Journal of Biomaterials and Nanobiotechnology, 2011, 2, 596-600
doi:10.4236/jbnb.2011.225071 Published Online December 2011 (http://www.scirp.org/journal/jbnb)
Role of Tat-Mediated PDZ Peptide Delivery in
Pain Therapy
Haiying Wu1,2, Feng Tao3*
1Department of Critical Care Medicine, The First Affiliated Hospital of Kunming Medical College, Kunming, China; 2Department of
Neurology, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA; 3Department of Anesthesiology and Critical
Care Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA.
Received September 13th, 2011; revised October 24th, 2011; accepted November 11th, 2011.
ABSTRACT
Delivery of therapeutic peptides or proteins into tissues is severely limited by the size and biochemical properties of the
molecules. Protein transduction domain (PTD)-mediated cargo transduction represents a novel and promising strategy
to deliver biologically active peptides in vivo. The first PTD was identified from the HIV-1 transactivating transcrip-
tional activator protein Tat in 1988. Since then, other PTDs have also been identified, including the third α-helix of the
antennapedia homeotic transcription factor and synthetic peptide carriers. However, Tat PTD (amino acids 47 - 57)
has shown markedly better ability for intracellular delivery than other PTDs. It has been demonstrated that fusion pep-
tides containing the Tat PTD enter the central nervous system after systemic administration. Our previous study has
shown that i.p. injected Tat-PSD-95 PDZ2 expresses in the central nervous system and significantly disrupts PDZ do-
main-mediated protein interactions between PSD-95 and N-methyl-D-aspartate receptor subunit NR2A/2B, thereby
alleviating chronic pain. Therefore, Tat-mediated intracellular delivery can be used for systemic administration of an-
algesics in pain management.
Keywords: Tat Peptides, Protein Transduction Domain, Protein Interactions, PDZ Domains, Chronic Pain
1. Introduction
The discovery of small cationic peptides (8 - 16 amino
acids in length) termed protein transduction domains
(PTDs) or cell-penetrating peptides [1,2], which cross
biological membranes, has emerged as a venerable Tro-
jan horse to transport large, biologically active molecules,
such as peptides, proteins, and oligonucleotides, into
mammalian cells in vitro, as well as in preclinical models
and clinical trials in vivo. Protein transduction was origi-
nally observed in 1988 after full-length HIV-1 transacti-
vating transcriptional activator protein (Tat) was shown
to enter mammalian cells, leading to transcriptional acti-
vation from an HIV-1 long-terminal repeat promoter
construct [3,4]. Since the initial discovery of Tat-medi-
ated transduction, other novel transduction domains have
been identified within several other proteins, including
the third α-helix of the antennapedia homeotic transcrip-
tion factor [5-7] and synthetic peptide carriers, such as
polylysine and polyarginine [8-10].
Chronic pain affects more than 50 million Americans
per year and costs more than $100 billion each year in
health care and lost productivity. It is often poorly man-
aged by current drugs, such as opioids and non-steroidal
anti-inflammatory drugs. Considerable evidence indi-
cates that the development of central hyperexcitability
and persistent pain involves the activation of N-methyl-
D-aspartate receptors (NMDARs), which play an impor-
tant role in the processing of nociceptive information
[11-14]. However, directly blocking the function of
NMDARs is therapeutically impractical because doing so
would also impede other vital synaptic transmissions in
the central nervous system (CNS). Postsynaptic density
protein-95 (PSD-95), a PDZ-containing scaffolding pro-
tein, has been identified to interact and attach NMDARs
to internal signaling molecules at neuronal synapses of
the CNS [15,16]. This function suggests that PSD-95
might be involved in physiological and pathophysiologi-
cal actions triggered via the activation of NMDARs in
the CNS. Therefore, targeting PSD-95 protein represents
a potential therapeutic approach for diseases that involve
NMDAR signaling. NMDAR-PSD-95 protein interac-
tions are mediated by a PDZ domain (a term derived
from the names of the first three proteins identified to

Role of Tat-Mediated PDZ Peptide Delivery in Pain Therapy597
contain the domain: PSD-95, Dlg, and ZO-1). PSD-95
possesses three PDZ domains. The second (PSD-95
PDZ2) interacts with NMDAR NR2 subunits at a
seven-amino acid, COOH-terminal domain that contains
a terminal tSXV motif (where S is serine, X is any amino
acid, and V is valine) [15].
