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Mechanisms of uptake of cell-penetrating peptides

Endocytosis versus direct translocation

It is somehow easy to understand why it is still so controversial the mechanisms of cellular uptake of cell-penetrating peptides. Although there is ample evidence that these peptides are capable of directly crossing the plasma membrane without any intermediate step, still several researchers claim that endocytosis is an intermediate step required for entry into the cells. In a nutshell, this last conclusion can be understood by looking at how most of the research on CPPs is conducted. Probably the most wide spread method is confocal microscopy and with this method it can be directly seen labeled peptides trapped into endosomes almost at the same time free peptide accumulates at the nucleus. This might be taken as a clear suggestion that CPPs uptake must be mediated by an initial endosomal uptake. The problem with this assumption is that usually peptides rich in arginine and lysine amino acids have a strong affinity for the cell plasma membrane and the cell is continuously endocyting for several other reasons that are not related to the CPPs, such as recycling the plasma membrane, absorbing extracellular fluids through macropinocytosis, etc.. Therefore, any peptide or molecule with affinity for the plasma membrane will be eventually internalized by endocytosis but this will not necessary imply that these molecules will be found freely diffusing in the cytoplasm and the nucleus as is the case for CPPs.  The next potential source of problems is how the different routes of endocytotic mediated uptake are tested. The standard way to test a route is to use a drug that interferes with the specific endocytotic pathway and see if CPPs are still able to cross the plasma membrane. It is well known that ionic interactions play a critical role for the binding to the plasma membrane and translocation of CPPs and when a drug is added to block a pathway it might also affect this binding by competing for critical binding sites at the membrane. Therefore, in this case cannot be directly interpreted that the reason the peptide is not translocating is just that the pathway is blocked.  These types of studies are standard in the field and based on them most researchers claim that macropinocytosis is required for CPPs entry. Furthermore, some researchers even go to the extreme to suggest that CPPs induce macropinocytosis. Even if endocytosis could be an intermediate step for CPPs internalization, what is completely missing in these studies is a clear justification on how these peptides are able to detach from the endosomal membrane and scape to reach the cytosol and the nucleolus.

Today there is ample and clear evidence that these peptides are capable of spontaneously crossing the plasma membrane. This has been shown in numerous ways such as on giant unilamellar vesicles (GUVs) composed of every sort of cell membrane components from pure phospholipids to actual cell membranes. It has been suggested and then shown experimentally that these peptides form small transient pores across the plasma membrane that could explain how these peptide are able to carry across the plasma membrane other cargoes covalently attached to them. It has also been suggested that these peptide might be able to move through the membrane without the involvement of a pore by getting complexed with negative groups at the plasma membrane. However, this seems to not explain how other cargoes could diffuse with the peptide across the membrane to the interior of the cell. The other issue with these conclusions is that they were obtained based on experiments that looked at the partition of the peptides between octanol and a water solution after the addition of a detergent, a detergent that is obviously not present in cells and that it would destroy the plasma membrane.

It is important to emphasize that there are no perfect experiments and that in this case it might require a large amount of experiments to reach a common agreement on the translocation pathway. Today it believed that CPPs might translocate directly across the plasma membrane and by macropinosytosis as an intermediate step. Important questions still remain for both pathways; such as if the peptides can actually induce macropinocytosis, if they can and how do they escape endosomes. In the case of direct translocation it is also not clear how the peptides detach from the plasma membrane to reach the cytosol and the nucleus. It has been shown that it is very difficult to remove membrane bound peptides and that to get rid of membrane bound CPPs, to look only at internalized peptides, the extracellular membrane bound peptides have to be cleaved using trypsin.

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