Transduced Tat-SOD fusion protein protects against ischemic brain injury

Reactive oxygen species (ROS) are implicated in reperfusion injury after transient focal cerebral ischemia. The antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD), is one of the major means by which cells counteract the deleterious effects of ROS after ischemia. Recently, we reported that when Tat-SOD fusion protein is transduced into pancreatic beta cells it protects the beta cells from destruction by relieving oxidative stress in ROS-implicated diabetes (Eum et al., 2004). In the present study, we investigated the protective effects of Tat-SOD fusion protein against neuronal cell death and ischemic insults. When Tat-SOD was added to the culture medium of neuronal cells, it rapidly entered the cells and protected them against paraquat-induced cell death. Immunohistochemical analysis revealed that Tat-SOD injected intraperitoneally (i.p.) into mice has access to various tissues including brain neurons. When i.p. injected into gerbils, Tat-SOD prevented neuronal cell death in the hippocampus in response to transient fore-brain ischemia. These results suggest that Tat-SOD provides a strategy for therapeutic delivery in various hu-man diseases, including stroke, related to this anti-oxidant enzyme or to ROS.     

Transduction efficacy of Tat-Cu,Zn-superoxide dismutase is enhanced by copper ion recovery of the fusion protein

We previously reported that Tat-Cu,Zn-superoxide dismutase (Tat-SOD) can be directly transduced into mammalian cells across the lipid membrane barrier. To enhance the therapeutic potential of Tat-SOD for the treatment of various disorders that are related to this antioxidant enzyme, the transduction efficacy of Tat-SOD should be heightened. Therefore, we investigated whether copper ion recovery of the fusion protein could enhance the transduction potential of Tat-SOD in cultured HeLa cells. The results showed that the transduction potential of Tat-SOD was markedly enhanced by copper ions, and moderately increased by zinc ions. Compared with Tat-SOD, the Tat-SOD that recovered the copper ion (CR-Tat-SOD) achieved a significant increase in intracellular concentration and enzymatic activity. Therefore, CR-Tat-SOD was transduced into HeLa cells in a rapid saturation manner, but Tat-SOD was shown in a time-dependent manner. With the higher transduction efficacy of CR-Tat-SOD than that of Tat-SOD, the transduced CR-Tat-SOD significantly increased the viability of HeLa cells that were pretreated with paraquat, an intracellular superoxide anion generator. Although the mechanism of the enhanced transduction of Tat-SOD by copper ions is still unanswered, these results indicate that copper ions facilitate the transduction of SOD. These then significantly increase the biological effectiveness of this antioxidant enzyme.     

9-polylysine protein transduction domain: enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin

Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), have been considered to have a beneficial effect against various diseases that are mediated by the reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against oxidative stresses, the lack of their transduction ability into cells resulted in a limited ability to detoxify intracellular ROS. To render the SOD enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant SOD proteins were generated. A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment that encodes the 9 amino acids Tat protein transduction domain (RKKRRQRRR) of HIV-1 and lysine rich peptide (KKKKKKKKK) in a bacterial expression vector in order to produce a genetic in-frame Tat-SOD and 9Lys-SOD fusion protein, respectively. The expressed and purified Tat-SOD and 9Lys-SOD fusion proteins can transduce into human fibroblast cells, and they were enzymatically active and stable for 24 h. The cell viability of the fibroblast cells that were treated with paraquat, an intracellular superoxide anion generator, was increased by the transduced Tat-SOD or 9Lys-SOD. The transduction efficacy of 9Lys-SOD was more efficient than that of Tat-SOD. We evaluated the ability of the SOD fusion pmteins to transduce into animal skin. This analysis showed that Tat-SOD and 9Lys-SOD fusion proteins efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin (judged by the immunohistochemistry and specific enzyme activities). The enzymatic activity of the transduced 9Lys-SOD was higher than that of Tat-SOD, indicating that the penetration of 9Lys-SOD was more efficient when put into the skin. These results suggest Tat-SOD and 9Lys-SOD fusion proteins can be used as anti-aging cosmetics, or in protein therapy, for various disorders that are related to this antioxidant enzyme and ROS.     

