Role of proteolysis in caspase-8 activation and stabilization
Role of proteolysis in caspase-8 activation and stabilization
复制标题
DOI：
10.1021/bi602623b
复制
发表时间：
2007-04-10
期刊：
影响因子：
2.9
通讯作者：
Salvesen, Guy S.
中科院分区：
文献类型：
--
作者：
Pop, Cristina;Fitzgerald, Patrick;Salvesen, Guy S.
胱天蛋白酶-8是一种凋亡蛋白酶，在细胞膜的胞质面被激活。活化依赖于衔接子诱导的单体胱天蛋白酶-8的二聚化，随后是分离催化结构域的两个亚基的接头的特异性有限的自蛋白水解。然而，这种自动蛋白水解的作用，直接激活刽子手半胱天冬酶-3和-7，是未知的顶端半胱天冬酶-8。我们已经产生了连接突变体的半胱天冬酶-8，可以在一个受控的方式由凝血酶或烟草蚀纹花叶病毒蛋白酶蛋白水解，我们用这些来定义的作用，蛋白水解酶的激活和稳定性。我们表明，蛋白水解是不足以产生重组半胱天冬酶-8的酶活性。使用Hoffmeister盐的动力学活化研究表明，活化是胱天蛋白酶二聚化的结果。然而，接头蛋白水解显著增强了半胱天冬酶-8二聚化的平衡，从而增加了二聚体的稳定性。动力学和荧光测量表明，半胱天冬酶-8激活的Hoffmeister盐至少是一个两步的事件，所需的步骤是二聚化，随后的分子内事件，进一步稳定的催化构象。半胱天冬酶-8的自身蛋白水解可能是增加二聚体酶从其在细胞膜上的活化复合物解离后的寿命的机制。
Caspase-8 is an apoptotic protease that is activated at the cytosolic face of the cell membrane. Activation relies on adaptor-induced dimerization of monomeric caspase-8 and is followed by specific limited autoproteolysis of the linker which separates the two subunits of the catalytic domain. However, the role of this autoproteolysis, which directly activates executioner caspases-3 and -7, is unknown for the apical caspase-8. We have generated linker mutants of caspase-8 that can be proteolyzed in a controlled manner by thrombin or tobacco etch mosaic virus protease, and we use these to define the role of proteolysis in the activation and stability of the enzyme. We show that proteolysis is insufficient for generating enzymatic activity in recombinant caspase-8. Kinetic activation studies using Hoffmeister salts demonstrate that activation is the result of caspase dimerization. However, linker proteolysis significantly enhances the equilibrium for caspase-8 dimerization, thereby increasing the stability of the dimer. Kinetic and fluorescence measurements demonstrate that caspase-8 activation by Hoffmeister salts is at least a two-step event, with the required step being dimerization, followed by an intramolecular event that further stabilizes the catalytic conformation. Autoproteolysis of caspase-8 may be a mechanism for increasing the lifetime of the dimeric enzyme following dissociation from its activating complex at the cell membrane.