2. PDZ Domain-Mediated Protein
Interactions in the CNS
PDZ domains were discovered from consensus se-
quences of 80 - 90 amino acid residues of three proteins:
the postsynaptic density protein PSD-95, the Drosophila
septate junction protein Dlg, and the tight junction pro-
tein ZO-1 [15]. The common structure of PDZ domains
comprises six β strands (βA-βF) and two α helices (αA
and αB). Peptide ligands from the extreme C-termini of
targeted proteins bind as an antiparallel β-strand in a
groove formed by the second α-helix (αB) and the second
β-strand (βB) of the PDZ domains. Amino acid residues
at the 0 and –2 positions of the carboxyl peptide play
dominant roles in the peptide’s binding to a cognate PDZ
domain, although residues at the –1 and –3 positions and
those further upstream also contribute to the binding.
Despite similarities in secondary structure and the com-
mon preference for C-terminal ligands, PDZ domains
display different binding specificity.
Generally, PDZ domains are classified into three types
according to their specificity for C-terminal peptide
ligands. In type I PDZ domains, such as those of PSD-95
and PSD-93, a serine or threonine residue occupies the
–2 position of the C-terminal ligand. This type of PDZ
domain is specific for the S/T-X-Ф target sequence (X:
unspecified amino acid; Ф: hydrophobic amino acid) [17].
For instance, the type I PDZ domains mediate the protein
interactions between the C-terminal ligand of NMDA
receptor subunit NR2A/2B and the second PDZ domain
of PSD-95 or PSD-93. In contrast, the type II PDZ do-
main (such as that of PICK1), specific for -X-Ф-X-Ф
sequence, is characterized by hydrophobic residues at
both the –2 position of the peptide ligand and the αB1
position of the PDZ domain [17]. For instance, the type
II domain mediates the protein interactions between the
C-terminal ligand of AMPA receptor subunit GluR2 and
the PDZ domain of PICK1. The type III PDZ domain,
such as that of neuronal nitric oxide synthase, is specific
for a -X-D/E-X-Ф pattern and prefers negatively charged
amino acids at the –2 position [18].
3. Mechanisms Underlying Tat-Mediated
Intracellular Delivery
Cell surface heparan sulphate proteoglycans (HSPGs)
have been shown to play a role in Tat-mediated intracel-
lular delivery [19,20]. Tat-linked cargoes bind to HSPGs
on the plasma membrane and are then taken up by endo-
cytosis [21,22]. In the endocytosed vesicles, heparan
sulphate is degraded by heparinase, which releases the
Tat-linked cargoes [23]. The involvement of heparan
sulphate in Tat-mediated intracellular delivery has been
evidenced in three ways [21]. 1) Enzymatic removal of
extracellular heparan sulphate drastically reduces cell
uptake of Tat-linked cargoes; 2) The co-administration of
exogenous heparan sulphate competitively inhibits the
following cellular effects: the Tat uptake itself, the for-
mation of aggregates on the cell membrane, and the re-
duction of the extracellular acidification rate; 3) The dif-
ferential interference contrast image contrast of these
aggregates on the membrane could be mimicked with a
source of binding of exogenous heparan sulphate to the
Tat.
Previous studies have suggested that Tat-linked car-
goes enter cells via an energy-dependent endocytic proc-
ess [24,25], because the membrane inhibitor sodium
azide inhibits ATP production and impairs endocytosis
[26]. The mechanism of entry by clathrin-coated vesicles
has been ruled out. The receptor-independent endocytosis
known as macropinocytosis has been demonstrated [27,
28]. It has been observed that Tat-linked cargoes are lo-
calized and sequestered in endosomes. Upon treatment
with endosomal releasing polymer, poly(propylacrylic
acid), the fusion cargoes are released into the cytoplasm
[29]. However, the particular intracellular delivery path-
way is dependent on characteristics of the cargo fused,
conformation attained after fusion with Tat, and experi-
mental conditions.
