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Functional characterization of HGF activator inhibitors which can mediate the regulation of apoptotic pathway via Fas.

HGF 激活剂抑制剂的功能表征，可通过 Fas 介导细胞凋亡途径的调节。

基本信息

批准号：
15570112
负责人：
DENDA Kimitoshi
金额：
$ 2.11万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Hepatocyte growth factor activator inhibitors type 1 (HAI-1) and type 2 (HAI-2) are Kunitz-type serine protease inhibitors, identified as potent inhibitors toward hepatocyte growth factor (HGF) activator. To clarify the physiological role of these regulatory factors on cellular apoptosis, we have performed the functional analyses of HAI-1 and HAI-2.1. To investigate the regulatory role of HAIs on cell proliferation, we examined inhibitory spectrum on individual functional domain in HAI molecules. Kunitz I in each HAI molecule appears to be the functional domain for inhibiting the HGF-converting activity of HGF activator. Furthermore, the presence of two Kunitz domains affected the inhibitory activity of HAI-1 against HGF activator. Thus serine protease binding sites of Kunitz I and Kunitz II are located close to each other, and proteolytic processing to generate HAI-1 with only one Kunitz domain regulates the activity of HAI-1. Identification of cognated serine proteases against Kunitz … More II will reveal the novel biological function of HAIs.2. In order to identify HAI secretases, we examined protein-protein interaction in the cytoplasmic regions of HAIs using a yeast two-hybrid screening system. Several cytoplasmic proteins, including cytosleletal proteins and intracelluiar enzymes were obtained. One of them was known to be involved in suppression on apoptosis, suggesting that HAI may play important role on the regulation of Fas-mediated death signaling. Analyses of this cellular event is now in progress.3. To clarify the physiological role of HAI molecules in male germ cells and tissue, we assessed these expression patterns in testicular tissues during sperm formation. We prepared the specific monoclonal antibodies of HAIs. Immunohistochemical analysis reveals that HAI-1 labeled by the monoclonal antibody were localized only in earlier stages in the three developmental phases, suggesting that HAI-1 appears in the morphologic change phase from spermatogonium to round spermatid, whereas HAI-2 was localized along almost entire surface of the cells in the tissues. Similarly, HGF activator was expressed extensively in the seminiferous tubule. HAI-1 and HAI-2 in testis might thus play an important role in the different stages of cellular differentiation, morphologic change or migration during spermatogenesis.Further analyses of the functional regulation by HAIs in the process of cellular growth will provide a novel mechanism of the regulation of Fas-meidated apoptosis. Less

肝细胞生长因子激活物抑制物1(HAI-1)和肝细胞生长因子激活物抑制物2(HAI-2)是一类库尼茨型丝氨酸蛋白酶抑制物，被认为是肝细胞生长因子(HGF)激活剂的有效抑制剂。为了阐明这些调控因子在细胞凋亡中的生理作用，我们对HAI-1和HAI-2.1进行了功能分析。为了研究HAI对细胞增殖的调节作用，我们检测了HAI分子中单个功能结构域的抑制谱。每个HAI分子中的kunitz I似乎是抑制HGF激活剂的HGF转化活性的功能结构域。此外，HAI-1对HGF激活剂的抑制活性受两个kunitz结构域的影响。因此，kunitz I和kunitz II的丝氨酸蛋白酶结合部位彼此靠近，而仅含有一个kunitz结构域的蛋白水解性过程调节了HAI-1的活性。昆尼茨…同源丝氨酸蛋白酶的鉴定更多的II将揭示HAIs的新的生物学功能。为了鉴定HAI分泌酶，我们使用酵母双杂交筛选系统检测了HAI胞浆区域的蛋白质-蛋白质相互作用。获得了多种细胞质蛋白，包括细胞骨架蛋白和胞内酶。其中一个已知参与了对细胞凋亡的抑制，提示HAI可能在Fas介导的死亡信号的调节中发挥重要作用。对这一细胞事件的分析目前正在进行中。为了阐明HAI分子在男性生殖细胞和组织中的生理作用，我们评估了这些在精子形成过程中在睾丸组织中的表达模式。我们制备了HAIs的特异性单抗。免疫组织化学分析表明，HAI-1仅定位于三个发育阶段的早期，提示HAI-1出现在从精原细胞到圆形精子细胞的形态变化期，而HAI-2几乎分布在组织细胞的整个表面。类似地，HGF激活物在生精小管中广泛表达。因此，HAI-1和HAI-2可能在精子发生过程中细胞分化、形态变化或迁移的不同阶段发挥重要作用，进一步分析HAI-1和HAI-2在细胞生长过程中的功能调控将为Fas介导的细胞凋亡的调控提供新的机制。较少