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

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ArticleTitle Development of the Protein Therapeutics Using the Super Anti-cell Death Factor FNK

AuthorList Shigeo Ohta

Affiliation Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School

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	Development of the Protein Therapeutics Using the Super Anti-cell Death Factor FNK
AuthorList	Shigeo Ohta
Affiliation	Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School
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