Use of the yeast three-hybrid system as a tool to study caspases
Use of the yeast three-hybrid system as a tool to study caspases
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DOI：
10.1006/abio.1998.2823
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发表时间：
1998-10-01
影响因子：
2.9
通讯作者：
Beyaert, R
中科院分区：
文献类型：
--
作者：
Van Criekinge, W;van Gurp, M;Beyaert, R
半胱天冬酶是一个异聚体（p20/p10）半胱氨酸蛋白酶家族，在细胞凋亡和炎症调节中具有重要功能。到目前为止，用于鉴定半胱天冬酶的新底物的工具大多限于随机筛选已知或假定在细胞凋亡中起作用的体外翻译蛋白。我们描述了使用酵母三杂交方法作为一种工具，适应经典的双杂交系统的需要异聚半胱天冬酶的功能解剖已知的相互作用或筛选生理底物和抑制剂。通过共表达p20（Cys 285 Ser）和p10 caspase-1亚基获得功能性异聚体caspase-1，所述p20（Cys 285 Ser）和p10 caspase-1亚基仅各自融合至Ga 14 DNA结合结构域。在Ga 14激活结构域和病毒半胱天冬酶-1假底物抑制剂CrmA或p35或原型生理半胱天冬酶-1底物白细胞介素-1 β原的第三杂交体共表达后，可以重建功能性Ga 14转录因子。相反，CrmA或p35与胱天蛋白酶-1的未成熟p45或p30前体形式之间没有发现相互作用。因此，三杂交系统可能允许筛选新的生理底物和异聚半胱天冬酶抑制剂。(C)北京：科学出版社.
Caspases are a family of heteromeric (p20/p10) cysteine proteases with important functions in the regulation of apoptosis and inflammation. Up to now, tools to identify new substrates for caspases have mostly been limited to the random screening of in vitro translated proteins that are known, or assumed, to play a role in apoptosis. We describe the use of a yeast three-hybrid approach as a tool that adapts the classical two-hybrid system to the needs of heteromeric caspases for functional dissection of known interactions or screening for physiological substrates and inhibitors. Functional heteromeric caspase-1 was obtained by coexpression of p20(Cys285Ser) and p10 caspase-1 subunits that mere each fused to the Ga14 DNA-binding domain. Upon coexpression of a third hybrid of the Ga14 activation domain and the viral caspase-1 pseudosubstrate inhibitors CrmA or p35, or the prototype physiological caspase-1 substrate prointerleukin-1 beta, a functional Ga14 transcription factor could be reconstituted. In contrast, no interaction was found between CrmA or p35 and the immature p45 or p30 precursor forms of caspase-1. Therefore, the three-hybrid system might allow screening for new physiological substrates and inhibitors of heteromeric caspases. (C) 1998 Academic Press.