Systemic treatment with ubiquitin carboxy terminal hydrolase L1 TAT protein ameliorates axonal injury and reduces functional deficits after traumatic brain injury in mice.

Traumatic brain injury (TBI) is often associated with axonal injury that leads to significant motor and cognitive deficits. Ubiquitin carboxy terminal hydrolase L1 (UCHL1) is highly expressed in neurons and loss of its activity plays an important role in the pathogenesis of TBI. Fusion protein was constructed containing wild type (WT) UCHL1 and the HIV trans-activator of transcription capsid protein transduction domain (TAT-UCHL1) that facilitates transport of the protein into neurons after systemic administration. Additional mutant proteins bearing cysteine to alanine UCHL1 mutations at cysteine 152 (C152A TAT-UCHL1) that prevents nitric oxide and reactive lipid binding of C152, and at cysteine 220 (C220A TAT-UCHL1) that inhibits farnesylation of the C220 site were also constructed. WT, C152A, and C220A TAT-UCHL1 proteins administered to mice systemically after controlled cortical impact (CCI) were detectable in brain at 1 h, 4 h and 24 h after CCI by immunoblot. Mice treated with C152A or WT TAT-UCHL1 decreased axonal injury detected by NF200 immunohistochemistry 24 h after CCI, but C220A TAT-UCHL1 treatment had no significant effect. Further study indicated that WT TAT-UCHL1 treatment administered 24 h after CCI alleviated axonal injury as detected by SMI32 immunoreactivity 7 d after CCI, improved motor and cognitive deficits, reduced accumulation of total and K48-linked poly-Ub proteins, and attenuated the increase of the autophagy marker Beclin-1. These results suggest that UCHL1 activity contributes to the pathogenesis of white matter injury, and that restoration of UCHL1 activity by systemic treatment with WT TAT-UCHL1 after CCI may improve motor and cognitive deficits. These results also suggest that farnesylation of the C220 site may be required for the protective effects of UCHL1.

TAT-MeCP2 protein variants rescue disease phenotypes in human and mouse models of Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by pathogenic variants leading to functional impairment of the MeCP2 protein. Here, we used purified recombinant MeCP2e1 and MeCP2e2 protein variants fused to a TAT protein transduction domain (PTD) to evaluate their transduction ability into RTT patient-derived fibroblasts and the ability to carry out their cellular function. We then assessed their transduction ability and therapeutic effects in a RTT mouse model. In vitro, TAT-MeCP2e2-eGFP reversed the pathological hyperacetylation of histones H3K9 and H4K16, a hallmark of abolition of MeCP2 function. In vivo, intraperitoneal administration of TAT-MeCP2e1 and TAT-MeCP2e2 extended the lifespan of Mecp2-/y mice by >50%. This was accompanied by rescue of hippocampal CA2 neuron size in animals treated with TAT-MeCP2e1. Taken together, these findings provide a strong indication that recombinant TAT-MeCP2 can reach mouse brains following peripheral injection and can ameliorate the phenotype of RTT mouse models. Thus, our study serves as a first step in the development of a potentially novel RTT therapy.

Suppression of epithelial to mesenchymal transition markers in mouse lens by a Smad7-based recombinant protein.

Cataracts, a clouding of the eye lens, are a leading cause of visual impairment and are responsible for one of the most commonly performed surgical procedures worldwide. Although generally safe and effective, cataract surgery can lead to a secondary lens abnormality due to transition of lens epithelial cells to a mesenchymal phenotype (EMT) and opacification of the posterior lens capsular bag. Occurring in up to 40% of cataract cases over time, posterior capsule opacification (PCO) introduces additional treatment costs and reduced quality of life for patients. Studies have shown that PCO pathogenesis is driven in part by TGF-ß, signaling through the action of the family of Smad coactivators to effect changes in gene transcription. In the present study, we evaluated the ability of Smad-7, a well characterized inhibitor of TGF-ß -mediated Smad signaling, to suppress the EMT response in lens epithelial cells associated with PCO pathogenesis. Treatment of lens epithelial cells with a cell-permeable form of Smad7 variant resulted in suppressed expression of EMT markers such as alpha smooth muscle actin and fibronectin. A single application of cell-permeable Smad7 variant in the capsular bag of a mouse cataract surgery model resulted in suppression of gene transcripts encoding alpha smooth muscle actin and fibronectin. These results point to Smad7 as a promising biotherapeutic agent for prevention or substantial reduction in the incidence of PCO following cataract surgery.

