Adrain Colin, Murphy Brona M, Martin Seamus J
Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland.
J Biol Chem. 2005 Feb 11;280(6):4663-73. doi: 10.1074/jbc.M410915200. Epub 2004 Nov 29.
Granzyme B is a major cytotoxic T lymphocyte/natural killer (CTL/NK) granule protease that can activate members of the caspase family of cysteine proteases through processing of caspase zymogens. However, the molecular order and relative importance of caspase activation events that occur in target cells during granzyme B-initiated apoptosis has not been established. Here, we have examined the hierarchy of granzyme B-initiated caspase activation events using a cell-free system where all caspases are present at physiological levels. We show that granzyme B initiates a two-tiered caspase activation cascade involving seven caspases, where caspase-3 is required for the second tier of caspase activation events. Using a two-dimensional gel-based proteomics approach we have also examined the scale of granzyme B-initiated alterations to the proteome in the presence or absence of effector caspase-3 or -7. These studies indicate that granzyme B targets a highly restricted range of substrates and orchestrates cellular demolition largely through activation of caspase-3.
颗粒酶B是一种主要的细胞毒性T淋巴细胞/自然杀伤细胞（CTL/NK）颗粒蛋白酶，它可以通过加工半胱天冬酶酶原激活半胱氨酸蛋白酶家族的成员。然而，在颗粒酶B引发的细胞凋亡过程中，靶细胞内发生的半胱天冬酶激活事件的分子顺序和相对重要性尚未确定。在这里，我们使用了一种无细胞系统来研究颗粒酶B引发的半胱天冬酶激活事件的层次结构，该系统中所有半胱天冬酶都处于生理水平。我们发现颗粒酶B启动了一个涉及七种半胱天冬酶的两级半胱天冬酶激活级联反应，其中半胱天冬酶-3是第二层半胱天冬酶激活事件所必需的。我们还使用基于二维凝胶的蛋白质组学方法，研究了在存在或不存在效应半胱天冬酶-3或-7的情况下，颗粒酶B引发的蛋白质组变化规模。这些研究表明，颗粒酶B靶向高度受限的底物范围，并主要通过激活半胱天冬酶-3来协调细胞破坏。
