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。

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570112/
• 关键词: hepatocyte growth factor (HGF); HGF activator; serine protease; blood coagulation; apoptosis; Kunitz domain; セリンプロテアーゼカスケード

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剂抑制剂 1 型 (HAI-1) 和 2 型 (HAI-2) 是 Kunitz 型丝氨酸蛋白酶抑制剂，被确定为肝细胞生长因子 (HGF) 激活剂的有效抑制剂，以阐明这些调节因子的生理作用。为了研究 HAI 对细胞增殖的调节作用，我们对 HAI-1 和 HAI-2.1 的各个功能域的抑制谱进行了分析。 HAI 分子。每个 HAI 分子中的 Kunitz I 似乎是抑制 HGF 激活剂的 HGF 转化活性的功能域。此外，两个 Kunitz 域的存在影响了 HGF-1 对 HGF 激活剂结合的抑制活性。 Kunitz I 和 Kunitz II 位点彼此靠近，仅具有一个 Kunitz 结构域的蛋白水解加工产生 HAI-1 调节 HAI-1 的活性识别。针对 Kunitz 的同源丝氨酸蛋白酶的研究将揭示 HAI 的新生物学功能。2. 为了鉴定 HAI 分泌酶，我们使用酵母双杂交筛选系统检查了 HAI 细胞质区域中的蛋白质-蛋白质相互作用。获得了多种蛋白质，包括细胞质蛋白和细胞内酶，其中一种已知参与细胞凋亡的抑制，这表明 HAI 可能在 Fas 介导的调节中发挥重要作用。对该细胞事件的分析正在进行中。3. 为了阐明 HAI 分子在男性生殖细胞和组织中的生理作用，我们评估了精子形成过程中睾丸组织中的这些表达模式。免疫组化分析显示，单克隆抗体标记的HAI-1仅定位于三个发育阶段的早期阶段，表明HAI-1出现在精原细胞向圆形的形态变化阶段。精子发生过程中，HAI-2几乎分布在细胞分化、形态变化或迁移的组织细胞的整个表面。进一步分析HAI在细胞生长过程中的功能调节将提供一种新的机制。 Fas 介导的细胞凋亡的调节较少。