Resident Microglia Activate before Peripheral Monocyte Infiltration and p75NTR Blockade Reduces Microglial Activation and Early Brain Injury after Subarachnoid Hemorrhage.

Early brain injury (EBI) after aneurysmal subarachnoid hemorrhage (SAH) contributes to high morbidity and mortality. Although it is well recognized that acute neuroinflammation reaction is one of the most important triggers of EBI, pharmacotherapy proved to be clinically effective against the initiating of neuroinflammation after SAH is lacking. The resident microglia and infiltrated peripheral monocyte are two main types of immune cells in central nervous system (CNS) and control the inflammation process in brain after SAH. But the time course and relative contributions of these two immune cell activations after SAH are unknown. The p75 neurotrophin receptor (p75NTR), member of TNF receptor superfamily, expresses on infiltrated peripheral monocytes and suppresses their proinflammatory action after brain insults. But the p75NTR expression on resident microglia in vivo is rarely explored and their function keeps elusive. Therefore, we designed this study to investigate the time course of resident microglia activation and peripheral monocyte infiltration, as well as the microglial expression of p75NTR by using CX3C-chemokine receptor 1 (Cx3cr1) and chemokine receptor 2 (Ccr2) double transgenic mice (Cx3cr1GFP/+Ccr2RFP/+) after SAH. The results showed activated microglia was observed in cortex as early as 24 h and further increased at 48 and 72 h post SAH, while the infiltrated monocyte was not found until 72h. In addition, activated microglia expressed p75NTR acutely and p75NTR specific antagonist TAT-Pep5 significantly reduced microglia activation, neuroinflammation and EBI from 24 to 72 h. Together, these data suggest that the early neuroinflammation reaction might be initiated and intensified mainly by resident microglia rather than infiltrated monocyte at least in the first 48 h after SAH and p75NTR blockading by TAT-Pep5P might alleviate EBI through mediating microglial activation.

In vitro and in vivo delivery of therapeutic proteins using cell penetrating peptides.

The failure of proteins to penetrate mammalian cells or target tumor cells restricts their value as therapeutic tools in a variety of diseases such as cancers. Recently, protein transduction domains (PTDs) or cell penetrating peptides (CPPs) have been shown to promote the delivery of therapeutic proteins or peptides into live cells. The successful delivery of proteins mainly depends on their physicochemical properties. Although, linear cell penetrating peptides are one of the most effective delivery vehicles; but currently, cyclic CPPs has been developed to potently transport bioactive full-length proteins into cells. Up to now, several small protein transduction domains from viral proteins including Tat or VP22 could be fused to other peptides or proteins to entry them in various cell types at a dose-dependent approach. A major disadvantage of PTD-fusion proteins is primary uptake into endosomal vesicles leading to inefficient release of the fusion proteins into the cytosol. Recently, non-covalent complex formation (Chariot) between proteins and CPPs has attracted a special interest to overcome some delivery limitations (e.g., toxicity). Many preclinical and clinical trials of CPP-based delivery are currently under evaluation. Generally, development of more efficient protein transduction domains would significantly increase the potency of protein therapeutics. Moreover, the synergistic or combined effects of CPPs with other delivery systems for protein/peptide drug delivery would promote their therapeutic effects in cancer and other diseases. In this review, we will describe the functions and implications of CPPs for delivering the therapeutic proteins or peptides in preclinical and clinical studies.

TAT-peroxiredoxin 2 Fusion Protein Supplementation Improves Sperm Motility and DNA Integrity in Sperm Samples from Asthenozoospermic Men.

