中山大学Zhou Songyang、Su Wu、Feng Liu团队取得一项新突破。他们利用快速接近标记系统PhastID发现ATP6AP1是Rheb的非常规鸟嘌呤核苷酸交换因子（GEF）。该研究于2024年3月6日发表于国际学术期刊《细胞研究》杂志。

研究人员建立了一种利用角越橘火球菌生物素蛋白连接酶（PhBPL）辅助生物素鉴定（PhastID）的快速、稳定的近距离标记系统，并详细阐述了PhastID鉴定Rheb-近距离蛋白网络在胰岛素刺激时发生的变化。

研究发现溶酶体V-ATPase亚基ATP6AP1可与Rheb发生动态相互作用。ATP6AP1可通过其末端12个氨基酸直接与Rheb结合，并利用其C末端高度保守的三个天冬氨酸基团来增强Rheb的GTP负载。靶向ATP6AP1 C末端可以阻断Rheb的活化，抑制癌细胞的增殖和迁移。

该研究结果突显了PhastID在绘制活细胞中瞬时PPIs图谱方面的多功能性，揭示了ATP6AP1作为Rheb非传统GEF的功能，凸显了ATP6AP1在Rheb整合mTORC1信号激活方面的重要性，填补了Rheb/mTORC1激活过程中缺失的环节。

据悉，Rheb是一种小G蛋白，它是雷帕霉素靶标复合体1（mTORC1）的直接激活剂，可诱导信号级联反应以响应营养物质和生长因子刺激。尽管进行了大量研究，但直接激活Rheb的GEF仍然不清楚，这部分是由于作为信号转导标志的蛋白质-蛋白质相互作用（PPI），具有动态性和瞬时性。

附：英文原文

Title: The rapid proximity labeling system PhastID identifies ATP6AP1 as an unconventional GEF for Rheb

Author: Feng, Ran, Liu, Feng, Li, Ruofei, Zhou, Zhifen, Lin, Zhuoheng, Lin, Song, Deng, Shengcheng, Li, Yingying, Nong, Baoting, Xia, Ying, Li, Zhiyi, Zhong, Xiaoqin, Yang, Shuhan, Wan, Gang, Ma, Wenbin, Wu, Su, Songyang, Zhou

Issue&Volume: 2024-03-06

Abstract: Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein–protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1’s role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

DOI: 10.1038/s41422-024-00938-z

Source: https://www.nature.com/articles/s41422-024-00938-z