Max Delbrück Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin, Germany.
Max-Planck-Institute for Coal Research, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany.
Nat Commun. 2016 Oct 20;7:13047. doi: 10.1038/ncomms13047.
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.
相互作用图谱是阐明蛋白质复合物及其调控因子的生物学功能的强大策略。在这里，我们报告了一种定量相互作用网络的产生，该网络直接将 14 个人类蛋白与 AAA+ ATPase p97 联系起来，p97 是一种具有多种细胞功能的必需六聚体蛋白。我们表明，高亲和力相互作用蛋白 ASPL 有效地促进 p97 六聚体解体，导致稳定的 p97:ASPL 杂四聚体的形成。高分辨率结构和生化研究表明，ASPL 中的扩展 UBX 结构域（eUBX）对于 p97 六聚体解体至关重要，并促进 p97:ASPL 杂四聚体的组装。这个自发过程伴随着 p97 中 D2 ATPase 结构域的重新定向和其活性的丧失。最后，我们证明 ASPL 的过表达会破坏 ERAD 中的 p97 六聚体功能，并且工程化的 eUBX 多肽可以诱导细胞死亡，为开发可能靶向 p97 活性的抗癌多肽抑制剂提供了理论依据。
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