PURPOSE: We compared levels of peroxiredoxin 2 in semen samples from normozoospermic and asthenozoospermic men. The potential effects of TAT-peroxiredoxin 2 fusion protein on sperm motility and DNA integrity were also evaluated. MATERIALS AND METHODS: Semen samples were obtained from 50 normozoospermic and 50 asthenozoospermic men. Lipid peroxidation of semen was determined using a commercial malondialdehyde kit. Sperm DNA fragmentation was evaluated by TUNEL assay. Western blot and immunofluorescence were performed to detect the amount of peroxiredoxin 2 protein in seminal plasma and spermatozoa. Sperm motility, DNA damage and levels of reactive oxygen species were evaluated after TAT-peroxiredoxin 2 fusion protein supplementation to the sperm suspension for 2 and 12 hours of incubation. RESULTS: In asthenozoospermic semen samples a significantly higher level of malondialdehyde and DNA damage was discovered. However, the expression of peroxiredoxin 2 was significantly lower in seminal plasma and spermatozoa compared with that of normozoospermic men. TAT-peroxiredoxin 2 fusion protein was successfully prepared and delivered to the spermatozoa. Interestingly adding TAT-peroxiredoxin 2 in asthenozoospermic sperm suspension effectively defended against the decrease in progressive motility and the increase in DNA damage. CONCLUSIONS: This study shows that supplementation of TAT-peroxiredoxin 2 fusion protein in the sperm suspension from asthenozoospermic men effectively improved sperm motility and DNA integrity by reducing levels of reactive oxygen species. Therefore, we speculate that peroxiredoxin 2 may have an important role as an antioxidant defense in semen and would provide new prevention and therapy alternatives for asthenozoospermia.

Hypoxic resistance of hypodermically transplanted pancreatic islets by using cell-absorbable antioxidant Tat-metallothionein.

Subcutaneous site is ideal for clinical islet transplantation because it has the advantage of simple operation procedure under local anesthesia and can be biopsied when needed. However, the transplantation outcomes at subcutaneous site have been disappointing due to hypoxia-induced oxidative stress by poor vascularization. We hypothesized that subcutaneously transplanted islets would have hypoxia resistance by using internalization of metallothionein (MT), an antioxidant scavenging enzyme, which was mediated by fusion between MT and cell penetrating Tat peptide. The Tat-MT was dose-dependently transduced into islets without any damage. Tat-MT-treated islets could be protected from oxidative stress induced by intracellular nitric oxide donor, sodium nitroprusside (SNP). When Tat-MT-treated islets were subcutaneously transplanted into diabetic nude mice, they normally controlled the blood glucose levels without severe fluctuation (median survival time (MST): >30 days), whereas most untreated islets were rejected (MST 17 days). From the intraperitoneal glucose tolerance test 5 days after posttransplantation, glucose responsiveness of Tat-MT-treated islets was similar to that of normal healthy mice, while untreated islets had delayed glucose responsiveness. From the results of immunohistochemical stain, Tat-MT-treated islets had strong anti-insulin positive cells and lower anti-HIF-1α positive cells. However, untreated islets had rare anti-insulin positive cells and strong anti-HIF-1α-positive cells. Collectively, these findings demonstrated that Tat-MT delivery into islet could offer a new strategy for successful islet transplantation under subcutaneous space.

Injectable microsphere/hydrogel hybrid system containing heat shock protein as therapy in a murine myocardial infarction model.

Heat shock proteins, acting as molecular chaperones, protect heart muscle from ischemic injury and offer a potential approach to therapy. Here we describe preparation of an injectable form of heat shock protein 27, fused with a protein transduction domain (TAT-HSP27) and contained in a hybrid system of poly(d,l-lactic-co-glycolic acid) microsphere and alginate hydrogel. By varying the porous structure of the microspheres, the release of TAT-HSP27 from the hybrid system was sustained for two weeks in vitro. The hybrid system containing TAT-HSP27 was intramyocardially injected into a murine myocardial infarction model, and its therapeutic effect was evaluated in vivo. The sustained delivery of TAT-HSP27 substantially suppressed apoptosis in the infarcted site, and improved the ejection fraction, end-systolic volume and maximum pressure development in the heart. Local and sustained delivery of anti-apoptotic proteins such as HSP27 using a hybrid system may present a promising approach to the treatment of ischemic diseases.

