Recently reported that the basic domain of human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) protein possesses the ability to traverse biological membranes efficiently in a process termed protein transduction. Although the mechanisms are still unclear, transduction oc...
Recently reported that the basic domain of human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) protein possesses the ability to traverse biological membranes efficiently in a process termed protein transduction. Although the mechanisms are still unclear, transduction occurs in receptor- and transporter-independent fashion that appears to target the lipid bilayer directly. Furthermore, HIV-1 Tat proteins have recently been shown to serve as carriers to direct uptake of heterologous proteins into the cells in vivo and in vitro. Thus, HIV-1 Tat proteins have tremendous potential to deliver large-sized compounds into the cells. The reactive-oxygen species are inevitably formed as by-products of various, normal cellular processes involving interaction with oxygen. These reactive-oxygen species damage macromolecules in the cells, and therefore, sometimes make significant contributions to the several pathological processes of human diseases. Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is among the key cellular enzymes by which cells detoxify free radicals and protect themselves from oxidative damage. Cu,Zn-SOD are thought to provide a primary line of defense by catalyzing the dismutation of reactive-oxygen species. In an effort to replenish the Cu,Zn-SOD activity in cells of various human disorders related to this antioxidant enzyme. Cu,Zn-SOD is a dimeric metalloenzyme of which active sites contain two copper and two zinc ions per molecules. The copper ion in Cu,Zn-SOD is essentially required to maintain dismutase activity of the enzyme. Whereas the zinc ion does not function in the catalytic cycle but helps stabilize the enzyme. However, it has been known that these metal ions of the Cu,Zn-SOD were reduced from 30 to 60% of that required of full occupancy of the four metal-binding sites per protein dimmer when recombinant Cu,Zn-SOD was overexpressed in bacteria. Reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). NO which is generated in the beta cell after cytokine stimulation is regarded as a hallmark of beta cell toxicity together with the generation of reactive oxygen species. Several antioxidants have been described as beneficial for ROS and NO-associated diseases. Cu,Zn-SOD is among the key cellular enzymes by which cells detoxify free radicals and protects themselves from oxidative damage. Results obtained from Chapter 1 (Transduction of Cu,Zn-SOD mediated by HIV-1 Tat protein basic domain into mammalian cells) were summarized as follow. 1. High level of expression of Tat-SOD fusion protein with enzyme activity achieved in E. coli BL21 by using a pTat-SOD expression vector. 2. Western blot analysis of cultured HeLa cells treated with denatured Tat-SOD fusion protein revealed that the fusion protein was successfully delivered into the cells in a time- and dose-dependent manner, whereas native Tat-SOD and control SOD were not delivered into the cells. 3. The enzyme activities of Cu,Zn-SOD in cultured HeLa cells treated with Tat-SOD were also increased concomitantly with the amounts of delivered protein into the cells. 4. The apparent degradation of transduced Tat-SOD was observed as a function of incubation time. However, significant levels of transduced protein and enzyme activity persisted in HeLa cells until 24 hrs. 5. Transduction of Tat-SOD was definitely effective against superoxide anion induced cytotoxicity in HeLa cells. To enhance the transduction potential of Tat-SOD, we prepared 3 different type of metal ion recovered Tat-SODs (Cu-recovered form, Zn-recovered form, Cu/Zn-recovered form) and examined their transduction potential. Results obtained from Chapter 2 (Transduction efficacy of Tat-Cu,Zn-SOD is enhanced by copper ion recovery of the fusion protein) were summarized as follow. 1. Copper ion content and dismutation activity of Tat-SOD are successfully regained by copper ion recovery method. 2. CR-Tat-SOD transduced into cells in an extremely rapid saturation manner whereas Tat-SOD transduced into cells in a time-dependent manner. 3. Transduction levels of CR-Tat-SOD were markedly increased as compared with those of Tat-SOD. 4. The low dose of CR-Tat-SOD significantly increased cell viability as compared with Tat-SOD. We have investigated the protective potential of cell-permeable fusion protein, Tat-SOD, against cytotoxicity induced by NO and oxygen free radicals in insulin-producing cell lines (MIN6N and RINm-5F cells) and primary islet cells. Results obtained from Chapter 3 (Protective effects of Tat-SOD on cytotoxicity induced by nitric oxide and oxygen free radicals in insulinoma cell lines and primary islet cells) were summarized as follow. 