Department of Cell Physiology and Metabolism , University of Geneva , 1211 Geneva 4 , Switzerland.
National Centre of Competence in Research (NCCR) in Chemical Biology , University of Geneva , Geneva , Switzerland.
ACS Chem Biol. 2020 Jan 17;15(1):243-253. doi: 10.1021/acschembio.9b00832. Epub 2019 Dec 18.
VCP/p97 belongs to the AAA+ ATPase family and has an essential role in several cellular processes ranging from cell division to protein homeostasis. Compounds targeting p97 inhibit the main ATPase domain and cause cell death. Here, using PNA-encoded chemical libraries, we have identified two small molecules that target the regulatory domain of p97, comprising the N-terminal and the D1 ATPase domains, and do not cause cell death. One molecule, , inhibits the degradation of a p97-dependent reporter, whereas the other, , increases it. ATPase assays show that and do not affect the main catalytic domain of p97. Mapping of the binding site using a photoaffinity conjugate points to a cleft at the interface of the N-terminal and the D1 ATPase domains. We have therefore discovered two new compounds that bind to the regulatory domain of p97 and modulate specific p97 cellular functions. Using these compounds, we have revealed a role for p97 in the regulation of mitotic spindle orientation in HeLa cells.
VCP/p97 属于 AAA+ ATP 酶家族，在从细胞分裂到蛋白质动态平衡等多种细胞过程中发挥着重要作用。靶向 p97 的化合物会抑制其主要的 ATP 酶结构域并导致细胞死亡。在这里，我们使用 PNA 编码的化学文库，鉴定出两种靶向 p97 调节结构域的小分子，它们包含 N 端和 D1 ATP 酶结构域，并且不会导致细胞死亡。其中一种分子 ，抑制依赖于 p97 的报告蛋白的降解，而另一种分子 ，则增加其降解。ATP 酶实验表明， 和 不影响 p97 的主要催化结构域。使用光亲和标记物进行结合位点映射表明，它们结合在 N 端和 D1 ATP 酶结构域的界面缝隙处。因此，我们发现了两种新的可与 p97 调节结构域结合并调节特定 p97 细胞功能的化合物。利用这些化合物，我们揭示了 p97 在调控 HeLa 细胞有丝分裂纺锤体方向中的作用。