Protective effects of protein transduction domain-metallothionein fusion proteins against hypoxia- and oxidative stress-induced apoptosis in an ischemia/reperfusion rat model.

Ischemic heart diseases caused by insufficient oxygen supply to the cardiac muscle require pharmaceutical agents for the prevention of the progress and recurrence. Metallothionein (MT) has a potential as a protein therapeutic for the treatment of this disease due to its anti-oxidative effects under stressful conditions. In spite of its therapeutic potential, efficient delivery systems need to be developed to overcome limitations such as low transduction efficiency, instability and short half-life in the body. To enhance intra-cellular transduction efficiency, Tat sequence as a protein transduction domain (PTD) was fused with MT in a recombinant method. Anti-apoptotic and anti-oxidative effects of Tat-MT fusion protein were evaluated under hyperglycemia and hypoxia stress conditions in cultured H9c2 cells. Recovery of cardiac functions by anti-apoptotic and anti-fibrotic effects of Tat-MT was confirmed in an ischemia/reperfusion (I/R) rat myocardial infarction model. Tat-MT fusion protein effectively protected H9c2 cells under stressful conditions by reducing intracellular ROS production and inhibiting caspase-3 activation. Tat-MT fusion protein inhibited apoptosis, reduced fibrosis area and enhanced cardiac functions in I/R. Tat-MT fusion protein could be a promising therapeutic for the treatment of ischemic heart diseases.

Inhibition of neutrophil exocytosis ameliorates acute lung injury in rats.

Exocytosis of neutrophil granules contributes to acute lung injury (ALI) induced by infection or inflammation, suggesting that inhibition of neutrophil exocytosis in vivo could be a viable therapeutic strategy. This study was conducted to determine the effect of a cell-permeable fusion protein that inhibits neutrophil exocytosis (TAT-SNAP-23) on ALI using an immune complex deposition model in rats. The effect of inhibition of neutrophil exocytosis by intravenous administration of TAT-SNAP-23 on ALI was assessed by albumin leakage, neutrophil infiltration, lung histology, and proteomic analysis of bronchoalveolar lavage fluid (BALF). Administration of TAT-SNAP-23, but not TAT-control, significantly reduced albumin leakage, total protein levels in the BALF, and intra-alveolar edema and hemorrhage. Evidence that TAT-SNAP-23 inhibits neutrophil exocytosis included a reduction in plasma membrane CD18 expression by BALF neutrophils and a decrease in neutrophil granule proteins in BALF. Similar degree of neutrophil accumulation in the lungs and/or BALF suggests that TAT-SNAP-23 did not alter vascular endothelial cell function. Proteomic analysis of BALF revealed that components of the complement and coagulation pathways were significantly reduced in BALF from TAT-SNAP-23-treated animals. Our results indicate that administration of a TAT-fusion protein that inhibits neutrophil exocytosis reduces in vivo ALI. Targeting neutrophil exocytosis is a potential therapeutic strategy to ameliorate ALI.

Sequential delivery of TAT-HSP27 and VEGF using microsphere/hydrogel hybrid systems for therapeutic angiogenesis.

Ischemic disease is associated with high mortality and morbidity rates, and therapeutic angiogenesis via systemic or local delivery of protein drugs is one potential approach to treat the disease. In this study, we hypothesized that combined delivery of TAT-HSP27 (HSP27 fused with transcriptional activator) and VEGF could enhance the therapeutic efficacy in an ischemic mouse model, and that sequential release could be critical in therapeutic angiogenesis. Alginate hydrogels containing TAT-HSP27 as an anti-apoptotic agent were prepared, and porous PLGA microspheres loaded with VEGF as an angiogenic agent were incorporated into the hydrogels to prepare microsphere/hydrogel hybrid delivery systems. Sequential in vitro release of TAT-HSP27 and VEGF was achieved by the hybrid systems. TAT-HSP27 was depleted from alginate gels in 7 days, while VEGF was continually released for 28 days. The release rate of VEGF was attenuated by varying the porous structures of PLGA microspheres. Sequential delivery of TAT-HSP27 and VEGF was critical to protect against muscle degeneration and fibrosis, as well as to promote new blood vessel formation in the ischemic site of a mouse model. This approach to controlling the sequential release behaviors of multiple drugs could be useful in the design of novel drug delivery systems for therapeutic angiogenesis.

