Changes of pyridoxal kinase expression and activity in the gerbil hippocampus following transient forebrain ischemia.

In the previous study, we observed chronological alterations of glutamic acid decarboxylase (GAD), which is the enzyme converting glutamate into GABA. GAD isoforms (GAD65 and GAD67) differ substantially in their interactions with cofactor pyridoxal 5'-phosphate, which is catalyzed by pyridoxal kinase (PLK). In the present study, we examined the chronological changes of PLK expression and activity in the hippocampus after 5 min transient forebrain ischemia in gerbils. PLK immunoreactivity in the sham-operated group was detected weakly in the hippocampus. Ischemia-related change of PLK immunoreactivity in the hippocampus was significant in the hippocampal cornu ammonis (CA1)region, not in the hippocampal CA2/3 region and dentate gyrus. PLK immunoreactivity was observed in non-pyramidal GABAergic neurons at 30 min to 3 h after ischemic insult. At 12 h after ischemic insult, PLK immunoreactivity was shown in many CA1 pyramidal cells as well as some non-pyramidal cells. At this time point, PLK immunoreactivity and protein content was highest after ischemia. Thereafter, PLK immunoreactivity and protein content is decreased time-dependently by 4 days after ischemic insult. Four days after ischemia, some astrocytes expressed PLK in the CA1 region. The specific PLK activity was not altered following ischemic insult up to 2 days after ischemic insult. Thereafter, the specific PLK activity decreased time-dependently. However, total activity of PLK was significantly increased 12-24 h after ischemic insult, and thereafter total activity of PLK decreased. Therefore, we suggest that the over-expression of PLK in the CA1 pyramidal cells at 12 h after ischemia may induce increase of GAD in the CA1 pyramidal cells, which plays an important role in delayed neuronal death via the increase of GABA or enhancement of GABA shunt pathway.

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.

Enhanced oxidative damage by the familial amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutants.

Some cases of familial amyotrophic lateral sclerosis (FALS), a degenerative disorder of motor neurons, is associated with mutation in the Cu,Zn-superoxide dismutase (SOD) gene SOD1. The purified FALS mutant and wild-type Cu,Zn-SODs expressed in Escherichia coli cells have identical dismutation activity whereas the hydroxyl radical formation of FALS mutants was enhanced relative to that of the wild-type enzyme. These higher free radical-generating activities of mutants facilitated the release of copper ions from their own molecules. The reaction of the mutants with hydrogen peroxide enhanced DNA strand breaks and lipid peroxidation. The results suggested that the enhanced oxidative damage of macromolecules is mediated in the Cu,Zn-SOD mutants and hydrogen peroxide system via the generation of hydroxyl radicals by a combination of the higher free radical-generating activities of mutants and a Fenton-like reaction of copper ions released from oxidatively damaged Cu,Zn-SODs. Carnosine has been proposed to act as antioxidant in vivo. We investigated whether carnosine could protect the oxidative damage induced by FALS mutants. Carnosine effectively inhibited the DNA cleavage and lipid peroxidation. These results suggest that the higher free radical-generating function of FALS mutants can lead to increased oxidative damage of macromolecules which further implicates free radical-mediated motor neuronal injury in the pathogenesis of FALS and carnosine may be explored as potential therapeutic agents for FALS patients.

Transduction of Cu,Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into mammalian cells.

A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment encoding the nine amino acid transactivator of transcription (Tat) protein transduction domain (RKKRRQRRR) of HIV-1 in a bacterial expression vector to produce a genetic in-frame Tat-SOD fusion protein. The expressed and purified Tat-SOD fusion protein in Escherichia coli can enter HeLa cells in a time- and dose-dependent manner when added exogenously in a culture media. Denatured Tat-SOD protein was transduced much more efficiently into cells than were native proteins. Once inside the cells, transduced Tat-SOD protein was enzymatically active and stable for 24 h. The cell viability of HeLa cells treated with paraquat, an intracellular superoxide anion generator, was increased by transduced Tat-SOD. These lines of results suggest that the transduction of Tat-SOD fusion protein may be one of the ways to replenish the Cu,Zn-SOD in the various disorders related to this antioxidant enzyme.

