| Popis: |
X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) has been implicated as a novel tumor suppressor, which was proposed to exert pro-apoptotic effect by antagonizing the anticaspase activity of XIAP. Here, we delineated the domain architecture of XAF1 by applying limited proteolysis and peptide mass fingerprinting analysis. Our results indicated that XAF1 has a distinct domain organization, with a highly compact N-terminal domain (XAF1NTD) followed by a middle domain (XAF1MD), a 42-residue unstructured linker and a C-terminal domain (XAF1CTD). The search of XIAP binding region within XAF1 revealed that a modest affinity XIAPRING binding site (dissociation constant, Kd, ∼18 μM) is located at the C-terminal portion of XAF1. This C-terminal region, embracing XAF1CTD and a flexible tail at C-terminus (residue Thr251-Ser301), is functionally identified as XIAPRING-binding domain of XAF1 (XAF1RBD) in the present study. We have also mapped the interaction sites for XAF1RBD on XIAPRING by using NMR spectroscopy. By applying in vitro ubiquitination assay, we observed that XAF1RBD/XIAP interaction is essential for the ubiquitination of GST-XAF1RBD fusion protein. In addition, the C-terminal XAF1 fragment harboring XAF1RBD was found to be substantially ubiquitinated by XIAPRING. Base on these observations, we speculate a possible role of XAF1RBD in targeting XAF1 for XIAP-mediated ubiquitination. |
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