Role of ADP-ribosylation in Stress Granules

ADP-核糖基化在应激颗粒中的作用

• 批准号: 10268197
• 负责人: Anthony K L Leung
• 金额: $ 40.21万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10268197
• 关键词: ADP ribosylation; Acids; Address; Affect; Amyotrophic Lateral Sclerosis; Binding; Binding Proteins; Biophysics; Cell physiology; Cells; Complex; Cytoplasm; Cytoplasmic Granules; DNA; DNA Repair; Data; Disease; Enzymes; Eukaryotic Cell; Event; Family; Funding; Goals; Heat-Shock Response; Higher Order Chromatin Structure; Hypoxia; In Vitro; Individual; Knowledge; Label; Length; Link; Liquid substance; Location; Malignant Neoplasms; Mediating; Membrane; Messenger RNA; Methods; Mitotic spindle; Modeling; Molecular; Mutagenesis; Mutation; Nerve Degeneration; Neurologic; Nucleic Acids; Nucleoplasm; Oils; Organelles; Oxidative Stress; Pathologic; Pharmacologic Substance; Phase; Physical condensation; Physiological; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymerase; Polymers; Post-Translational Protein Processing; Protein Dynamics; Proteins; Proteomics; RNA; Regulation; Repair Complex; Ribonucleoproteins; Ribosomes; Role; Scaffolding Protein; Series; Stress; Structure; Techniques; Testing; Therapeutic; Time; Translation Initiation; Virus Diseases; Water; base; biological adaptation to stress; biophysical techniques; design; inhibitor/antagonist; innovation; insight; live cell imaging; mutant; nervous system disorder; next generation; recruit; response; scaffold; stress granule; stressor; tool

Project Summary Membrane-less structures are prevalent in cells and executing unique functions (e.g., DNA repair foci and nucleoli for making ribosomal subunits). The mechanisms by which their formation, size, number, and dynamics are regulated, however, remain unclear. Emergent studies revealed that their formation can be partly explained by a biophysical phenomenon known as liquid-liquid phase separation, whereby the nucleoplasm and cytoplasm are considered complex fluids that stably segregate like oil and water. Phase separation is often triggered when proteins bind to a common scaffold such as the nucleic acids DNA and RNA, resulting in the condensation of proteins to form higher-order structures. We previously discovered that an under-studied nucleic acid called poly(ADP-ribose) (PAR) is critical for the formation of a class of membrane-less organelles implicated in cancer, virus infection and neurodegeneration called stress granules. Stress granules are cytoplasmic RNA-protein assemblies formed in different sizes in response to stressors such as hypoxia, oxidative stress and heat shock. Most granule components dynamically exchange with the surrounding cytoplasm, and individual granules grow in size over time through fusion. Notably, stress granules in models of neuropathological diseases, such as amyotrophic lateral sclerosis (ALS), have slower exchange dynamics and are less able to fuse. However, the molecular factors that control the stress granule dynamics and fusion (which affects size and number of granules) remain poorly understood. In this proposal, we will (1) determine how PAR regulates phase separation in stress granules using innovative techniques to define critical parameters of ADP- ribosylation for stress granule formation in cells and in vitro, and (2) determine whether PAR- protein interactions regulate stress granule fusion using mutagenesis, live-cell imaging, biophysical methods and proteomics. Projected Impact: Besides its role in stress granules, PAR is also critical for the formation of time- and location-specific membrane-less structures, including DNA repair complexes and nucleoli. This proposal will thus advance the field by defining critical parameters for physiologically relevant PAR-mediated phase separation and by identifying ADP-ribosylated proteins required for these phenomena. Given that PARPs are druggable and actively targeted by pharmaceutical companies, next-generation inhibitors may be designed to modulate the formation and dynamics of physiological and pathological membrane-less structures in neurological or other diseases.

项目摘要 无膜结构在细胞中普遍存在，并执行独特的功能（例如，DNA 修复灶和用于制造核糖体亚基的核仁）。它们的机制 然而，其形成、大小、数量和动力学是否受到调控仍不清楚。紧急 研究表明，它们的形成可以部分地用生物物理现象来解释， 称为液-液相分离，由此核质和细胞质被分离。 被认为是像油和水一样稳定分离的复杂流体。相分离通常 当蛋白质结合到共同的支架如核酸DNA和RNA时触发， 导致蛋白质缩合形成高级结构。我们之前 发现一种研究不足的核酸称为聚（ADP-核糖）（PAR）是关键的 形成一类与癌症、病毒感染和 称为应激颗粒的神经变性。应激颗粒是细胞质RNA蛋白 反应于应激源如缺氧、氧化应激 和热休克。大多数颗粒成分与周围环境动态交换 细胞质，并且单个颗粒通过融合随着时间的推移而在尺寸上生长。值得注意的是， 在神经病理学疾病，如肌萎缩侧索硬化症（ALS）， 交换动力较慢，融合能力较弱。然而，分子因素， 控制应力颗粒动力学和融合（影响颗粒的大小和数量） 仍然知之甚少。在这个建议中，我们将（1）确定PAR如何调节相位 使用创新技术分离应力颗粒，以确定ADP的关键参数， 核糖基化用于细胞和体外应激颗粒形成，和（2）确定PAR- 蛋白质相互作用利用诱变，活细胞成像， 生物物理方法和蛋白质组学。预计的影响：除了在应激颗粒中的作用，PAR 也是形成时间和位置特异性无膜结构的关键， 包括DNA修复复合物和核仁。因此，该提案将通过以下方式推动该领域的发展： 定义生理学相关PAR介导的相分离的关键参数， 鉴定这些现象所需的ADP核糖基化蛋白。鉴于PARP是 下一代抑制剂可用于药物治疗，并被制药公司积极瞄准， 设计用于调节生理和病理的 神经系统或其他疾病中的无膜结构。