Fragmentation of human ceruloplasmin induced by hydrogen peroxide.

We investigated the fragmentation of human ceruloplasmin induced by H2O2 to study its oxidative damage. When ceruloplasmin was incubated with H2O2, the frequency of the protein fragmentation increased in a proportion to the concentration of H2O2. It also increased in a time-dependent manner and was accompanied by gradual loss of the oxidase activity. Hydroxyl radical scavengers such as azide and mannitol inhibited the fragmentation of ceruloplasmin. The deoxyribose assay showed that hydroxyl radicals were generated in the reaction of ceruloplasmin with H2O2. Incubation of ceruloplasmin with H2O2 resulted in a time-dependent release of copper ions. The released copper ion may participate in a Fenton-like reaction to produce hydroxyl radical, which enhanced the fragmentation. The protection of the fragmentation by copper chelators such as diethylenetriaminepentaacetic acid and bathocuproine indicates a role for copper ion in the reaction. These results suggest that the fragmentation of ceruloplasmin induced by H2O2 is due to hydroxyl radicals formed by a copper-dependent Fenton-like reaction.

Release of copper ions from the familial amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutants.

Point mutations of Cu,Zn-superoxide dismutase (SOD) have been linked to familial amyotrophic lateral sclerosis (FALS). We reported that the Swedish FALS Cu,Zn-SOD mutant, D90A, exhibited an enhanced hydroxyl radical-generating activity, while its dismutation activity was identical to that of the wild-type enzyme (Kim et al. 1998a; 1998b). Transgenic mice that express a mutant Cu,Zn-SOD, Gly93 --> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the cDNA for the FALS G93A mutant, overexpressed the protein in E. coli cells, purified the protein, and studied its enzymic activities. Our results showed that the G93A, the D90A, and the wild-type enzymes have identical dismutation activity. However, the hydroxyl radical-generating activity of the G93A mutant was enhanced relative to those of the D90A and the wild-type enzyme (wild-type < D90A < G93A). These higher free radical-generating activities of mutants facilitated the release of copper ions from their own molecules (wild-type < D90A < G93A). The released copper ions can enhance the Fenton-like reaction to produce hydroxyl radicals and play a major role in the oxidative damage of macromolecules. Thus, the FALS symptoms may be associated with the enhancements in both the free radical-generating activity and the releasing of copper ions from the mutant enzyme.

Expression, purification, and characterization of a familial amyotrophic lateral sclerosis-associated D90A Cu,Zn-superoxide dismutase mutant.

Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS). We cloned the FALS mutant, D90A, and wild-type of human Cu,Zn-SOD, overexpressed them in E. coli, purified the proteins, and studied their properties. We investigated their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with H2O2 as a substrate. Our results showed that both wild-type and mutant enzymes have identical dismutation activities. However, the hydroxyl radical-generating function of the D90A mutant, as measured using a 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate), was enhanced relative to that of the wild-type enzyme. Catalysis of this reaction by D90A was more sensitive to inhibition by the copper chelators, penicillamine and diethyldithiocarbamate, than was catalysis by wild-type Cu,Zn-SOD. Our study suggests that this gain-of-function of FALS mutant may, in part, be responsible for the development of FALS symptoms.

The free radical-generating function of a familial amyotrophic lateral sclerosis-associated D90A Cu,Zn-superoxide dismutase mutant.

The free radical-generating functions of the D90A Cu,Zn-superoxide dismutase (SOD) associated with Swedish familial amyotrophic lateral sclerosis (FALS) patients are investigated. The results show that both the wild-type and mutant enzymes have identical dismutase activity, while the free radical-generating activity of the D90A mutant is enhanced relative to that of the wild-type enzyme. The studies suggest that the active channel of the D90A mutant is larger than that of the wild-type enzyme. A higher free radical-generating activity of the mutant enzyme led to the release of copper ions from the damaged protein. The generation of strand breaks in plasmid DNA was enhanced more effectively by the D90A mutant Cu,Zn-SOD than by the wild-type enzyme. The results suggest that the pathology of FALS may be attributed to oxidative damage caused by the gain-of-function of FALS Cu,Zn-SOD mutant.