ReviewThe taming of the cell penetrating domain of the HIV Tat: Myths and realities
Introduction
The past decade has witnessed tremendous advances in the field of protein transduction, aiming to correct defects for proteins involved in a variety of disease processes. At present, it is possible to produce a given protein molecule by recombinant DNA technology for in vivo therapeutic applications. Nevertheless, it still remains a challenge to deliver the recombinant proteins to desired targets in vivo, although small molecules or peptides capable of crossing cellular membranes have been successfully designed to deliver small or moderately large proteins. Despite developments in the area of protein transduction peptides, the classical delivery methods of protein coding genes via adeno-associated virus (AAV) [1], [2], adenovirus (AV) [3], [4], lentivirus [5], herpes virus (HSV) [6], [7] vectors, and plasmid expression vectors [8], [9] remain the preferred choice for expression of proteins.
Because of their natural abilities in delivering the specific genes to permissive cells, viral vector-mediated gene expression is considered the most efficient and reliable approach for expressing functional proteins de novo in mitotically active or post-mitotically blocked cell types (HIV viral vectors). Nonetheless, viral vectors invariably are required in large doses to achieve therapeutic expression levels of intended protein (s). Moreover, viral vectors integrate with the host chromatin material. These properties may have consequences from long term effects on host genetic systems, and therefore, safety remains a serious concern for their ultimate clinical application [10], [11], [12], [13].
An alternative approach that appears to be the safest is to produce recombinant proteins exogenously and then deliver them systemically or by localized injections into the target organs. The delivery and bioavailability of recombinant proteins into cells or tissues need further improvements, however. Discovery of the HIV Tat protein transduction domain (PTD) has opened avenues for directing in vitro and in vivo delivery of proteins into cells. Several studies have shown the potential of PTD in drug delivery [14], [15] and transduction of proteins as large as 110 kDa into different cells [16]. In vivo injection of fusion proteins systemically has demonstrated the effectiveness of the PTD in protein delivery [15], [16]. In the present review we discuss the current status of the protein transduction focusing mainly on the PTD domain of HIV Tat.
Section snippets
Cell penetrating peptides (CPPs) for protein delivery
Various approaches have been designed to develop CPPs for introducing recombinant proteins into the cells. Penetratin [17], polylysine [18], [19], polyarginine [20], Tat-PTD [16], [21], HSV VP22 [22], [23], [24], Kaposi FGF [25], Syn B1 [26], FGF-4 [27], [28], nuclear localization signal (NLS) [29], and anthrax toxin derivative 254-amino acids (aa) peptide segment [30], diphtheria toxin ‘R’ binding domain [31], MPG (HIV gp41/SV40 Tag NLS) [32], pep-1 [33], WR peptide [34], and exotoxin A [35]
Potential of PTD fusion protein transduction in vitro
Several years ago, Frankel and Pabo [48] and Green and Lowenstein [49] demonstrated that extracellular HIV Tat can cross the plasma membrane and enter the cell, reaching the nucleus. The successful entry of extracellular Tat was investigated by others and has become a common test to monitor HIV-LTR promoter activity. Subsequently, more detailed analysis of Tat protein [21], [47], [50], [51] identified a PTD of 9–11 aa residues, a basic domain that can transduce itself, as well as the bigger
Potential of PTD fusion protein transduction in vivo
In 1999 Schwarze et al. [16] showed that PTD-beta-gal fusion protein applied intraperitoneally entered the brain after crossing the blood–brain barrier (BBB). Subsequently, several other studies have also reported effective biodistribution of therapeutically important proteins in vivo by using PTD [52], [69], [70], [71], [72], [73], [74], [75]. In a previous study, however, no PTD-mediated transduction in the brain was observed [46], possibly because beta-gal was chemically conjugated to PTD
Mechanism of PTD internalization
