Role of Tat-Mediated PDZ Peptide Delivery in Pain Therapy

doi:10.4236/jbnb.2011.225071

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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.

E-mail: *[email protected]

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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