Active component of Fatsia japonica enhances the transduction efficiency of Tat-SOD fusion protein both in vitro and in vivo

It has been reported that Tat-SOD can be directly transduced into mammalian cells and skin and acts as a potential therapeutic protein in various diseases. To isolate the compound that can enhance the transduction efficiency of Tat-SOD, we screened a number of natural products. 3-O-[beta-D-Glucopyranosyl(1-->4)-alpha-L-arabinopyranosyl]- hederagenin (OGAH) was identified as an active component of Fatsia japonica and is known as triterpenoid glycosides (hederagenin saponins). OGAH enhanced the transduction efficiencies of Tat-SOD into HeLa cells and mice skin. The enzymatic activities in the presence of OGAH were markedly increased in vitro and in vivo when compared with the controls. Although the mechanism is not fully understood, we suggest that OGAH, the active component of Fatsia japonica, might change the conformation of the membrane structure and it may be useful as an ingredient in antiaging cosmetics or as a stimulator of therapeutic proteins that can be used in various disorders related to reactive oxygen species (ROS).     

In vivo protein transduction: biologically active intact pep-1-superoxide dismutase fusion protein efficiently protects against ischemic insult

Reactive oxygen species (ROS) are implicated in reperfusion injury after transient focal cerebral ischemia. The antioxidant enzyme Cu,Zn-superoxide dismutase (SOD) is one of the major means by which cells counteract the deleterious effects of ROS after ischemia. Recently, we reported that denatured Tat-SOD fusion protein is transduced into cells and skin tissue. Moreover, PEP-1 peptide, which has 21 amino acid residues, is a known carrier peptide that delivers full-length native proteins in vitro and in vivo. In the present study, we investigated the protective effects of PEP-1-SOD fusion protein after ischemic insult. A human SOD gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-SOD fusion protein. The expressed and purified fusion proteins were efficiently transduced both in vitro and in vivo with a native protein structure. Immunohistochemical analysis revealed that PEP-1-SOD injected intraperitoneally (i.p.) into mice can have access into brain neurons. When i.p.-injected into gerbils, PEP-1-SOD fusion proteins prevented neuronal cell death in the hippocampus caused by transient forebrain ischemia. These results suggest that the biologically active intact forms of PEP-1-SOD provide a more efficient strategy for therapeutic delivery in various human diseases related to this antioxidant enzyme or to ROS, including stroke.     

HIV-1 Tat-mediated protein transduction of Cu,Zn-superoxide dismutase into pancreatic beta cells in vitro and in vivo

Reactive oxygen species (ROS) are considered an important mediator in pancreatic beta cell destruction, thereby triggering the development of insulin-dependent diabetes mellitus. In the present study, we investigated the HIV-1 Tat protein transduction domain-mediated transduction of Cu,Zn-superoxide dismutase (SOD), which supplies SOD activity exogenously in pancreatic beta cells under oxidative stress. Tat-SOD fusion protein was successfully delivered into insulin-producing RINm5F cells and rat islet cells. The intracellular dismutation activities of SOD were found to increase in line with the amount of protein delivered into the cells. ROS, nitric oxide-induced cell death, lipid peroxidation, and the DNA fragmentation of insulin-producing cells were found to be significantly reduced when the cells were pretreated with Tat-SOD. Next, we examined the in vivo transduction of Tat-SOD into streptozotocin-induced diabetic mice. A single intraperitoneal injection of Tat-SOD resulted in the delivery of this biologically active enzyme to the pancreas. Moreover, increased radical scavenging activity in the pancreas was induced by multiple injections of Tat-SOD, and this enhanced the tolerance of pancreatic beta cells to oxidative stress. These results suggest that the transduction of Tat-SOD offers a new strategy for protecting pancreatic beta cells from destruction by relieving oxidative stress in ROS-implicated diabetes.     