4. Tat-Mediated PDZ Peptide Delivery in
Chronic Pain Treatment
The ability of Tat-linked cargoes to cross the blood-brain
barrier has encouraged us to use this system in develop-
ing potential targets for chronic pain treatment (Figure
1). Our previous studies have demonstrated the roles of
PDZ-containing scaffolding proteins (such as PSD-95) in
the spinal transduction of NMDA receptor signaling in
chronic pain states and found that deficiency of spinal
PSD-95 significantly inhibits the development and main-
tenance of chronic pain [30,31]. To define further the
role of PDZ domain-mediated NMDAR-PSD-95 protein
interactions in chronic pain, we constructed a peptide
comprising the PSD-95 PDZ2 and rendered it cell per-
meable by fusing it to Tat PTD to obtain the fusion pep-
tide Tat-PSD-95 PDZ2. We injected mice intraperito-
neally (systemically) or intrathecally (locally) with this
fusion peptide and then assessed their behavioral re-
sponses to intraplantar injection of complete Freund’s
adjuvant (CFA) [32]. Importantly, we showed that Tat-
PSD-95 PDZ2 was delivered into the spinal cord after

Role of Tat-Mediated PDZ Peptide Delivery in Pain Therapy 598
(a) (b)
Figure 1. Tat-linked PDZ peptide disrupts NMDA receptor signaling in the central nervous system. (a) Under physiological
condition, the scaffolding protein PSD-95 attaches NMDA receptors to internal signaling molecules at neuronal synapses by
PDZ domain mediated protein-protein interactions with NMDA receptors and neuronal nitric oxide synthase (nNOS). Thus,
PSD-95 might be involved in physiological and pathophysiological actions triggered via the activation of NMDA receptors; (b)
The second PDZ domain of PSD-95 (PSD-95 PDZ2) interacts with the seven-amino acid, COOH-terminal domain containing
a terminal tSXV motif (where S is serine, X is any amino acid, and V is valine) common to NR2 subunits of NMDA receptors.
The PSD-95 PDZ2 also forms a heterodimeric PDZ-PDZ interaction with the PDZ domain of nNOS. Thus, Tat-linked PDZ
peptide Tat-PSD-95 PDZ2 can disrupt these interactions and NMDA receptor signaling in the central nervous system. Be-
cause the activation of NMDA receptors plays an important role in the processing of nociceptive information, the disruption
of PDZ domain-mediated protein-protein interactions within NMDA receptor signaling may inhibit the development of
chronic pain.
intraperitoneal injection. Furthermore, the fusion peptide
dose-dependently disrupted the protein-protein interac-
tions between NMDAR NR2 subunits and PSD-95 and
significantly inhibited CFA-induced chronic inflamma-
tory pain [32]. These results suggest that PDZ domain-
mediated protein interactions at spinal synapses might
play an important role in the molecular mechanisms of
chronic inflammatory pain behaviors. Our study provides
novel insight into the molecular mechanisms that under-
lie chronic inflammatory pain states and a new approach
for chronic inflammatory pain therapy. Thus, cell-per-
meable Tat peptides can treat chronic pain by disrupting
PDZ domain-mediated protein-protein interactions. The
Tat-linked PDZ peptides might be ready for clinical trials
to develop a specific drug for chronic pain therapy.
5. Perspectives
In the last decade, the applications of the Tat-mediated
intracellular delivery system have been expanded due to
its several advantages, such as size-independent and non-
viral transportation. This system can be improved if we
modify the Tat PTD in order to make its delivery more
specific, thereby widening its therapeutic potential. The
ability to specifically deliver Tat-linked cargoes could
theoretically reduce the side effects produced by the de-
livery of cargo to undesired organs and reduce the total
amount of Tat-fused peptide drugs for CNS diseases in-
cluding chronic pain.
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