Journal of Nippon Medical School: Vol.70 No.5 page.442

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ArticleTitle 細胞死抑制活性強化因子を用いた蛋白質治療法の開発

AuthorList 太田成男

Affiliation 日本医科大学院加齢科学系専攻細胞生物学分野

Language JA

Volume 70

Issue 5

Year 2003

Page 442-446

Received

Accepted

Keywords apoptosis, protein transduction, protein therapy, Bcl-2 family, site-directed mutagenesis

Abstract
A powerful artificial anti-apoptotic factor will be useful for the reproductive therapies for many diseases by prolonging survival of stem cells. For constructing it, we designed the super anti-apoptotic factor by disturbing three intramolecular polar interactions among α-helix structures of Bcl-xL. The resultant mutant Bcl-xL, named FNK, was expected to make the pore-forming domain more mobile and flexible than the wild-type. When overexpressed in Jurkat cells, FNK was markedly more potent in prolonging survival following apoptosis-inducing treatment with a kind of cell death cytokines (anti-Fas), a protein kinase inhibitor (staurosporine), cell cycle inhibitors (TN-16, camptothecin, hydroxyurea and trichostatin A) or oxidative stress (hydrogen peroxide and paraquat) than wild-type Bcl-xL. Furthermore, the transfectants of FNK became more resistant against a calcium ionophore and even a heat treatment than wild-type Bcl-xL. In addition, FNK showed marked anti-apoptotic activity in CHO and Jurkat cells deprived of serum. Thus, FNK may be the first mutant generated by site-directed mutagenesis of Bcl-xL with an enhance gain-of-function phenotype.

Next, we tried to transduce the FNK protein into cells. Protein therapeutics has the advantage of delivering proteins in a short period of time. We have engineered the anti-apoptotic bcl-x gene to generate the super anti-apoptotic factor, FNK, with a more powerful cytoprotective activity. In this study, we fused the protein transduction domain (PTD) of the HIV/Tat protein to FNK, and used the construct in an animal model of ischemic brain injury. When added into culture media of human neuroblastoma cells and rat neocortical neurons, PTD-FNK rapidly transduced into cells and localized to mitochondria within 1 hr. It protected the neuroblastomas and neurons against staurosporine-induced apoptosis and glutamate-induced excitotoxicity, respectively. The cytoprotective activity of PTD-FNK was found at concentrations as low as 0.3 pM. Additionally, PTD-FNK affected the cytosolic movement of calcium ions, which may relate to its neuroprotective action. Immunohistochemical analysis revealed that myc-tagged PTD-FNK (PTD-myc-FNK) injected intraperitoneally into mice can have access into brain neurons. When injected intraperitoneally into gerbils, PTD-FNK prevented delayed neuronal death in the hippocampus caused by transient global ischemia. These results suggest that PTD-FNK has a potential for clinical utility as a novel protein therapeutic strategy to prevent cell death in the brain.

Thus, the protein delivery system will be useful to make cells survived for a long time during the differentiation of stem cells in the reproductive therapies.

Correspondence to

ArticleTitle	細胞死抑制活性強化因子を用いた蛋白質治療法の開発
AuthorList	太田成男
Affiliation	日本医科大学院加齢科学系専攻細胞生物学分野
Language	JA
Volume	70
Issue	5
Year	2003
Page	442-446
Received
Accepted
Keywords	apoptosis, protein transduction, protein therapy, Bcl-2 family, site-directed mutagenesis
Abstract	A powerful artificial anti-apoptotic factor will be useful for the reproductive therapies for many diseases by prolonging survival of stem cells. For constructing it, we designed the super anti-apoptotic factor by disturbing three intramolecular polar interactions among α-helix structures of Bcl-xL. The resultant mutant Bcl-xL, named FNK, was expected to make the pore-forming domain more mobile and flexible than the wild-type. When overexpressed in Jurkat cells, FNK was markedly more potent in prolonging survival following apoptosis-inducing treatment with a kind of cell death cytokines (anti-Fas), a protein kinase inhibitor (staurosporine), cell cycle inhibitors (TN-16, camptothecin, hydroxyurea and trichostatin A) or oxidative stress (hydrogen peroxide and paraquat) than wild-type Bcl-xL. Furthermore, the transfectants of FNK became more resistant against a calcium ionophore and even a heat treatment than wild-type Bcl-xL. In addition, FNK showed marked anti-apoptotic activity in CHO and Jurkat cells deprived of serum. Thus, FNK may be the first mutant generated by site-directed mutagenesis of Bcl-xL with an enhance gain-of-function phenotype. Next, we tried to transduce the FNK protein into cells. Protein therapeutics has the advantage of delivering proteins in a short period of time. We have engineered the anti-apoptotic bcl-x gene to generate the super anti-apoptotic factor, FNK, with a more powerful cytoprotective activity. In this study, we fused the protein transduction domain (PTD) of the HIV/Tat protein to FNK, and used the construct in an animal model of ischemic brain injury. When added into culture media of human neuroblastoma cells and rat neocortical neurons, PTD-FNK rapidly transduced into cells and localized to mitochondria within 1 hr. It protected the neuroblastomas and neurons against staurosporine-induced apoptosis and glutamate-induced excitotoxicity, respectively. The cytoprotective activity of PTD-FNK was found at concentrations as low as 0.3 pM. Additionally, PTD-FNK affected the cytosolic movement of calcium ions, which may relate to its neuroprotective action. Immunohistochemical analysis revealed that myc-tagged PTD-FNK (PTD-myc-FNK) injected intraperitoneally into mice can have access into brain neurons. When injected intraperitoneally into gerbils, PTD-FNK prevented delayed neuronal death in the hippocampus caused by transient global ischemia. These results suggest that PTD-FNK has a potential for clinical utility as a novel protein therapeutic strategy to prevent cell death in the brain. Thus, the protein delivery system will be useful to make cells survived for a long time during the differentiation of stem cells in the reproductive therapies.
Correspondence to