MinireviewsNonclassical Transport Proteins and Peptides: An Alternative to Classical Macromolecule Delivery Systems
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INTRODUCTION
Despite continuing advances in the formulation and development of macromolecular therapeutics, the passage of large therapeutic molecules through cell membranes remains a significant obstacle to their delivery. The best understood route of macromolecule entry into cells is endocytosis. Utilizing this route of transport for the delivery of macromolecules, however, has significant limitations. The primary problem is that the endosome/lysosome pathway is a major site of degradation within the cell;
VP22
The VP22 protein is a major component of the tegument of herpes simplex virus 1.26 The function of VP22 in herpes virus infection has not been definitively established, although it has been shown to undergo phosphorylation,27 nucleotidylation,28 and has been found to interact with cellular components such as chromatin,25,29 microtubules,25,30 nonmuscle myosin IIA,31 and oligonucleotides.32 A recent report by Sciortino and colleagues suggests that VP22 may play a role in shuttling infected cell
FIBROBLAST GROWTH FACTORS
The fibroblast growth factors (FGFs) are a family of proteins currently thought to contain at least 23 different members. FGFs exhibit a wide variety of biological functions primarily related to the stimulation of cell proliferation;48 FGF-1, -2, and -9 exhibit strong general mitogenic activity49,50 while FGF-7 and -10 (also known as keratinocyte growth factors 1 and 2, or KGFs) specifically stimulate dermal proliferation. Interestingly, at least FGFs 1,2, 9, 16, and 20 exhibit nonclassical
HIV-1 TAT
The Tat protein is a 101 residue nuclear transcription activating protein of the HIV-1 retrovirus,74 initially studied because of its role in the HIV infection process. In 1988, Frankel and Green independently demonstrated that a purified 86 residue form is efficiently internalized by cells in culture.75,76 Subsequent studies showed that this internalization process is accompanied by nuclear localization after internalization.77 Tat is actively secreted in the absence of a conventional signal
HOMEOPROTEINS
The homeoproteins are a class of transactivating factors that interact with DNA via a highly conserved, helical 60-amino acid sequence called the homeodomain.98 As first demonstrated in a study of the Drosophila antennapedia (Antp) homeodomain by Perez and colleagues, the homeoproteins exhibit nonclassical, receptor-independent internalization and nuclear localization.99,100 Antp translocation activity, initially attributed to the 60-residue homeodomain, was later localized to a 16-amino acid
CELL-PENETRATING PEPTIDES
Both HIV-1 Tat and the Penetratin peptide are often included in a class of vectors known as cell-penetrating peptides. A significant increase in interest in these peptides has occurred in recent years, to such an extent that a complete overview is beyond the scope of this review. For a more detailed discussion, see reviews by Lindgren et al.,13 Wadia,112 and others.113., 114., 115., 116. The majority of cell-penetrating peptides can be divided into two groups. The first consists of short (<20
CURRENT THEORIES OF NONCLASSICAL TRANSPORT
Given the diversity of agents that can mediate the non-classical transport of various macromolecules into and out of cells, it is tempting to speculate that a variety of different mechanisms may be involved in this process. We feel that this is unlikely, however, because two common properties are possessed by virtually all of the nonclassical agents. The first of these is the polycationic nature of the transport peptides or regions of the transducing proteins. That cationic character is
POSSIBLE ROLE OF “NON-NATIVE” STATES
Lipid bilayers contain both polar and apolar domains, with the apolar interior providing an apparently impenetrable barrier to the passage of highly polar molecules like the cationic polymers of interest here. Some small molecules are thought to circumvent this barrier by possessing at least partial lipophilic character. Interestingly, it has been proposed that a similar result can be achieved in globular proteins by their adaptation of partially unfolded or molten globule-like conformations.
THE ROLE OF POLYANIONS
A common factor among nonclassical transport vectors is a seemingly disproportionate abundance of clustered cationic residues, suggesting potential interaction with the profusion of negatively charged proteoglycans and lipids on the cell surface, as well as other polyanions throughout the cell. Polyanion binding has been demonstrated for VP22,32 both FGF-1 and -2,53,143,144 as well as the full-length Tat protein91 and various basic peptides, and can be confidently inferred for other vectors
THE UNIQUE PROPERTIES OF THE GUANIDINIUM CATION
The high degree of similarity between cationic cell-penetrating peptides with regard to both charge density and amino acid content is one of the strongest pieces of evidence that a common internalization mechanism exists for at least these vectors.93 Inhibition of internalization by other cationic vectors supports this position.93 The marked prevalence of arginine residues in both peptide and protein vectors suggests that the arginine residue itself may be a key factor for highly efficient
INVERTED HEXAGONAL PHASES
Prochiantz and colleagues have proposed a model for internalization of the Penetratin peptide102,157,158 in which an initial electrostatic interaction between the peptide and the bilayer causes bilayer disruption and induction of an inverted hexagonal lipid phase. This results in movement of the peptide into the bilayer, where it becomes entrapped in an inverted micelle,158,159 which passes to the opposite side of the bilayer and releases its contents. Formation of such an inverted hexagonal
SECRETION
Many proteins that demonstrate nonclassical import activity are also leaderless secretory proteins, potentially enhancing their utility as macromolecule delivery vectors. As in import, the mechanism by which export of these proteins is achieved, often with protein cargo attached, is unclear. Thus, it is uncertain whether the import and export mechanisms are related. It seems possible, however, that the vector properties which are responsible for import may at least play a role in secretion. In
ADDITIONAL PARTICIPANTS
Although the mechanisms discussed above each describe a potentially complete transport pathway, none specifically exclude the involvement of other cell components. This may be especially relevant to studies of these pathways and others in simple lipid models, where a lack of additional, key cellular components may result in poor transport activity.98,167 It is therefore important to recall the complexity of the cell, and the variety of molecules that a vector can come in contact with that may
A UNIFYING HYPOTHESIS
With the plethora of results, observations, and hypotheses currently available concerning nonclassical transport processes, is it possible to propose a general mechanism that can provide a basis for further experimental work? We believe that it is and do so below, with the following caveat: the details of the underlying mechanism(s) remain to be elucidated. With this in mind, direct attacks on the problem in terms of the ideas we present should provide a good starting point, although there may
CONCLUSIONS
The presence of an endogenous pathway in cells through which macromolecules can both enter and exit freely is a potentially quite exciting discovery in the field of macromolecular drug delivery. As the study of vehicles that utilize this nonclassical transport pathway has progressed, a number of new delivery entities have been developed or discovered, increasing the possibility that a viable formulation incorporating such vehicles will reach the clinic in the near future. In fact, in vivo
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