Medical Proteomics Research Center, KRIBB, Daejeon 305-806, Republic of Korea.
Biochem Biophys Res Commun. 2010 Mar 19;393(4):794-9. doi: 10.1016/j.bbrc.2010.02.084. Epub 2010 Feb 18.
Caspase-3 is an important executor caspase that plays an essential role in apoptosis. Recently, HS1-associated protein X1 (HAX-1) was found to be a substrate of caspase-3. Although HAX-1 has serve multifunctional roles in cellular functions such as cell survival and calcium homeostasis, the detailed functional mechanism of HAX-1 remains still unclear. In this study, we performed proteomic experiments to identify the HAX-1 interactome. Through immunoprecipitation and 2D gel electrophoresis, we identified X-linked inhibitor of apoptosis protein (XIAP) as a novel HAX-1-interacting protein. By performing the GST pull-down assay, we defined the interaction domains in HAX-1 and XIAP, showing that HAX-1 binds to the BIR2 and BIR3 domains of XIAP whereas XIAP binds to the C-terminal domain of HAX-1. In addition, surface plasma resonance experiments showed that both BIR2 and BIR3 domains of XIAP bind to HAX-1 with affinity similar to that of full-length XIAP, indicating that either domain is necessary and sufficient for tight binding to HAX-1. Taken together with the observation that HAX-1 suppresses the polyubiquitination of XIAP, the cell viability assay results suggest that the formation of the HAX-1-XIAP complex inhibits apoptosis by enhancing the stability of XIAP against proteosomal degradation.
Caspase-3 是一种重要的执行 Caspase，在细胞凋亡中发挥着重要作用。最近，HS1 相关蛋白 X1（HAX-1）被发现是 caspase-3 的底物。尽管 HAX-1 在细胞存活和钙稳态等细胞功能中具有多种功能，但 HAX-1 的详细功能机制仍不清楚。在这项研究中，我们进行了蛋白质组学实验来鉴定 HAX-1 的相互作用组。通过免疫沉淀和 2D 凝胶电泳，我们鉴定出 X 连锁凋亡抑制蛋白（XIAP）是 HAX-1 的一种新型相互作用蛋白。通过 GST 下拉实验，我们确定了 HAX-1 和 XIAP 中的相互作用域，表明 HAX-1 与 XIAP 的 BIR2 和 BIR3 结构域结合，而 XIAP 与 HAX-1 的 C 末端结构域结合。此外，表面等离子体共振实验表明，XIAP 的 BIR2 和 BIR3 结构域均与 HAX-1 具有相似的亲和力结合，表明任一结构域对于与 HAX-1 的紧密结合都是必要和充分的。结合 HAX-1 抑制 XIAP 的多泛素化的观察结果，细胞活力测定结果表明，HAX-1-XIAP 复合物的形成通过增强 XIAP 对蛋白酶体降解的稳定性来抑制细胞凋亡。