1. Tat-SOD fusion protein was successfully delivered into insulin-producing (MIN6N and RINm-5F) cells and the intracellular dismutation activities of SOD were also increased concomitantly with the amount of delivered protein into the cells. 2. MV and SNP decreased viability of insulinoma cells in time- and dose-dependent manner. Lipid peroxidation and DNA fragmentation in the cells were also induced by treatments of MV and SNP. 3. Transduced Tat-SOD fusion protein significantly decreased MV- and SNP-induced cell death, lipid peroxidation and DNA fragmentation in insulinoma cells. 4. Transduction of Tat-SOD into the cells under oxidative stress markedly induced the expression of Bcl-2 and heat shock protein 70 (hsp70) which are well known as an anti-apoptic protein and as an intracellular chaperone, respectively. 5. Tat-SOD fusion protein was delivered into the islet cells and SOD activities were also increased in islet cells. MV and SNP-induced cell death and DNA fragmentation were decreased when Tat-SOD was pre-treated. 6. In the intraperitoneally injected mice, Tat-SOD fusion protein was delivered in pancreas, kidney and brain tissues and SOD activities were also increased in the tissues. Immunohistochemistry analysis demonstrated that Tat-SOD fusion protein was delivered into isolated islet cells and injected mice pancreas. It is concluded from the above results that exogenous human Cu,Zn-SOD fused with Tat protein can be directly transduced into HeLa cells and delivered enzymatically active Tat-SOD shows the cellular protective functions against the oxidative stress. Copper ion recovered Tat-SOD (CR-Tat-SOD) transduced into HeLa cells in a rapid saturation manner and significantly increased the viability of HeLa cells pre-treated with MV. Tat-SOD fusion protein delivered into insulinoma cells and pancreatic islet cells, Also, transduced Tat-SOD efficiently protects on SNP and MV-induced cell death. Therefore, this transduction may allow the therapeutic delivery of Cu,Zn-SOD for the various disorders related to Cu,Zn-SOD and to protect the destruction of insulin-producing cells in autoimmune diabetes.
Recently reported that the basic domain of human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) protein possesses the ability to traverse biological membranes efficiently in a process termed protein transduction. Although the mechanisms are still unclear, transduction occurs in receptor- and transporter-independent fashion that appears to target the lipid bilayer directly. Furthermore, HIV-1 Tat proteins have recently been shown to serve as carriers to direct uptake of heterologous proteins into the cells in vivo and in vitro. Thus, HIV-1 Tat proteins have tremendous potential to deliver large-sized compounds into the cells. The reactive-oxygen species are inevitably formed as by-products of various, normal cellular processes involving interaction with oxygen. These reactive-oxygen species damage macromolecules in the cells, and therefore, sometimes make significant contributions to the several pathological processes of human diseases. Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is among the key cellular enzymes by which cells detoxify free radicals and protect themselves from oxidative damage. Cu,Zn-SOD are thought to provide a primary line of defense by catalyzing the dismutation of reactive-oxygen species. In an effort to replenish the Cu,Zn-SOD activity in cells of various human disorders related to this antioxidant enzyme. Cu,Zn-SOD is a dimeric metalloenzyme of which active sites contain two copper and two zinc ions per molecules. The copper ion in Cu,Zn-SOD is essentially required to maintain dismutase activity of the enzyme. Whereas the zinc ion does not function in the catalytic cycle but helps stabilize the enzyme. However, it has been known that these metal ions of the Cu,Zn-SOD were reduced from 30 to 60% of that required of full occupancy of the four metal-binding sites per protein dimmer when recombinant Cu,Zn-SOD was overexpressed in bacteria. Reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). NO which is generated in the beta cell after cytokine stimulation is regarded as a hallmark of beta cell toxicity together with the generation of reactive oxygen species. Several antioxidants have been described as beneficial for ROS and NO-associated diseases. Cu,Zn-SOD is among the key cellular enzymes by which cells detoxify free radicals and protects themselves from oxidative damage. Results obtained from Chapter 1 (Transduction of Cu,Zn-SOD mediated by HIV-1 Tat protein basic domain into mammalian cells) were summarized as follow. 