Suppression of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mice by transduced Tat-Annexin protein.

We examined that the protective effects of ANX1 on 12-O-tetradecanoylphorbol- 13-acetate (TPA)-induced skin inflammation in animal models using a Tat-ANX1 protein. Topical application of the Tat-ANX1 protein markedly inhibited TPAinduced ear edema and expression levels of cyclooxygenase-2 (COX-2) as well as pro-inflammatory cytokines such as interleukin- 1 beta (IL-1 ß), IL-6, and tumor necrosis factor-alpha (TNF-α). Also, application of Tat-ANX1 protein significantly inhibited nuclear translocation of nuclear factor-kappa B (NF-κ B) and phosphorylation of p38 and extracellular signalregulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TPA-treated mice ears. The results indicate that Tat-ANX1 protein inhibits the inflammatory response by blocking NF-κ B and MAPK activation in TPA-induced mice ears. Therefore, the Tat-ANX1 protein may be useful as a therapeutic agent against inflammatory skin diseases.

Anti-inflammatory effects of Tat-Annexin protein on ovalbumin-induced airway inflammation in a mouse model of asthma.

Chronic airway inflammation is a key feature of bronchial asthma. Annexin-1 (ANX1) is an anti-inflammatory protein that is an important modulator and plays a key role in inflammation. Although the precise action of ANX1 remains unclear, it has emerged as a potential drug target for inflammatory diseases such as asthma. To examine the protective effects of ANX1 protein on ovalbumin (OVA)-induced asthma in animal models, we used a cell-permeable Tat-ANX1 protein. Mice sensitized and challenged with OVA antigen had an increased amount of cytokines and eosinophils in their bronchoalveolar lavage (BAL) fluid. However, administration of Tat-ANX1 protein before OVA challenge significantly decreased the levels of cytokines (interleukin (IL)-4, IL-5, and IL-13) and BAL fluid in lung tissues. Furthermore, OVA significantly increased the activation of mitogen-activated protein kinase (MAPK) in lung tissues, whereas Tat-ANX1 protein markedly reduced phosphorylation of MAPKs such as extracellular signal-regulated protein kinase, p38, and stress-activated protein kinase/c-Jun N-terminal kinase. These results suggest that transduced Tat-ANX1 protein may be a potential protein therapeutic agent for the treatment of lung disorders including asthma.

Protective effects of intraperitoneal injection of TAT-SOD against focal cerebral ischemia/reperfusion injury in rats.

AIMS: The intracellular superoxide anion has been shown to be involved in brain injury. TAT-Superoxide dismutase (TAT-SOD) can be transduced across the cell membrane to scavenge superoxide. This protein's unique properties make it a promising therapeutic candidate to attenuate cerebral damage. In this study, we sought further the understanding of the fusion protein's cerebral protective effects and the mechanism which is exerted in these effects. MAIN METHODS: Male Sprague Dawley rats (n=100, 230±20 g) were divided randomly into five experimental groups: a sham group, a cerebral Ischemia/Reperfusion (I/R) group treated with saline (20 ml/Kg, i.p.), and three cerebral I/R groups treated with TAT-SOD (25 KU/ml/Kg, i.p.) at either 2h before I/R, 2h after I/R or 4h after I/R. Cerebral I/R injury was facilitated by inducing ischemia for two hours followed by 24h reperfusion. The levels of SOD, Malondialdehyde (MDA), and ATPase in cerebral tissues were determined. The apoptotic indexes were evaluated, and apoptosis genes were analyzed immunohistochemically. KEY FINDINGS: TAT-SOD treatment significantly increased cerebral SOD and ATPase activities, decreased MDA content, and remarkably reduced apoptosis indexes. TAT-SOD treatments 2h before or after I/R significantly reduced caspase-3 and bax proteins and boosted bcl-2 protein, while the treatment at 4h after I/R showed no influence on the three proteins. SIGNIFICANCE: TAT-SOD treatment effectively enhanced cerebral antioxidant ability, reduced lipid peroxidation, preserved mitochondrial ATPase and thus inhibited nerve cell apoptosis. The effective treatment window extended from 2h before to 2h after I/R.