It has been observed that histones and cationic polyamines such as polylysine stimulate the uptake of albumin by tumor cells in culture [85], [86]. But, the cellular uptake mechanism(s) of CPPs is currently unknown. CPPs are structurally diverse and highly variable in nature. Nonetheless, their common feature is the high density of basic amino acid residues (Arg and Lys): the presence of basic amino acids in the PTD is considered the hallmark of transduction peptides. There are exceptions,
Role of lysosomotropic agents in PTD-mediated protein transduction
Some studies have reported inefficient delivery of PTD-based fusion proteins in vitro [66], [113], [133]. The failure of PTD-mediated protein transduction was explained in two ways. First, nuclear translocation of fusion proteins deposited on the plasma membrane after fixation of cells has been the common explanation, and hence PTD actually does not transduce proteins. Second, deposits of PTD fusion proteins on the cell surface with no biological activity were demonstrated. These studies may
Transcellular property of PTD
Successful protein transductions have resulted in the investigation of the transcellular effect on bystander cells. Limited studies have shown that PTD is conferred with transcellular property [125], [141]. Indeed, this behavior is extremely valuable in delivering therapeutic proteins to surrounding cells. Therefore, it is important to know whether a preformed PTD fusion protein or a DNA expression vector for the PTD fusion protein is involved in transcellular transduction. Of note, Tat-PTD is
Validation of true PTD-mediated protein transduction
PTDs are promising tools for transducing presynthesized proteins across the plasma membrane. Nonetheless, because artifacts result from fixation and endosomal entrapment, true cytosolic distribution or targeting to nucleus is hampered by the use of nonvisual methods [134]. There are limited numbers of approaches that can verify the true nature of PTD fusion protein transduction in vitro. The one most commonly employed is trypsin treatment, which removes the surface-bound fusion proteins and
Cytotoxicity of PTD
There are limited toxicity studies on PTD peptide. Tat peptide of 48–85 aa encompassing a PTD domain did not show neurotoxicity in cultures in vitro [166], but prolonged exposure (24 h) of Hela cells to Tat peptide containing alpha helical sequence (37–60) resulted in necrosis of 60% of cells [47] while Tat peptide (43–60) revealed 10–15% toxicity. Short exposure of cell cultures with 20–100 μM concentrations of PTD did not exhibit any adverse effects [47], [167], whereas a 500 μM dose of PTD
Limitations in PTD-mediated protein transduction
Since the inception of protein delivery studies, cell-to-cell movement of transduced fusion protein has not been thoroughly studied. Furthermore, in reality, PTD-based delivery of fusion proteins will invariably result in nuclear targeting [63], [135], [142], [143], [144], [145], [146], [147], [148], [168] which may not be required in every case. It is useful, however, in some cases such as expression of single chain antibody fragment, where the product is required in the nucleus to inhibit
Challenges and future of PTD
PTD fusion proteins have great potential, especially for in vitro studies. Application of PTD-directed protein delivery in vivo could prove useful under certain situations, where immediate administration of presynthesized proteins is required. But because of limited evidence and lack of a uniform means of producing proteins, and the poor protein transduction property of PTD, its wide scale use is likely to be delayed. Furthermore, because of inherent variations in the properties of different
Conclusions
PTD of HIV Tat protein is a nuclear localization signal which is conferred with mild protein transduction property. PTD delivers the cargo to the nucleus, but lacks transcellular property. The taming of the PTD has resulted in more potent PTDs for cytoplasmic delivery. Finally, PTD may be envisioned as a universal protein and nucleic acid transducer but obviously not in the present form.
Acknowledgements
The work is supported by NIH grant (RO1 NS050064) to AC. AC also thanks Drs. Krister Kristensson, Department of Neuroscience, Karolinska Institute, Stockholm, Sweden; Suzanne Gartner, Avindra Nath and Pamela Talalay, Department of Neurology, Johns Hopkins University, Baltimore, USA, for discussions and facilities.
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