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

肝细胞生长因子激活因子抑制剂1型（HAI-1）和2型（HAI-2）是kunitz型丝氨酸蛋白酶抑制剂，被认为是肝细胞生长因子（HGF）激活因子的有效抑制剂。为了阐明这些调节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HAIs对细胞增殖的调节作用，我们检测了HAIs分子中单个功能域的抑制谱。每个HAI分子中的Kunitz I似乎是抑制HGF激活剂转化HGF活性的功能域。此外，两个Kunitz结构域的存在影响了HAI-1对HGF激活剂的抑制活性。因此，Kunitz I和Kunitz II丝氨酸蛋白酶结合位点相互靠近，蛋白水解过程产生的只有一个Kunitz结构域的HAI-1调节了HAI-1的活性。鉴定抗Kunitz . More II的同源丝氨酸蛋白酶将揭示hais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HAIs细胞质区域的蛋白-蛋白相互作用。获得了多种细胞质蛋白，包括胞壁蛋白和胞内酶。其中一个已知参与抑制细胞凋亡，提示HAI可能在fas介导的死亡信号调控中发挥重要作用。对这一细胞事件的分析正在进行中。为了阐明HAI分子在男性生殖细胞和组织中的生理作用，我们评估了精子形成过程中睾丸组织中的这些表达模式。制备HAIs特异性单克隆抗体。免疫组化分析显示，该单克隆抗体标记的HAI-1仅定位于三个发育阶段的早期阶段，表明HAI-1出现在从精原细胞到圆形精母细胞的形态变化阶段，而HAI-2几乎定位于组织细胞的整个表面。同样，HGF激活因子在精小管中广泛表达。因此，睾丸中的HAI-1和HAI-2可能在精子发生过程中细胞分化、形态改变或迁移的不同阶段发挥重要作用。进一步分析HAIs在细胞生长过程中的功能调控，将为fas介导的细胞凋亡调控提供新的机制。少

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