A new synthetic protein, TAT-RH, inhibits tumor growth through the regulation of NFkappaB activity.

BACKGROUND: Based on its role in angiogenesis and apoptosis, the inhibition of NFkappaB activity is considered an effective treatment for cancer, hampered by the lack of selective and safe inhibitors. We recently demonstrated that the RH domain of GRK5 (GRK5-RH) inhibits NFkappaB, thus we evaluated its effects on cancer growth. METHODS: The role of GRK5-RH on tumor growth was assessed in a human cancer cell line (KAT-4). RH overexpression was induced by adenovirus mediated gene transfer; alternatively we administered a synthetic protein reproducing the RH domain of GRK5 (TAT-RH), actively transported into the cells. RESULTS: In vitro, adenovirus mediated GRK5-RH overexpression (AdGRK5-NT) in human tumor cells (KAT-4) induces IkappaB accumulation and inhibits NFkappaB transcriptional activity leading to apoptotic events. In BALB/c nude mice harboring KAT-4 induced neoplasias, intra-tumor delivery of AdGRK5-NT reduces in a dose-dependent fashion tumor growth, with the highest doses completely inhibiting it. This phenomenon is paralleled by a decrease of NFkappaB activity, an increase of IkappaB levels and apoptotic events. To move towards a pharmacological setup, we synthesized the TAT-RH protein. In cultured KAT-4 cells, different dosages of TAT-RH reduced cell survival and increased apoptosis. In BALB/c mice, the anti-proliferative effects of TAT-RH appear to be dose-dependent and highest dose completely inhibits tumor growth. CONCLUSION: Our data suggest that GRK5-RH inhibition of NFkappaB is a novel and effective anti-tumoral strategy and TAT-RH could be an useful tool in the fighting of cancer.

TAT-Hsp70-mediated neuroprotection and increased survival of neuronal precursor cells after focal cerebral ischemia in mice.

Cerebral ischemia stimulates endogenous neurogenesis within the subventricular zone and the hippocampal dentate gyrus of the adult rodent brain. However, such newly generated cells soon die after cerebral ischemia. To enhance postischemic survival of neural precursor cells (NPC) and long-lasting neural regeneration, we applied the antiapoptotic chaperone heat shock protein 70 (Hsp70) fused to a cell-penetrating peptide derived from the HIV TAT to ensure delivery across the blood-brain barrier and the cell membrane. After transient focal cerebral ischemia in mice, TAT-Hsp70 was intravenously injected concomitant with reperfusion and additionally on day 14 after stroke. TAT-Hsp70 treatment resulted in smaller infarct size (27.1+/-9.0 versus 109.0+/-14.0 and 88.5+/-26.0 mm(3) in controls) and in functional improvement as assessed by the rota rod, tight rope, and water maze tests when compared with saline- and TAT-hemagglutinin-treated controls. In addition, postischemic survival of endogenous doublecortin (Dcx)-positive NPC was improved within the lesioned striatum of TAT-Hsp70-treated animals for up to 4 weeks after stroke without changing overall cell proliferation of BrdU(+) cells. Thus, TAT-Hsp70 treatment after stroke may be a promising tool to act neuroprotective and improve postischemic functional outcome, and also to increase survival of endogenous NPC after stroke.

TAT-Bcl-x(L) improves survival of neuronal precursor cells in the lesioned striatum after focal cerebral ischemia.

