Investigatiions on the functional regulation in the process of processing from the membrane-integrated form of HGF activator inhibitors

HGF激活剂抑制剂膜整合型加工过程中的功能调控研究

• 批准号: 11680603
• 负责人: DENDA Kimitoshi
• 金额: $ 2.18万
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680603/
• 关键词: hepatocyte growth factor (HGF); HGF activator; serine protease; blood coagulation; Kunitzu 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 strong inhibitors toward hepatocyte growth factor (HGF) activator. Each HAI molecule possesses the hydrophobic region at the C-terminus, suggesting that they are produced primarily in a membrane-integrated form with two Kunitz domains in its extracellular region, and ectodomain shedding releases several secreted forms. In order to clarify the physiological role of these regulatory factors, we have performed the functional analyzes of HAI-1 and HAI-2.1. Immunoblotting analysis revealed that HAI-1 is first produced in a 66 kDa membrane-integrated form, and subsequent ectodomain shedding releases two major secreted forms from the cell surface into the extracellular space, their sizes being 40/39 kDa and 58 kDa. Whereas the former containing one Kunitz domain shows strong inhibitory activity against the HGF-converting activity of HGF activator, the latte … More r containing two Kunitz domains shows markedly weaker inhibitory activity. Thus, the presence of the C-terminal Kunitz domain (Kunitz II) in the 58 kDa HAI-1 may interfere with the binding of HGF activator to the reactive site of the N-terminal Kunitz domain (Kunitz I), while elimination of this region by proteolytic processing could lead to strong binding of HGF activator to HAI-1.2. To determine roles of the Kunitz domains in the inhibitory activity of HAIs against serine proteases, we constructed various HAI mutants and examined their inhibitory activity against HGF activator. 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. These results suggest that 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 II will reveal the novel biological function of HAIs.3. In order to search for HAI secretases, we have 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 death signaling (manuscripts in preparation).Further analyzes on the functional regulation in the process of the proteolytic processing of HAIs will provide a novel mechanism of the regulation on HGF activation. Less

肝细胞生长因子激活物抑制剂1型（HAI-1）和2型（HAI-2）是Kunitz型丝氨酸蛋白酶抑制剂，被鉴定为肝细胞生长因子（HGF）激活物的强抑制剂。每个HAI分子在C-末端具有疏水区，表明它们主要以膜整合形式产生，在其胞外区具有两个Kunitz结构域，并且胞外域脱落释放几种分泌形式。为了阐明这些调节因子的生理作用，我们对HAI-1和HAI-2.1进行了功能分析。免疫印迹分析显示，HAI-1首先以66 kDa的膜整合形式产生，随后的胞外域脱落将两种主要的分泌形式从细胞表面释放到细胞外空间中，它们的大小为40/39 kDa和58 kDa。前者含有一个Kunitz结构域，对HGF激活剂的HGF转化活性有很强的抑制作用，而后者含有一个Kunitz结构域， ...更多信息 含有两个Kunitz结构域的r显示出明显较弱的抑制活性。因此，58 kDa HAI-1中C-末端Kunitz结构域（Kunitz II）的存在可能干扰HGF激活剂与N-末端Kunitz结构域（Kunitz I）的反应位点的结合，而通过蛋白水解加工消除该区域可能导致HGF激活剂与HAI-1.2的强结合。为了确定Kunitz结构域在HAI对丝氨酸蛋白酶的抑制活性中的作用，我们构建了各种HAI突变体，并检测了它们对HGF激活剂的抑制活性。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，两个Kunitz结构域的存在影响HAI-1对HGF激活剂的抑制活性。这些结果表明，Kunitz I和Kunitz II的丝氨酸蛋白酶结合位点彼此靠近，并且产生仅具有一个Kunitz结构域的HAI-1的蛋白水解加工调节HAI-1的活性。针对Kunitz II的同源丝氨酸蛋白酶的鉴定将揭示HAIs的新生物学功能。为了寻找HAI分泌酶，我们使用酵母双杂交筛选系统研究了HAI细胞质区域中的蛋白质-蛋白质相互作用。获得了几种胞质蛋白，包括胞浆蛋白和胞内酶。其中一个已知参与细胞凋亡的抑制，提示HAI可能在死亡信号的调控中发挥重要作用（文稿正在准备中），进一步分析HAI在蛋白水解过程中的功能调控，将为HAI调控HGF活化提供新的机制。少

项目成果

Kataoka, H., Itoh, H., Nuki, Y., Hamasuna, R., Naganujma, S.,Kitamura, N., and Shimomura, T: "Mouse Hepatocyte Growth Factor (HGF) Activator Inhibitor Type 2 Lacking the First Kunitz Domain Potently Inhibits the HGF Activator"biochem. Biophys. Res. commun

Kataoka, H.、Itoh, H.、Nuki, Y.、Hamasuna, R.、Naganujma, S.、Kitamura, N. 和 Shimomura, T：“小鼠肝细胞生长因子 (HGF) 激活剂抑制剂 2 型缺乏第一个

Itoh, H., Kataoka, H., Yamaguchi, M., Naganuma, S., Akiyama, Y., Nuki, Y., Shimomura, T., Miyazawa, K., Kitamura, N., and Koono, M.: "Identification of hepatocyte growth factor activator inhibitor type 2 (HAI-2)-related small peptide (H2RSP) : its nuclear

Itoh, H.、Kataoka, H.、Yamaguchi, M.、Naganuma, S.、Akiyama, Y.、Nuki, Y.、Shimomura, T.、Miyazawa, K.、Kitamura, N. 和 Koono, M.

Shimomura, T. et al.: "Multiple sites of proteolytic cleavage to release soluble forms of hepatocyte growth factor activator inhibitor type 1 from a transmembrane form"J. Biochem.. 126. 821-828 (1999)

Shimomura, T. 等人：“多位点蛋白水解裂解从跨膜形式释放可溶形式的肝细胞生长因子激活剂抑制剂 1 型”J.

Shimomura, T., Denda, K., Kawaguchi, T., Matsumoto, K., Miyazawa, K., and Kitamura, N.: "Multiple sites of proteolytic cleavage to release soluble forms of hepatocyte growth factor activator inhibitor type 1 from a transmembrane form"J. biochem. 126(5). 8

Shimomura, T.、Denda, K.、Kawaguchi, T.、Matsumoto, K.、Miyazawa, K. 和 Kitamura, N.：“通过多个蛋白水解位点释放可溶形式的肝细胞生长因子激活剂抑制剂 1 型

Denda, K. et al.: "Functional characterization of Kunitz domains in Hepatocyte growth factor activator inhibitor type 1"J. Biol. Chem.. 277(in press). (2002)

Denda, K. 等人：“肝细胞生长因子激活剂抑制剂 1 型中 Kunitz 结构域的功能特征”J.