cargo into several cell lines was achieved with the help of the dfTAT peptide.
The structures of the modified peptides as reported by Erazo-Oliveras et al. are shown below.
ckTAT
fk(TMR)TAT
dfTAT
Figure 1: Structures of ckTAT, fK(TMR)TAT and dfTAT: CKRKKRRQRRRG (Upper panel); CK(ε-NH-TMR)RKKRRQRRRG (Middle panel); [CK(ε-NH-TMR)RKKRRQRRRG]2 (Lower panel) are depicted. The arginine side chains are shown in blue, the TMR fluorophore is shown in red and the disulfide bond forming the dimer is shown in magenta.
Scientists observed that the delivery did not require a binding interaction between the peptide and the proteins. In addition, this type of delivery did not noticeably affect cell viability, cell proliferation or gene expression. However, only the future will tell if this intracellular delivery method becomes a useful application for cell-based assays, cellular imaging and/or ex vivo manipulations (manipulations performed outside) of cells. On the other hand, if this approach could be used to selectively deliver a tumor suppressor protein into cancer cells, a new method to treat cancer with potentially less side effects could be available soon.
How does endosome mediated cell delivery work?
Endosomes are smooth-surfaced membrane containing vesicles or compartments found inside eukaryotic cells. Endosomes are the intermediate transporters of particles brought into a cell from outside and are part of the endocytosis pathway. Endocytosis is an energy-using process which allows cells to take materials from the outside into the cell by engulfing and fusing them with the cell or plasma membrane. Furthermore, molecules internalized from the plasma membrane can follow this pathway all the way to lysosomes, the last compartment of the endocytic pathway, where they are degraded. However, in an opposite process called exocytosis, these molecules can be recycled back to the plasma membrane.
Endocytosis has been proposed as one of the primary mechanisms which allow cell-penetrating peptides (CPP) and their cargos to enter a cell. Unfortunately, one major limitation of this pathway is the entrapment of the CPP-cargo in intracellular vesicles. To reach the targets located in the cytoplasm the cargo needs to escape the vesicles in order to exert its biological function.
Endocytosis can be divided into two major categories:
- Phagocytosis - the ingestion of a smaller cell or cell fragments, a microorganism, or foreign particles, involves the uptake of large particles
- Pinocytosis - the transport of fluids into a cell, involving solute uptake.
Also, pinocytosis can be further divided into macropinocytosis, clathrin-dependent, caveolin-dependent and clathrin/caveolin-independent pathways. Experimental data suggest that numerous factors appear to influence the route of cellular uptake of the CPPs, some of which may need further elucidation.
Over the years, several strategies for the controlled cellular delivery of bioactive macromolecules with therapeutic potential have been developed. Among these, several non-viral carrier systems such as liposomes, polycationic carrier, nanomaterials and peptides have been investigated for their ability to penetrate or transduce cell membranes efficiently.
Protein transduction is an emerging technology with potential applications in gene therapy. It can be described as the internalization of proteins from the outside into the cell. For many current gene therapy strategies, for example for the gene therapy of cancer, a sustained and regulated expression of the transgene is not necessarily required. Therefore, it appears possible that a short term delivery of the gene product, rather than the gene itself, maybe all that is needed. The observation that some proteins, when added to the outside of the cell, can be taken up by the cell has resulted in detailed studies of the fundamental mechanism that allows this to happen. As a result protein transduction domains were identified. Additionally, it was found that these domains when fused to other proteins allowing these proteins to enter the cell as well, and sometimes even the nucleus.
The Drosophila antennapedia peptide, the herpes simplex virus VP22 protein and the HIV TAT protein transduction motif are the three most widely studied transduction motifs. Furthermore, there are indications that other proteins may have similar properties. Some of these are peptide motifs present in haemagglutinin from influenza, lactoferrin, fibroblast growth factor and the homeodomain (HD) of engrailed, Hoxa-5, Hoxb-4 and Hoxc-8 proteins.
The cytosolic delivery of proteins into several cells with the help of the dfTAT peptide appears to be similar to the transfection of cells with lipofectamine. Lipofectamine, or Lipofectamine 2000, a common transfection reagent is produced and sold by Invitrogen. It helps to increase the transfection efficiency of RNA, including mRNA and siRNA, or plasmid DNA into in vitro cell cultures. This is called lipofection. Lipid subunits present in the reagent can form liposomes in aqueous environments that can entrap the transfection materials, for example DNA plasmids. The DNA-containing liposomes having a positive charge on their surfaces, can fuse with the negatively charged plasma membrane of living cells, allowing nucleic acid to cross into the cytoplasm now available to the cell for replication or expression.
Several approaches for the cytosolic delivery of liposomal macromolecules have also been developed.
These include:
1) The co-encapsulation of fusogenic peptides into targeted drug-containing liposomes
2) The coupling of HIV-1 derived cell penetrating peptide TAT to the surface of liposomes
3) Photochemical internalization, based on photochemically inducible permeabilisation of
endocytic vesicles.
In addition, several endosome-disrupting peptides that destabilize endosome membranes have been derived from certain pathogens in recent years, including viruses and bacterial toxins. These membrane-disrupting peptides are known to promote endosomal escape after endosomal acidification and already some of them have been used for the design of fusion peptides.
Table 1: Sequences of cell-penetrating peptide based tags for fusion peptides
References
Alfredo Erazo-Oliveras, Kristina Najjar, Laila Dayani, Ting-Yi Wang, Gregory A Johnson & Jean-Philippe Pellois; Protein delivery into live cells by incubation with an endosomolytic agent. nature methods | ADVANCE ONLINE PUBLICATION Received 31 May 2013; accepted 9 May 2014; published online 15 June 2014; doi:10.1038/nmeth.2998.
KG Ford, BE Souberbielle, D Darling and F Farzaneh; Protein transduction: an alternative to genetic Intervention. Review. Gene Therapy (2001) 8, 1–4. http://www.nature.com/gt/journal/v8/n1/full/3301383a.html
Marjan M. Fretz, Enrico Mastrobattista, Gerben A. Koning Wim Jiskoot and Gert Storm; Strategies for cytosolic delivery of liposomal macromolecules. Published in the International Journal of Pharmaceutics. 298: 305-309 (2005).
Ji-Sing Lioua, Betty Revon Liua, Adam L. Martin, Yue-Wern Huangd, Huey-Jenn Chiang, Han-Jung Lee; Protein transduction in human cells is enhanced by cell-penetrating peptides fused with an endosomolytic HA2 sequence. Peptides 37 (2012) 273–284.
Astrid Walrant, Isabelle Correia, Chen-Yu Jiao, Olivier Lequin, Eric H. Bent, Nicole Goasdoué, Claire Lacombe, Gérard Chassaing, Sandrine Sagan, Isabel D. Alves; Different membrane behaviour and cellular uptake of three basic arginine-rich peptides. Biochimica et Biophysica Acta 1808 (2011) 382–393.