1. High level of expression of Tat-SOD fusion protein with enzyme activity achieved in E. coli BL21 by using a pTat-SOD expression vector. 2. Western blot analysis of cultured HeLa cells treated with denatured Tat-SOD fusion protein revealed that the fusion protein was successfully delivered into the cells in a time- and dose-dependent manner, whereas native Tat-SOD and control SOD were not delivered into the cells. 3. The enzyme activities of Cu,Zn-SOD in cultured HeLa cells treated with Tat-SOD were also increased concomitantly with the amounts of delivered protein into the cells. 4. The apparent degradation of transduced Tat-SOD was observed as a function of incubation time. However, significant levels of transduced protein and enzyme activity persisted in HeLa cells until 24 hrs. 5. Transduction of Tat-SOD was definitely effective against superoxide anion induced cytotoxicity in HeLa cells. To enhance the transduction potential of Tat-SOD, we prepared 3 different type of metal ion recovered Tat-SODs (Cu-recovered form, Zn-recovered form, Cu/Zn-recovered form) and examined their transduction potential. Results obtained from Chapter 2 (Transduction efficacy of Tat-Cu,Zn-SOD is enhanced by copper ion recovery of the fusion protein) were summarized as follow. 1. Copper ion content and dismutation activity of Tat-SOD are successfully regained by copper ion recovery method. 2. CR-Tat-SOD transduced into cells in an extremely rapid saturation manner whereas Tat-SOD transduced into cells in a time-dependent manner. 3. Transduction levels of CR-Tat-SOD were markedly increased as compared with those of Tat-SOD. 4. The low dose of CR-Tat-SOD significantly increased cell viability as compared with Tat-SOD. We have investigated the protective potential of cell-permeable fusion protein, Tat-SOD, against cytotoxicity induced by NO and oxygen free radicals in insulin-producing cell lines (MIN6N and RINm-5F cells) and primary islet cells. Results obtained from Chapter 3 (Protective effects of Tat-SOD on cytotoxicity induced by nitric oxide and oxygen free radicals in insulinoma cell lines and primary islet cells) were summarized as follow. 1. Tat-SOD fusion protein was successfully delivered into insulin-producing (MIN6N and RINm-5F) cells and the intracellular dismutation activities of SOD were also increased concomitantly with the amount of delivered protein into the cells. 2. MV and SNP decreased viability of insulinoma cells in time- and dose-dependent manner. Lipid peroxidation and DNA fragmentation in the cells were also induced by treatments of MV and SNP. 3. Transduced Tat-SOD fusion protein significantly decreased MV- and SNP-induced cell death, lipid peroxidation and DNA fragmentation in insulinoma cells. 4. Transduction of Tat-SOD into the cells under oxidative stress markedly induced the expression of Bcl-2 and heat shock protein 70 (hsp70) which are well known as an anti-apoptic protein and as an intracellular chaperone, respectively. 5. Tat-SOD fusion protein was delivered into the islet cells and SOD activities were also increased in islet cells. MV and SNP-induced cell death and DNA fragmentation were decreased when Tat-SOD was pre-treated. 6. In the intraperitoneally injected mice, Tat-SOD fusion protein was delivered in pancreas, kidney and brain tissues and SOD activities were also increased in the tissues. Immunohistochemistry analysis demonstrated that Tat-SOD fusion protein was delivered into isolated islet cells and injected mice pancreas. It is concluded from the above results that exogenous human Cu,Zn-SOD fused with Tat protein can be directly transduced into HeLa cells and delivered enzymatically active Tat-SOD shows the cellular protective functions against the oxidative stress. Copper ion recovered Tat-SOD (CR-Tat-SOD) transduced into HeLa cells in a rapid saturation manner and significantly increased the viability of HeLa cells pre-treated with MV. Tat-SOD fusion protein delivered into insulinoma cells and pancreatic islet cells, Also, transduced Tat-SOD efficiently protects on SNP and MV-induced cell death. Therefore, this transduction may allow the therapeutic delivery of Cu,Zn-SOD for the various disorders related to Cu,Zn-SOD and to protect the destruction of insulin-producing cells in autoimmune diabetes.
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#세포투과성 Tat-Cu Zn-Superoxide dismutase
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