Activation of the native 45-kDa precursor form of interleukin-1-converting enzyme
Activation of the native 45-kDa precursor form of interleukin-1-converting enzyme
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DOI：
10.1074/jbc.271.22.13273
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发表时间：
1996-05-31
影响因子：
4.8
通讯作者：
Miller, DK
中科院分区：
文献类型：
--
作者：
Yamin, TT;Ayala, JM;Miller, DK
活性白细胞介素 1 β 转化酶 (ICE) 由 20 和 10 kDa 多肽（p20 和 p10）组成，这些多肽衍生自胞质 45 kDa 前体蛋白 (p45) 的加工。天然 p45 ICE 前体的裂解和激活已通过使用识别 ICE 各个区域的特异性抑制剂和抗体来表征。THP.1 单核细胞胞质提取物中 p45 的体外加工仅受抑制 ICE 的蛋白酶抑制剂抑制，而不受其他蛋白酶类别抑制剂的抑制。在这些提取物中添加 L-742,395（一种生物素化的不可逆 ICE 抑制剂）仅标记 p45，同时抑制 p45 加工，表明 p45 具有催化活性。 p45 在成为 p20 COOH 末端的位点处裂解后，形成了一种活性更强的中间体，该中间体在 SDS 聚丙烯酰胺凝胶电泳上迁移，分子量为 35 kDa（ED(50) 类似于 0.1 mu M L-742,395 标记，而 p45 为 5 mu M）。这种新的活性更高的 ICE 形式既可以作为裂解 p45 的中间酶，也可以作为形成最终活性 ICE 的底物（p20 的 ED(50) 为 1 nM L-742,395 标记，p22 为 p20 的 NH2 末端延伸形式），虽然 p45 的初始切割可以在对应于 p22 和 p20 NH2 末端的位点找到，但这些片段不能被 L-742,395 标记，因此是无活性的，p45 不是在对应于 p10 的 NH2 末端的位点进行处理。根据生物素化片段的 SDS 聚丙烯酰胺凝胶电泳上的条带位移确定，不到 50% 的 p45 被切割成活性 p20 或 p22 ICE，这表明体外激活效率非常低。 ICE 碎裂是通过分子间过程发生的，并且对稀释高度敏感。外源性 p20/p10 ICE 对 p45 的裂解与内源性 p45 裂解活性的不同之处在于，p20/p10 活性对盐更加敏感，并且它产生不同模式的裂解片段，主要是 35-和 12-kDa 片段。这些结果表明，当 p45 被加工成酶的 p20/p10 形式时，ICE 活性的性质发生变化。
Active interleukin-1 beta-converting enzyme (ICE) is composed of 20- and 10-kDa polypeptides (p20 and p10) derived from the processing of a cytosolic 45-kDa precursor protein (p45). The cleavage and activation of the native p45 ICE precursor have been characterized by use of specific inhibitors and antibodies recognizing various regions of ICE, The processing of p45 in vitro in THP.1 monocytic cell cytoplasmic extracts is inhibited only by protease inhibitors that inhibit ICE and not by inhibitors of other protease classes. The addition of L-742,395, a biotinylated irreversible ICE inhibitor, to these extracts labels only p45 and simultaneously inhibits p45 processing, demonstrating that the p45 has catalytic activity. Following a cleavage of p45 at a site that becomes the COOH terminus of p20, a more active intermediate is formed which migrates on SDS-polyacrylamide gel electrophoresis with an molecular mass of 35 kDa (ED(50) of similar to 0.1 mu M L-742,395 labeling versus 5 mu M for p45), This new more active ICE form serves both as an intermediate enzyme to cleave p45 as well as a substrate for the formation of the final active ICE (ED(50) of 1 nM L-742,395 labeling of p20 and for p22, an NH2-terminally extended form of p20), While initial cleavage of p45 can be found at the sites corresponding to both the NH2 termini of p22 and p20, these fragments cannot be labeled by L-742,395 and are hence inactive, p45 is not processed at the site corresponding to the NH2 terminus of the p10. Less than 50% of the p45 is cleaved down to active p20 or p22 ICE as determined by band shift on SDS-polyacrylamide gel electrophoresis of the biotinylated fragments, indicating that the in vitro activation is highly inefficient. The ICE fragmentation occurs by an intermolecular process and is highly dilution sensitive. Cleavage of p45 by exogenous p20/p10 ICE differs from that of the endogenous p45 cleavage activity in that the p20/p10 activity is more salt sensitive, and it produces a different pattern of cleavage fragments, principally 35- and 12-kDa fragments, These results indicate that the nature of the ICE activity changes as p45 is processed down to the p20/p10 form of the enzyme.