Wang K K, Posmantur R, Nadimpalli R, Nath R, Mohan P, Nixon R A, Talanian R V, Keegan M, Herzog L, Allen H
Department of Neuroscience Therapeutics, Division of Warner-Lambert Company, Parke-Davis Pharmaceutical Research, 2800 Plymouth Road, Ann Arbor, Michigan, 48105, USA.
Arch Biochem Biophys. 1998 Aug 15;356(2):187-96. doi: 10.1006/abbi.1998.0748.
Two cysteine protease families (caspase and calpain) participate in apoptosis. Here we report that the endogenous calpain inhibitor calpastatin is fragmented by caspase(s) to various extents during early apoptosis in two cell types. In anti-fas or staurosporine-treated Jurkat T-cells, the high-molecular-weight form (HMW) of calpastatin (apparent Mr 110 K) was extensively degraded to immunoreactive fragments of Mr 75 K and 30 K In apoptotic SH-SY5Y human neuroblastoma cells, HMW calpastatin was degraded to a major immunoreactive fragment of 75 K. In both cell types, fragmentation of HMW calpastatin was blocked by a caspase-specific inhibitor carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene. In vitro translated HMW calpastatin was sensitive to proteolysis by recombinant caspase-1, -3, and -7. By contrast, in vitro translated LMW calpastatin (which lacks domains L and I) was cleaved into multiple fragments only by caspase-1 and was relatively resistant to caspase-3, -7, and other caspases tested. Consistently with that, purified erythroid LMW calpastatin was also highly susceptible to caspase-1 digestion. Recombinant human calpastatin spanning domain I through III (CAST(DI-III)) was found cleaved by caspase-1 at at least three sites, located in either the A or the C helix of domains I and III (ALDD137L, LSSD203F and ALAD404S), while only a single site (ALDD137L) was cleaved by caspase-3. These findings suggest that both HMW and LMW calpastatins are more vulnerable to caspase-1 than to caspase-3. Surprisingly, both erythroid LMW calpastatin and recombinant CAST(DI-III) fragmented by caspase-1 suffered only a less than twofold reduction of inhibitory activity toward calpain. We propose that the proteolysis of calpastatin in early apoptosis might have yet unidentified effects on the cross-talk between the two protease systems.
两个半胱氨酸蛋白酶家族（胱天蛋白酶和钙蛋白酶）参与细胞凋亡过程。在此我们报告，在内源性钙蛋白酶抑制剂钙蛋白酶抑制蛋白在两种细胞类型的早期凋亡过程中会不同程度地被胱天蛋白酶裂解。在抗 Fas 或星形孢菌素处理的 Jurkat T 细胞中，钙蛋白酶抑制蛋白的高分子量形式（HMW，表观 Mr 110K）被广泛降解为 Mr 75K 和 30K 的免疫反应性片段。在凋亡的 SH-SY5Y 人神经母细胞瘤细胞中，HMW 钙蛋白酶抑制蛋白被降解为一个主要的 75K 免疫反应性片段。在这两种细胞类型中，HMW 钙蛋白酶抑制蛋白的裂解都被一种胱天蛋白酶特异性抑制剂苄氧羰基 - 天冬氨酸 - CH2OC(O)-2,6 - 二氯苯所阻断。体外翻译的 HMW 钙蛋白酶抑制蛋白对重组胱天蛋白酶 -1、-3 和 -7 的蛋白水解敏感。相比之下，体外翻译的低分子量钙蛋白酶抑制蛋白（LMW，缺乏结构域 L 和 I）仅被胱天蛋白酶 -1 裂解为多个片段，并且对胱天蛋白酶 -3、-7 和其他测试的胱天蛋白酶相对耐药。与此一致，纯化的红细胞 LMW 钙蛋白酶抑制蛋白也对胱天蛋白酶 -1 的消化高度敏感。发现跨越结构域 I 至 III 的重组人钙蛋白酶抑制蛋白（CAST(DI - III)）被胱天蛋白酶 -1 在至少三个位点裂解，这些位点位于结构域 I 和 III 的 A 或 C 螺旋中（ALDD137L、LSSD203F 和 ALAD404S），而仅一个位点（ALDD137L）被胱天蛋白酶 -3 裂解。这些发现表明，HMW 和 LMW 钙蛋白酶抑制蛋白对胱天蛋白酶 -1 比胱天蛋白酶 -3 更敏感。令人惊讶的是，红细胞 LMW 钙蛋白酶抑制蛋白和被胱天蛋白酶 -1 裂解的重组 CAST(DI - III)对钙蛋白酶的抑制活性仅降低不到两倍。我们提出，早期凋亡过程中钙蛋白酶抑制蛋白的蛋白水解可能对这两种蛋白酶系统之间的相互作用有尚未明确的影响。
