Nikolovska-Coleska Zaneta, Meagher Jennifer L, Jiang Sheng, Kawamoto Steven A, Gao Wei, Yi Han, Qin Dongguang, Roller Peter P, Stuckey Jeanne A, Wang Shaomeng
Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.
Anal Biochem. 2008 Mar 1;374(1):87-98. doi: 10.1016/j.ab.2007.10.032. Epub 2007 Nov 20.
XIAP (X-chromosome-linked inhibitor of apoptosis protein) is an inhibitor of apoptosis by binding to and inhibition of caspase-3 and caspase-7 through its BIR2 domain and caspase-9 through its BIR3 domain. Smac (second mitochondria-derived activator of caspases) protein is an endogenous antagonist of XIAP. Smac forms a dimer and concurrently binds both the BIR2 and BIR3 domains in XIAP, functioning as a highly efficient and potent cellular inhibitor of XIAP. In this article, we have designed and synthesized a bivalent Smac-based ligand (Smac-1) and its fluorescent labeled analogue (Smac-1F) and characterized their interaction with different constructs of XIAP. Our study demonstrates that bivalent Smac-based ligands bind concurrently to both the BIR2 and BIR3 domains of XIAP and are more than 500 times more potent than the corresponding monovalent Smac-based ligands. Bivalent Smac-based ligands also function as much more potent antagonists of XIAP than do the corresponding monovalent Smac-based ligands in cell-free functional assays. Using Smac-1F and XIAP containing both BIR2 and BIR3 domains, we also developed and validated a new fluorescence polarization-based assay. Hence, our designed bivalent Smac-based peptides mimic the mode of dimeric Smac protein in their interaction with XIAP containing both BIR2 and BIR3 domains and achieve extremely high potency in binding and functional assays. Our study provides new insights into the mode of action of bivalent Smac ligands targeting XIAP and a basis for the design and development of cell-permeable, bivalent Smac mimetics.
XIAP（X染色体连锁凋亡抑制蛋白）是一种凋亡抑制因子，通过其BIR2结构域与半胱天冬酶-3和半胱天冬酶-7结合并抑制它们，通过其BIR3结构域与半胱天冬酶-9结合并抑制它。Smac（线粒体衍生的半胱天冬酶激活因子-2）蛋白是XIAP的内源性拮抗剂。Smac形成二聚体并同时结合XIAP中的BIR2和BIR3结构域，作为XIAP的一种高效且强效的细胞抑制剂发挥作用。在本文中，我们设计并合成了一种基于Smac的二价配体（Smac-1）及其荧光标记类似物（Smac-1F），并表征了它们与XIAP不同构建体的相互作用。我们的研究表明，基于Smac的二价配体同时结合XIAP的BIR2和BIR3结构域，其效力比相应的基于Smac的单价配体高500倍以上。在无细胞功能测定中，基于Smac的二价配体作为XIAP的拮抗剂也比相应的基于Smac的单价配体更有效。使用Smac-1F和同时包含BIR2和BIR3结构域的XIAP，我们还开发并验证了一种基于荧光偏振的新测定方法。因此，我们设计的基于Smac的二价肽在与同时包含BIR2和BIR3结构域的XIAP相互作用时，模拟了二聚体Smac蛋白的作用模式，并在结合和功能测定中达到了极高的效力。我们的研究为靶向XIAP的二价Smac配体的作用模式提供了新的见解，并为可细胞穿透的二价Smac模拟物的设计和开发奠定了基础。
