Regulation of ARF tumor suppressor function by biological phase separation

项目总结 这项拟议的研究旨在确定相分离如何调节肿瘤抑制因子ARF蛋白。 在人类癌症中经常经历功能丧失。在致癌应激期间，ARF隔离 核仁中的必需蛋白质，以阻止细胞周期或诱导细胞凋亡。具体地说，ARF 将Hdm2隔离在核仁中，激活依赖于P53的细胞周期停滞或凋亡。ARF也 将NPM1隔离在核仁中，扰乱核糖体组装，这也会导致细胞周期停滞 不依赖于P53。然而，ARF将其靶标隔离在核仁中的机制并不是 明白了。 最近的观察表明，核仁具有类似液体的性质，这种性质通过 它们的组成蛋白质和RNA的相分离。最近，我们的实验室和Brangwynne小组一起， 结果表明，核仁的液状特征部分取决于NPM1的相分离性质。 我假设p14ARF通过以下方式破坏颗粒成分的类似液体的属性 其多个富含精氨酸的基序与NPM1的疏水自结合和多价相互作用， 导致核糖体生物发生的抑制。 这项研究将确定(1)p14ARF的物理化学性质如何使其能够隔离其靶标 相分离小体中的蛋白质，(2)相分离p14ARF-NPM1的结构和动力学 以及(3)p14ARF表达对NPM1动态、核糖体生物发生和生长的影响 在活细胞中。这项研究的结果将为ARF的核仁肿瘤抑制功能提供新的见解。 在更广泛的背景下，核仁的流体特征是如何在致癌应激事件中改变的。

PROJECT SUMMARY The proposed study aims to determine how phase separation regulates the ARF protein, a tumor suppressor that frequently experiences loss of function in human cancers. During oncogenic stress, ARF sequesters essential proteins in the nucleolus in order to arrest the cell cycle or induce apoptosis. Specifically, ARF sequesters HDM2 in the nucleolus, which activates p53-dependent cell cycle arrest or apoptosis. ARF also sequesters NPM1 in the nucleolus, disrupting ribosome assembly, which also induces cell cycle arrest independently of p53. However, the mechanism by which ARF sequesters its targets in the nucleolus is not understood. Recent observations have demonstrated that nucleoli possess liquid-like properties, which manifest through phase separation of their constituent proteins and RNAs. Recently, our lab along with the Brangwynne group, showed that the liquid like features of the nucleolus depends in-part on the phase separation properties of NPM1. I hypothesize that p14ARF disrupts the liquid-like properties of the granular component through hydrophobic self-association and multivalent interactions of its multiple arginine-rich motifs with NPM1, resulting in inhibition of ribosome biogenesis. This study will determine (1) how p14ARF’s physicochemical properties allow it to sequester its target proteins within phase separated bodies, (2) the structure and dynamics of phase separated p14ARF-NPM1 complexes, and (3) the effect of p14ARF expression on NPM1 dynamics, ribosome biogenesis and growth in live cells. Results from this study will provide novel insights into ARF’s nucleolar tumor suppressor function, and in a broader context, how the fluid-features of nucleoli are altered during oncogenic stress events.