Preservation of caspase-3 subunits from degradation contributes to apoptosis evoked by lactacystin: Any single lysine or lysine pair of the small subunit is sufficient for ubiquitination
Preservation of caspase-3 subunits from degradation contributes to apoptosis evoked by lactacystin: Any single lysine or lysine pair of the small subunit is sufficient for ubiquitination
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DOI：
10.1124/mol.64.2.334
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发表时间：
2003-08-01
影响因子：
3.6
通讯作者：
Smith, JB
中科院分区：
文献类型：
--
作者：
Chen, L;Smith, L;Smith, JB
半胱天冬酶原-3（p32）由上游半胱天冬酶加工成p12和p20亚基，它们异源二聚体化。伴随着活性异源四聚体的形成，p20被自动加工成p17。用蛋白酶体的选择性抑制剂lactacystin处理HL-60细胞，以指数方式增加caspase-3样水解活性并诱导凋亡，但对上游caspase-8、caspase-9或颗粒酶B的活性几乎没有影响。Lactacystin处理降低了p32酶原，并诱发了p17和p12亚基的积累。用蛋白酶体抑制剂处理转染的人视网膜母细胞911细胞引起表位标记的p12、p17和p20的积累，但对p32酶原没有影响。这一结果表明，caspase-3亚基，在酶原相反，是不稳定的，因为降解的泛素-蛋白酶体系统。p12和p17的泛素缀合物在用p12和p17的半胱天冬酶失活突变体共转染的细胞中积累。精氨酸取代所有八个赖氨酸的p12几乎取消其泛素化。任何单个赖氨酸或赖氨酸对都足以使p12泛素化。Lactacystin处理HL-60细胞诱导X连锁凋亡抑制因子（XIAP）的蛋白水解加工，并减少全长XIAP，已知其对活性半胱天冬酶-3具有泛素-蛋白连接酶活性。这些研究结果表明，caspase-3亚基可以被泛素-蛋白酶体系统降解，并表明lactacystin诱导凋亡的部分通过禁用XIAP的泛素-蛋白连接酶功能和稳定活性caspase-3亚基。
Procaspase-3 (p32) is processed by upstream caspases to p12 and p20 subunits, which heterodimerize. Concomitant with formation of the active heterotetramer, p20 is autoprocessed to p17. Treatment of HL-60 cells with lactacystin, a selective inhibitor of the proteasome, exponentially increased caspase-3-like hydrolytic activity and induced apoptosis but had little or no effect on the activity of upstream caspase-8, caspase-9, or granzyme B. Lactacystin treatment decreased the p32 zymogen and evoked the accumulation of the p17 and p12 subunits. Treatment of transfected human retinoblast 911 cells with a proteasome inhibitor evoked the accumulation of epitope-tagged p12, p17, and p20 but had no effect on p32 zymogen. This result suggests that caspase-3 subunits, in contrast to the zymogen, are unstable because of degradation by the ubiquitin-proteasome system. Ubiquitin conjugates of p12 and p17 accumulated in cells that were cotransfected with p12 and a caspase inactive mutant of p17. Substitution of arginine for all eight lysines of p12 almost abolished its ubiquitination. Any single lysine or lysine pair was sufficient for p12 ubiquitination. Lactacystin treatment of HL-60 cells induced proteolytic processing of the X-linked inhibitor of apoptosis (XIAP) and decreased full-length XIAP, which is known to have ubiquitin-protein ligase activity for active caspase-3. These findings indicate that caspase-3 subunits can be degraded by the ubiquitin-proteasome system and suggest that lactacystin induces apoptosis in part by disabling the ubiquitin-protein ligase function of XIAP and by stabilizing active caspase-3 subunits.