Cell-penetrating peptides – mechanism of transduction and

Cell-penetrating peptides – mechanism of
transduction and synthesis – short review∗
Marcin T. KAWCZYŃSKI† , Joanna KRECZKO‡
Keywords: peptides; cell-penetrating peptides;
Abstract: Potential antimicrobial agents may be effective if
they are delivered efficiently to their site of action. In many
cases, lack of permeability through the cell membrane is the
main problem. One of the possible solutions could be a novel
intracellular delivery system involving cell-penetrating peptides
(CPPs), which may improve the efficiency of many new biopharmaceuticals. Among other things, CPPs are capable of transporting molecules that vary in structure and size such as: small
molecules, RNA and DNA nucleotides, nanoparticles, peptides,
proteins and even liposomes. CPP-cargo conjugates are internalised by endocytosis and other mechanisms of intracellular delivery not involving any receptors and not causing any significant cell membrane damage. Due to their relatively small size,
CPPs are relatively easy to synthesize using the solid phase-based
methodology.
1. Introduction and history
Many peptides and proteins do not possess the ability to pass through cell membranes.
This fact often constitutes a great problem when designing therapeutic molecules. On
the other hand, several years ago several classes of peptides able to penetrate biological
membranes were discovered. A crucial development was the discovery of the human
immunodeficiency virus type 1 (HIV-1) encoded transactivator of the transcription
(Tat) protein, which was the first molecule found to be able to gain intracellular access (Green and Loewenstein, 1988). To study its properties, a 36- amino- acid-long
fragment was chosen (Tat[37-72]). It was proven that this fragment conjugated to βgalactosidase can be promoted and then taken up by mammalian cells (Fawell et al.,
1994). Another peptide with similar properties which was discovered at that time
was Penetratin, a fragment of homeoprotein of Drosophila. Cell penetrating properties of this peptide were found during studies on the role of homeodomain in neuronal
morphogenesis (Joliot et al., 1991). Later studies on other parts of the homeodomain
∗ This work is supported by an internal grant from the Faculty of Chemistry, University of Gdańsk
DS/530-8454-D194-12
† Gdańsk University of Technology, Faculty of Chemistry, Department of Organic Chemistry
‡ University of Gdańsk, Faculty of Chemistry, Laboratory of Polypeptide Chemistry
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peptide led to the conception of cell-penetrating peptides (CPPs), also known as
peptide transduction domains (PTD), Trojan peptides or membrane translocating sequences (MTS). It is hard to give one definition of CPPs. Simply, they are relatively
short peptides (5-40 amino acids), with the ability to gain access to the cell interior
by means of different mechanisms, and with the possibility of promoting the intracellular delivery of conjugated (covalently or noncovalently) biologically active cargoes
(Langel, 2007).
After the discovery that the Tat protein sequence could be shortened to a peptide
a few amino acids long, the number of synthetic cell-penetrating peptides increased
dramatically. So far, many of them have been found to be very useful in drug delivery.
2. Cell-penetrating peptide families
Most CPPs contain many cationic amino acid residues and are derived from viral,
insect or mammal proteins. CPPs can be categorised by their origin, so next to the Tat
and penetratin families are Chimeric CPPs (compiled sequences from two different
peptides with penetrating properties) or Antimicrobial-derived CPPs. Representative
examples of families (by origin) are summarized in Tab. 1.
Tab. 1. Representative CPP families (Foerg and Merkle, 2008)
Example
Family
Origin
Sequence
peptide
Tat(48-60)
HIV-1 protein
GRKKRRQRRRPPQQ
Oligoarginine
Tat derivatives
Antennapediahomeodomain
Rn
Tat family
Penetratin
family
pAntp
pIsl
Chimeric
CPPs
Transportan
Pep-1
Antimicrobialderived CPPs
hCT-derived
peptides
Buforin 2
Isl-1
homeodomain
Galaninmastoparan
RVIRVWFQNKRCKDKK
Trp-rich
motif-SV40
KETWWETWWTE
WSQPKKKRKV
Toad stomach
PAF26
hCT(9-32)
RQIKIWFQNRRMKWKK
Human
calcitonin
GWTLNSAGYLLGK
INLKALAALAKKIL
TRSSRAGLQW
PVGRVHRLLRK
RKKWFW
LGTYTQDFNKFH
TFPQTAIGVGAP
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3. Mechanisms of membrane penetration
The complete mechanism of CPP entry into cells still remains unknown. There are
a few possible mechanisms of CPP entry into the cell and probably each peptide uses
more than one. Which mode of action is dominant depends mostly on the CPP’s
properties: its concentration, length, charge, hydrophobicity and other chemical and
physical features. However, it also depends on the cargo’s characteristics: type, size
and charge of a molecule (Fonseca et al., 2009).
At the beginning of studies on CPP, scientists postulated a passive, temperatureindependent mechanism, insensitive to endocytosis inhibitors (Vivès et al., 2003).
Recently, these experiments are being questioned because of inadequate methodology
and experimental difficulties in distinguishing cell surface-associated CPP from CPP
internalized in cytoplasm (Richard et al., 2003).
To this day, many mechanisms have been proposed for different CPPs and different cargoes, but there is a consensus that the endocytosis mechanism takes part in
the translocation of most CPPs (Madani et al., 2011).