Cerebral ischemia activates endogenous neurogenesis in the subventricular zone (SVZ) and the dentate gyrus. Consecutively, SVZ-derived neural precursors migrate towards ischemic lesions. However, functional relevance of activated neurogenesis is limited by poor survival of new-born precursors. We therefore employed the HI-virus-derived fusion protein TAT-Bcl-x(L) to study the effects of acute anti-apoptotic treatment on endogenous neurogenesis and functional outcome after transient cerebral ischemia in mice. TAT-Bcl-x(L) treatment led to significantly reduced acute ischemic cell death (128+/-23 vs. 305+/-65 TUNEL+ cells/mm(2) in controls) and infarct volumes resulting in less motor deficits and improved spatial learning. It significantly increased survival of doublecortin (Dcx)-positive neuronal precursors (389+/-96 vs. 213+/-97 Dcx+ cells in controls) but did not enhance overall post-ischemic cell proliferation or lesion-specific neuronal differentiation 28 days after ischemia. Our data demonstrate that post-stroke TAT-Bcl-x(L)-treatment results in acute neuroprotection, improved functional outcome, and enhanced survival of lesion-specific neuronal precursor cells after cerebral ischemia in mice.

TAT-mediated intracellular delivery of NPM-derived peptide induces apoptosis in leukemic cells and suppresses leukemogenesis in mice.

Nucleophosmin (NPM) is frequently overexpressed in leukemias and other tumors. NPM has been reported to suppress oncogene-induced senescence and apoptosis and may represent a therapeutic target for cancer. We fused a NPM-derived peptide to the HIV-TAT (TAT-NPMDeltaC) and found that the fusion peptide inhibited proliferation and induced apoptotic death of primary fibroblasts and preleukemic stem cells. TAT-NPMDeltaC down-regulated several NF-kappaB-controlled survival and inflammatory proteins and suppressed NF-kappaB-driven reporter gene activities. Using an inflammation-associated leukemia model, we demonstrate that TAT-NPMDeltaC induced proliferative suppression and apoptosis of preleukemic stem cells and significantly delayed leukemic development in mice. Mechanistically, TAT-NPMDeltaC associated with wild-type NPM proteins and formed complexes with endogenous NPM and p65 at promoters of several antiapoptotic and inflammatory genes and abrogated their transactivation by NF-kappaB in leu-kemic cells. Thus, TAT-delivered NPM peptide may provide a novel therapy for inflammation-associated tumors that require NF-kappaB signaling for survival.

Anti-apoptotic therapy with a Tat fusion protein protects against excitotoxic insults in vitro and in vivo.

A number of gene therapy approaches have been developed for protecting neurons from necrotic neurological insults. Such therapies are limited by the need for transcription and translation of the protective protein, delaying therapeutic impact. As an alternative, we explore the neuroprotective potential of protein therapy, using a fusion protein comprised of the death-suppressing BH4 domain of the Bcl-xL protein and the protein transduction domain of the human immunodeficiency virus Tat protein. This fusion protein decreased neurotoxicity caused by the excitotoxins glutamate and kainic acid in primary hippocampal cultures, and decreased hippocampal damage in vivo in an excitotoxic seizure model.

Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study.

The use of CPPs (cell-penetrating peptides) as delivery vectors for bioactive molecules has been an emerging field since 1994 when the first CPP, penetratin, was discovered. Since then, several CPPs, including the widely used Tat (transactivator of transcription) peptide, have been developed and utilized to translocate a wide range of compounds across the plasma membrane of cells both in vivo and in vitro. Although the field has emerged as a possible future candidate for drug delivery, little attention has been given to the potential toxic side effects that these peptides might exhibit in cargo delivery. Also, no comprehensive study has been performed to evaluate the relative efficacy of single CPPs to convey different cargos. Therefore we selected three of the major CPPs, penetratin, Tat and transportan 10, and evaluated their ability to deliver commonly used cargos, including fluoresceinyl moiety, double-stranded DNA and proteins (i.e. avidin and streptavidin), and studied their effect on membrane integrity and cell viability. Our results demonstrate the unfeasibility to use the translocation efficacy of fluorescein moiety as a gauge for CPP efficiency, since the delivery properties are dependent on the cargo used. Furthermore, and no less importantly, the toxicity of CPPs depends heavily on peptide concentration, cargo molecule and coupling strategy.