Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA.
Mol Cell. 2013 Jan 24;49(2):273-82. doi: 10.1016/j.molcel.2012.10.022. Epub 2012 Nov 29.
Inhibitors of Apoptosis Protein (IAPs) are guardian ubiquitin ligases that keep classic proapoptotic proteins in check. Systematic identification of additional IAP substrates is challenged by the heterogeneity and sheer number of ubiquitinated proteins (>5,000). Here we report a powerful catalytic tagging tool, the NEDDylator, which fuses a NEDD8 E2-conjugating enzyme, Ubc12, to the ubiquitin ligase, XIAP or cIAP1. This permits transfer of the rare ubiquitin homolog NEDD8 to the ubiquitin E3 substrates, allowing them to be efficiently purified for LC-MS/MS identification. We have identified >50 potential IAP substrates of both cytosolic and mitochondrial origin that bear hallmark N-terminal IAP binding motifs. These substrates include the recently discovered protein phosphatase PGAM5, which we show is proteolytically processed, accumulates in cytosol during apoptosis, and sensitizes cells to death. These studies reveal mechanisms and antagonistic partners for specific IAPs, and provide a powerful technology for labeling binding partners in transient protein-protein complexes.
凋亡蛋白抑制因子（IAPs）是守护泛素连接酶，可控制经典促凋亡蛋白。由于泛素化蛋白（>5000 种）的异质性和数量庞大，系统鉴定其他 IAP 底物具有挑战性。本研究报道了一种强大的催化标记工具，即 NEDDylator，它将 NEDD8 E2 连接酶 Ubc12 融合到泛素连接酶 XIAP 或 cIAP1 上。这允许将稀有泛素类似物 NEDD8 转移到泛素 E3 底物上，从而可以有效地对其进行纯化，以进行 LC-MS/MS 鉴定。我们已经鉴定出 >50 种具有细胞质和线粒体起源的潜在 IAP 底物，它们具有标志性的 N 端 IAP 结合基序。这些底物包括最近发现的蛋白磷酸酶 PGAM5，我们发现它被蛋白水解加工，在凋亡过程中在细胞质中积累，并使细胞对死亡敏感。这些研究揭示了特定 IAP 的作用机制和拮抗伙伴，并提供了一种强大的技术，可用于标记瞬时蛋白-蛋白复合物中的结合伙伴。
