Regulation of ARF tumor suppressor function by biological phase separation

通过生物相分离调节ARF肿瘤抑制功能

• 批准号: 10065965
• 负责人: Eric B Gibbs
• 金额: $ 6.74万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-09 至 2021-07-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10065965
• 关键词: Affect; Amino Acid Sequence; Apoptosis; Arginine; Behavior; Binding; Biogenesis; Biological Assay; Biological Models; Biological Process; Biomolecular Nuclear Magnetic Resonance; CDKN2A gene; Cancer Cell Growth; Cell Count; Cell Cycle Arrest; Cell Nucleolus; Cells; Cellular Stress; Cellular biology; Characteristics; Complex; Event; Exhibits; Fluorescence; Fluorescence Recovery After Photobleaching; Future; Growth; Homeostasis; Human; Hydrophobicity; Image; Imaging Techniques; In Vitro; Interphase; Length; Link; Liquid substance; MDM2 gene; Maintenance; Malignant - descriptor; Malignant Neoplasms; Maps; Mediating; Methods; Microscopy; Modeling; Molecular; Monitor; Mouse Protein; Mus; NMR Spectroscopy; NPM1 gene; Neutrons; Normal Cell; Nuclear Export; Nucleolar Proteins; Oncogenes; Oncogenic; Phase; Phenotype; Play; Production; Property; Protein Biosynthesis; Proteins; RNA; Reading; Regulation; Ribosomal RNA; Ribosomes; Site; Site-Directed Mutagenesis; Solid; Spectrum Analysis; Stress; Structure; System; TP53 gene; Techniques; Testing; Time; Training; Tumor Suppressor Genes; Tumor Suppressor Proteins; biological adaptation to stress; biophysical model; cancer cell; cell growth; experience; experimental study; fluorescence imaging; frontier; insight; live cell imaging; loss of function; molecular modeling; mutant; novel; novel therapeutic intervention; nucleophosmin; p19ARF; particle; polysome profiling; prevent; reconstitution; sensor; solid state nuclear magnetic resonance; structural biology; targeted cancer therapy; tumor

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.

项目摘要 这项研究的目的是确定相分离如何调节肿瘤抑制因子ARF蛋白 在人类癌症中经常发生功能丧失。在致癌应激期间， 在核仁中的必需蛋白质，以阻止细胞周期或诱导凋亡。具体而言，ARF 将HDM 2隔离在核仁中，其激活p53依赖性细胞周期停滞或凋亡。ARF还 将NPM 1隔离在核仁中，破坏核糖体组装，这也诱导细胞周期停滞 与p53无关。然而，ARF将其靶点隔离在核仁中的机制并不明确。 明白 最近的观察表明，核仁具有类似液体的性质，这表现在 其组成蛋白质和RNA的相分离。最近，我们的实验室沿着与布兰温集团， 表明核仁的液体状特征部分取决于NPM 1的相分离性质。 我假设p14 ARF通过以下途径破坏了颗粒组分的液体样性质： 其多个富含甘氨酸的基序与NPM 1的疏水自缔合和多价相互作用， 导致核糖体生物合成的抑制。 本研究将确定（1）p14 ARF的物理化学性质如何使其能够螯合其靶点 （2）相分离的p14 ARF-NPM 1的结构和动力学 p14 ARF表达对NPM 1动力学、核糖体生物合成和生长的影响 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， 以及在更广泛的背景下，在致癌应激事件期间，核仁的流体特征如何改变。