3.1. Endocytosis
At the beginning of studies on CPP, the concept of the endocytosis mechanism was
rejected (Vivès et al., 2003). More recent studies clearly demonstrate the involvement
of endocytosis with Tat family peptides (Richard et al., 2003) and penetratin (Drin
et al., 2003). Endocytosis might involve several pathways. Experiments on Tat peptide penetration involving inhibitors of clathrin-dependent endocytosis (Fig. 1), show
much lower uptake of this peptide (Richard et al., 2003). Studies on other conjugates
suggest different mechanisms, such as macropinocytosis and endocytosis via lipid rafts
(clathrin-independent) (Puri et al., 2001).
No matter what type of endocytosis or pinocytosis accompanied the penetration of
the peptide into the cell, the problem is peptide escape from the endosome on its way
to liposome. One proposal is to move the peptide to other organelles, and finally into
the cytoplasm. This takes place on the basis of early endosome reverse transport to
the Golgi apparatus. Then to the endoplasmic reticulum and then to the cytoplasm.
(Fischer et al., 2004).
Fig. 1. Clathrin-dependent endocytosis
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3.2. Inverted micelle
This mechanism has been proposed mainly for the penetratin family. These peptides
are arginine and lysine-rich molecules and an alanine scan shows that these residues
play an important role in cellular uptake. Also, studies show that the presence of tryptophan residue is crucial for a peptide’s penetrating activity (Langel, 2007). Members
of the penetratin family of CPPs also cross the membrane at low temperature with
good efficiency, which suggests an energy-independent mechanism. Various analogues
of penetratin have been synthesized (like D-amino acid residue containing analogues
and retro-peptides) which had efficiency similar to penetratin. These experiments
lead to the conclusion that the mechanism is independent of any membrane receptor. However, cyclic analogues do not gain access to cytosol, which indicates that the
linear structure of peptide is an important factor in this case.
Based on the foregoing information, a mechanism for the penetratin’s uptake into
the cell was proposed. At the beginning, the CPP is stabilized on the cell surface
as a result of electrostatic interactions of positively charged residues (Lys and Arg)
with the negatively charged ”heads” of phospholipids. At this point, the residue
of the tryptophan initiates formation of reverse micelles around the CPP (with or
without the conjugate), which moves it to the other side of the cell membrane (Fig. 2).
In another possible variant, the reverse micelle formation disturbs the structure of
the membrane, which allows CPPs to penetrate the membrane (Prochiantz, 2000).
Obviously, other mechanisms are still possible (like endocytosis described above).
3.3. Carpet-like mechanism
This mechanism is observed, for example, for some cationic antimicrobial peptides
(CAMP). Most CAMP have the ability to penetrate the cell membrane of microorganisms, which, in effect, leads to cell lysis. However, some of them are able to
penetrate in a more subtle way through the membrane. These are the penetrating
peptides (Marcos and Gandia, 2009). These peptides are usually short and have
two domains – one with cationic residues and the second, hydrophobic (containing
tryptophan) (Munoz et al., 2006).
In the carpet-like mechanism, positively charged domain of CAMP interact with
negatively charged ”heads” of membrane phospholipids. On the surface of the cell
membrane a ”carpet” is formed, which, after reaching a certain concentration, locally
destabilizes the membrane, leading to pore formation (Fig. 3). The peptide can pass
through these pores into the cell, with or without a cargo (Shai, 2002).
Fig. 2. Inverse micelle mechanism of uptake of CPP-cargo conjugates
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Fig. 3. Pore formation in cell membrane in carpet-like mechanism of CPPs
4. CPP synthesis
The CPP-cargo sequences could be easily expressed in Escherichia coli and then
purified, but for laboratory purposes (low quantity), it is much easier to synthesize it
by solid phase peptide synthesis (SPPS).
Peptides are synthesized using the Fmoc/But strategy. Peptides containing a free
C-terminal could be synthesized on 2-chlorotrityl chloride resin; for sequences containing a C-terminal amide, Rink Amide Resin might be used. After synthesis is
completed, the peptides will be cleaved from the resin(s) (with simultaneous deprotection of side chain groups of each amino acid residue) with a mixture based on
trifluoroacetic acid (TFA). The detailed composition of the mixture is determined depending on the peptide sequence, but usually contains TFA, phenol, water and triisopropylsilane. The cleaved peptides are precipitated with diethyl ether and lyophilized.
They could be purified by high pressure liquid chromatography (HPLC).
A great advantage of this method is the possibility of covalently bonding the cargo
to the peptide during the synthesis even if the cargo does not have an amino acid-like
structure. In the case of CPP being expressed in bacteria, non-peptide cargo have to
be introduced in another step.
SPPS also has disadvantages – some types of cargo are labile in reaction conditions
and a special approach is needed.
5. Summary
The ability to transport different types of molecules through the cell membrane has
made CPPs very useful in drug design. CPPs have already found applications in
medicine as drug delivery agents in the treatment of various diseases. These peptides
could also help reinvent some molecules because of their capacity to support the cellular delivery of therapeutics that do not normally cross the plasma membrane. In
conjugates with CPP, those drugs might pass the membrane barrier and have the
desired therapeutic effect.
Unfortunately, many aspects of CPPs are still unknown. Studies on the mechanism
of transduction, ways of transport and intracellular activity are still being conducted.
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