Apoptosis via new serine proteases and its intracellular signaling events.

我们使用HepG2细胞作为人肝细胞的模型系统。我们研究了扑热息痛诱导HepG2细胞凋亡的细胞内信号转导事件。对乙酰氨基酚以剂量和时间依赖的方式诱导HepG2细胞凋亡。半胱氨酸天冬氨酸氨基转移酶抑制剂N-benzyloxycarbonyl-Asp-Glu-Val-fluoromethylketone(zVAD-FINK)和丝氨酸蛋白酶抑制剂N-对甲苯磺酰-L-赖氨酸氯甲基酮(TLCK)均能有效抑制该酶活性。线粒体膜电位(ΔΨm)损失在处理24 h后达到平台期，TLCK可部分抑制ΔΨm降低。用Boc-Glu-Lys-Lys-MCA作为测定细胞裂解物中丝氨酸蛋白酶活性的底物，具有较高的灵敏度和特异性。用亲和层析法从醋氨酚处理的HepG2细胞中纯化丝氨酸蛋白酶。用SBTI固定化琼脂糖亲和层析不能得到任何具有较高活性的组分。接下来使用赖氨酸固定化琼脂糖凝胶，因为在醋氨酚处理的细胞提取液中，纤溶酶特异性的多肽-MCA底物显示出很高的蛋白酶活性。获得了一种高活性的蛋白水解酶，并对其进行了SDS-PAGE和银染。这些数据表明，扑热息痛诱导的肝细胞凋亡是由TLCK敏感的丝氨酸蛋白酶启动的，这些酶可能在结构上类似于纤溶酶原。

We used HepG2 cells as a model system for human liver cells. We investigated intracellular signaling events of acetaminophen-induced apoptosis in HepG2 cells. Acetaminophen induces apoptosis in HepG2 cells in a dose- and time-dependent manner. It was efficiently inhibited by the caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-fluoromethylketone (zVAD-fink) and the serine protease inhibitor N-p-tosyl-_L-lysine chloromethyl ketone (TLCK). Loss of the mitochondrial transmembrane potential (ΔΨm) reached a plateau after treatment for 24 h. TLCK could inhibit reduction of ΔΨm partially. For measurement of serine protease activity in cell lysates, Boc-Glu-Lys-Lys-MCA was selected as the best substrate among various peptidyl-MCA substrates because of its high sensitivity and specificity. Affinity chromatography was carried out in order to purify serine proteases from acetaminophen-treated HepG2 cells. We could not obtain any fraction with high protease activity by affinity chromatography on SBTI-immobilized Sepharose. Lysine-immobilized Sepharose was next utilized, because plasmin-specific peptidyl-MCA substrates showed high protease activity in acetaminophen-treated cell extract. One fraction of high protease activity was obtained and submitted to SDS-PAGE and silver staining. These data suggest that acetaminophen-induced apoptosis in liver cells is initiated activation of TLCK-sensitive serine proteases and that these proteases might mimic plasminogen structurally.