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.
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