Asoh Sadamitsu, Ohsawa Ikuroh, Mori Takashi, Katsura Ken-Ichiro, Hiraide Tomoharu, Katayama Yasuo, Kimura Megumi, Ozaki Daiya, Yamagata Kumi, Ohta Shigeo
Department of Biochemistry and Cell Biology, Institute of Gerontology, Nippon Medical School, Kawasaki-city, Kanagawa 211-8533, Japan.
Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):17107-12. doi: 10.1073/pnas.262460299. Epub 2002 Dec 10.
Preventing massive cell death is an important therapeutic strategy for various injuries and disorders. Protein therapeutics have the advantage of delivering proteins in a short period. We have engineered the antiapoptotic bcl-x gene to generate the super antiapoptotic factor, FNK, with a more powerful cytoprotective activity. In this study, we fused the protein transduction domain (PTD) of the HIVTat 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 h. 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 i.p. into mice can have access into brain neurons. When injected i.p. 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 protein therapeutic strategy to prevent cell death in the brain.
预防大规模细胞死亡是针对各种损伤和疾病的重要治疗策略。蛋白质疗法具有在短时间内递送蛋白质的优势。我们对抗凋亡的bcl-x基因进行了改造，以产生具有更强细胞保护活性的超级抗凋亡因子FNK。在本研究中，我们将HIV Tat蛋白的蛋白质转导结构域（PTD）与FNK融合，并将构建体用于缺血性脑损伤的动物模型。当添加到人神经母细胞瘤细胞和大鼠新皮质神经元的培养基中时，PTD-FNK在1小时内迅速转导进入细胞并定位于线粒体。它分别保护神经母细胞瘤和神经元免受星形孢菌素诱导的凋亡和谷氨酸诱导的兴奋性毒性。在低至0.3 pM的浓度下就发现了PTD-FNK的细胞保护活性。此外，PTD-FNK影响钙离子的胞质运动，这可能与其神经保护作用有关。免疫组织化学分析显示，腹腔注射到小鼠体内的带有myc标签的PTD-FNK（PTD-myc-FNK）可以进入脑神经元。当腹腔注射到沙鼠体内时，PTD-FNK可预防短暂性全脑缺血引起的海马延迟性神经元死亡。这些结果表明，PTD-FNK作为一种预防脑内细胞死亡的蛋白质治疗策略具有临床应用潜力。