Suppression of TNF-alpha-induced MMP-9 expression by a cell-permeable superoxide dismutase in keratinocytes

Up-regulation of selected matrix metalloproteinases (MMPs) such as MMP-9 contributes to inflammatory processes during the development of various skin diseases, such as atopic dermatitis. In this study, we examined the effect of a cell-permeable superoxide dismutase (Tat-SOD) on TNF-α-induced MMP-9 expression in human keratinocyte cells (HaCaT). When Tat-SOD was added to the culture medium of HaCaT cells, it rapidly entered the cells in dose- and time-dependent manners. Tat-SOD decreased TNF-α-induced reactive oxygen species (ROS) generation. Tat-SOD also inhibited TNF-α-induced NF-κB DNA binding activity. Treatment of HaCaT cells with Tat-SOD significantly inhibited TNF-α-induced mRNA and protein expression of MMP-9, as measured by RT-PCR and Western blot analysis. In addition, Tat-SOD suppressed TNF-α-induced gelatinolytic activity of MMP-9. Taken together, our results indicate that Tat-SOD can suppress TNF-α-induced MMP-9 expression via ROS-NF-κB-dependent mechanisms in keratinocytes, and therefore can be used as an immunomodulatory agent against inflammatory skin diseases related to oxidative stress.     

Enhanced transduction of Cu,Zn-superoxide dismutase with HIV-1 Tat protein transduction domains at both termini

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD) is responsible for highly efficient protein transduction across plasma membranes. In a previous study, we showed that Tat-Cu,Zn-superoxide dismutase (Tat-SOD) can be directly transduced into mammalian cells across the lipid membrane barrier. In this study, we fused the human SOD gene with a Tat PTD transduction vector at its N- and/or C-terminus. The fusion proteins (Tat-SOD, SOD-Tat, Tat-SOD-Tat) were purified from Escherichia coli and their ability to enter cells in vitro and in vivo compared by Western blotting and immunohistochemistry. The transduction efficiencies and biological activities of the SOD fusion protein with the Tat PTD at either terminus were equivalent and lower than the fusion protein with the Tat PTD at both termini. The availability of a more efficient SOD fusion protein provides a powerful vehicle for therapy in human diseases related to this anti-oxidant enzyme and to reactive oxygen species.     

Kinetic analysis of the interaction of tissue transglutaminase with a nonpeptidic slow-binding inhibitor

Tissue transglutaminase (TGase) is a Ca2+-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues and is allosterically regulated by GTP. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate as part of these disorders. Given the role that TGase plays in neurodegenerative disorders, we initiated a research program to discover inhibitors of this enzyme that might ultimately be developed into therapeutic agents. To identify such inhibitors, we screened 110,000 druglike compounds for their ability to inhibit TGase [Case, A., et al. (2005) Anal. Biochem. 338, 237-244]. In this paper, we report the kinetics of interaction of human TGase with one of the inhibitors that we identified, LDN-27219. We found that this compound is a reversible, slow-binding inhibitor that appears not to bind at the enzyme's active site but rather at the enzyme's GTP site, or a site that regulates binding of GTP. Interestingly, the potency and kinetics of inhibition are dependent on substrate structure and suggest a novel mechanism of inhibition that involves differential binding of LDN-27219 to multiple conformational states of this enzyme.     

