Apoptosis via new serine proteases and its intracellular signaling events.

我们使用HepG 2细胞作为人肝细胞的模型系统。我们研究了对乙酰氨基酚诱导HepG 2细胞凋亡的细胞内信号事件。对乙酰氨基酚以剂量和时间依赖性方式诱导HepG 2细胞凋亡。半胱氨酸蛋白酶抑制剂N-苄氧羰基-Asp-Glu-Val-氟甲基酮（zVAD-fink）和丝氨酸蛋白酶抑制剂N-对甲苯磺酰基-L-赖氨酸氯甲基酮（TLCK）均能有效抑制该酶的活性。处理24 h后线粒体跨膜电位（Δ Δ m）的损失达到平台。TLCK可部分抑制Δ λ m的降低。对于细胞裂解物中丝氨酸蛋白酶活性的测量，在各种肽基-MCA底物中选择Boc-Glu-Lys-Lys-MCA作为最佳底物，因为其高灵敏度和特异性。进行亲和层析以从对乙酰氨基酚处理的HepG 2细胞中纯化丝氨酸蛋白酶。我们不能得到任何部分具有高蛋白酶活性的亲和层析SBTI固定化琼脂糖凝胶。赖氨酸固定的琼脂糖凝胶，其次利用，因为纤溶酶特异性肽基-MCA底物显示出高的蛋白酶活性，在对乙酰氨基酚处理的细胞提取物。获得一个高蛋白酶活性的级分，并进行SDS-PAGE和银染色。这些数据表明，对乙酰氨基酚诱导的肝细胞凋亡启动激活TLCK敏感的丝氨酸蛋白酶，这些蛋白酶可能模拟纤溶酶原结构。

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.