The potential of stem cells for the treatment of brain tumors and globoid cell leukodystrophy

Stem cells of different origin are under careful scrutiny as potential new tools for the treatment of several neurological diseases. The major focus of these reaserches have been neurodegenerative disorders, such as Huntington Chorea or Parkinson Disease (Shihabuddin et al., 1999). More recently attention has been devoted to their use for brain repair after stroke (Savitz et al., 2002). In this review we will focus on the potential of stem cell treatments for glioblastoma multiforme (Holland, 2000), the most aggressive primary brain tumor, and globoid cell leukodystrophy (Krabbe disease), a metabolic disorder of the white matter (Berger et al., 2001). These two diseases may offer a paradigm of what the stem cell approach may offer in term of treatment, alone or in combination with other therapeutic approaches. Two kinds of stem cells will be consideredhere: neural stem cells and hematopoietic stem cells, both obtained after birth. The review will focus on experimental models, with an eye on clinical perspectives. This revised version was published online in July 2006 with corrections to the Cover Date.     

Skinny dipping for stem cells

Stem cells have been big news for the past couple of years and yet they remain remarkably inscrutable in terms of declaring their true nature and identity. On pages 778-784 of this issue, Toma et al. describe the identification of a new type of stem cell from the dermis of ths skin, called skin-derived precursor (SKP) cells. These can be converted into several differentiated cell types in vitro, including neurons, and might become a source of cells for therapeutic tissue repair.     

Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells.

Antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) have been considered to have a beneficial effect against various diseases mediated by reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against the oxidative stresses, the lack of their transduction ability into cells resulted in limited ability to detoxify intracellular ROS. To render the catalase enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant catalase proteins were generated. A human liver catalase gene was cloned and fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) and arginine-rich peptides (RRRRRRRRR) in a bacterial expression vector to produce genetic in-frame Tat-CAT and 9Arg-CAT fusion proteins, respectively. The expressed and purified fusion proteins can be transduced into mammalian cells (HeLa and PC12 cells) in a time- and dose-dependent manner when added exogenously in culture medium, and transduced fusion proteins were enzymatically active and stable for 60 h. When exposed to H(2)O(2), the viability of HeLa cells transduced with Tat-CAT or 9Arg-CAT fusion proteins was significantly increased. In combination with transduced SOD, transduced catalase also resulted in a cooperative increase in cell viability when the cells were treated with paraquat, an intracellular antioxide anion generator. We then evaluated the ability of the catalase fusion proteins to transduce into animal skin. This analysis showed that Tat-CAT and 9Arg-CAT fusion proteins efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal skin, as judged by immunohistochemistry and specific enzyme activities. These results suggest that Tat-CAT and 9Arg-CAT fusion proteins can be used in protein therapy for various disorders related to this antioxidant enzyme.     

Regulation of skin inflammation and angiogenesis by EC-SOD via HIF-1α and NF-κB pathways

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that breaks down superoxide anion into oxygen and hydrogen peroxide in extracellular spaces and plays key roles in controlling pulmonary and vascular diseases in response to oxidative stresses. We aimed to investigate the role of EC-SOD in angiogenesis and inflammation in chronic inflammatory skin disorders such as psoriasis. Overexpressed EC-SOD reduced expression of angiogenic factors and proinflammatory mediators in hypoxia-induced keratinocytes and in ultraviolet B-irradiated mice, whereas the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase-1 and anti-inflammatory cytokine interleukin-10 were increased. EC-SOD decreased new vessel formation, epidermal edema, and inflammatory cell infiltration in UVB-irradiated transgenic mice. Moreover, cells treated with recombinant human EC-SOD showed inhibited endothelial tube formation and cell proliferation. Overall, the antiangiogenic and anti-inflammatory effects of EC-SOD might be due to suppression of hypoxia-inducible factor-1α, protein kinase C, and nuclear factor-κB expression. Furthermore, EC-SOD expression in tissue from psoriasis patients was markedly decreased in psoriatic lesional and nonlesional skins from psoriasis patients in comparison to normal skin from healthy volunteers. Together, these results suggest that EC-SOD may provide a novel therapeutic approach to treating angiogenic and inflammatory skin diseases such as psoriasis.     

Antioxidant and bioenergetic coupling between neurons and astrocytes

Oxidative and nitrosative stress underlie the pathogenesis of a broad range of human diseases, in particular neurodegenerative disorders. Within the brain, neurons are the cells most vulnerable to excess reactive oxygen and nitrogen species; their survival relies on the antioxidant protection promoted by neighbouring astrocytes. However, neurons are also intrinsically equipped with a biochemical mechanism that links glucose metabolism to antioxidant defence. Neurons actively metabolize glucose through the pentose phosphate pathway, which maintains the antioxidant glutathione in its reduced state, hence exerting neuroprotection. This process is tightly controlled by a key glycolysis-promoting enzyme and is dependent on an appropriate supply of energy substrates from astrocytes. Thus brain bioenergetic and antioxidant defence is coupled between neurons and astrocytes. A better understanding of the regulation of this intercellular coupling should be important for identifying novel targets for future therapeutic interventions.     

ATP-dependent chromatin remodeling complex SWI/SNF in cardiogenesis and cardiac progenitor cell development

The recent identification of cardiac progenitor cells (CPCs) provides a new paradigm for studying and treating heart disease.To realize the full potential of CPCs for therapeutic purposes,it is essenti...     

Epidermal stem cells: interactions in developmental environments

Homeostasis of continuously renewing adult tissues, such as the epidermis of the skin, is maintained by epidermal stem cells (EpiSC), which are a small population of undifferentiated, self-renewing basal keratinocyte cells that produce daughter transit amplifying (TA) cells to make up the majority of the proliferative basal cell population in the epidermis. We have isolated EpiSC from neonatal and adult skin, and shown that these cells can regenerate an epidermis that lasts long term in vitro and in vivo, and that permanently expresses a recombinant gene in the regenerated tissue (Bickenbach and Dunnwald, 2000; Dunnwald et al., 2001). When we injected murine EpiSC into the developing blastocyst environment of the mouse, we found that both neonatal and adult EpiSC retained some ability to participate in the formation of tissues from all three germ layers (Liang and Bickenbach, 2002; Bickenbach and Chinnathambi, 2004; Liang et al., 2004). Although it appears evident that EpiSC act as pluripotent stem cells, how this reprogramming takes place is not understood. EpiSC might directly transdifferentiate into other cell types or they might first dedifferentiate into a more primitive cell type, and then proceed to develop along a cell lineage pathway. To begin to unravel this, we co-cultured EpiSC with embryonic stem (ES) cells, and found that EpiSC could alter their cell lineage protein expression to that of a more primitive cell type. We also placed EpiSC in a wounded environment and found that EpiSC interacted with the mesenchymal cells repopulating the wound bed. Our findings indicate that the population of cells that we isolate as EpiSC has a pluripotent capability. This has led us to postulate a paradigm shift for somatic stem cells. We propose that tissues maintain a sequestered population of uncommitted stem cells that retain a regenerative response which is enhanced when the cells are exposed to developmental or stress influences.     

Regulation of APC/C-Cdh1 and Its Function in Neuronal Survival

Neurons are post-mitotic cells that undergo an active downregulation of cell cycle-related proteins to survive. The activity of the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that regulates cell cycle progression in proliferating cells, plays a relevant role in post-mitotic neurons. Recent advances in the study of the regulation of APC/C have documented that the APC/C-activating cofactor, Cdh1, is essential for the function(s) of APC/C in neuronal survival. Here, we review the normal regulation of APC/C activity in proliferating cells and neurons. We conclude that in neurons the APC/C-Cdh1 complex actively downregulates the stability of the cell cycle protein cyclin B1 and the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3. Keeping these proteins destabilized is critical both for preventing the aberrant reentry of post-mitotic neurons into the cell cycle and for maintaining their reduced antioxidant status. Further understanding of the pathophysiological regulation of these proteins by APC/C-Cdh1 in neurons will be important for the search for novel therapeutic targets against neurodegeneration.