A solid state conceptualization of information transfer from gene to message to protein

从基因到消息到蛋白质的信息传递的固态概念化

• 批准号: 10083747
• 负责人: STEVEN L MCKNIGHT
• 金额: $ 48.6万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10083747
• 关键词: Adopted; Amyloid; Applications Grants; Back; Basophils; Binding Sites; Biochemistry; Biology; C9ORF72; California; Cells; Chemistry; Cytoplasmic Granules; Dipeptides; Electron Spin Resonance Spectroscopy; Eukaryotic Cell; Event; Filament; Gene Transfer; Genes; Heart; Human Genetics; Hydrogels; Intermediate Filament Proteins; Liquid substance; Mediating; Membrane; Messenger RNA; Methods; Molecular Conformation; Molecular Structure; Morphology; Mutagenesis; NMR Spectroscopy; Neurons; Nuclear; Organelles; Paper; Pathogenesis; Pathogenicity; Pharmacology; Phase; Phase Transition; Polymers; Proteins; Publications; Publishing; RNA; Research; Resolution; Science; Specific qualifier value; Structure; Tertiary Protein Structure; Transcript; Translations; Universities; Work; interest; organizational structure; prion-like; solid state; solid state nuclear magnetic resonance

Project Summary/Abstract: Since 2012 the McKnight lab has studied an enigmatic class of protein domains specified by low complexity (LC) sequences. Certain LC sequences assemble into amyloid-like polymers, leading to formation of liquid-like droplets that sequentially mature into hydrogels. Although morphologically indistinguishable from pathogenic, prion-like amyloids, polymers formed from LC sequences disassemble upon dilution. Atomic resolution of the structure of FUS polymers has recently revealed the basis of LC domain polymer lability (11). In the six years since publication of our back-to-back 2012 papers in Cell (9,10), studies of RNA granules, membrane-less organelles and LC domains have exploded. That LC domains are involved in the formation of meso-scaled puncta not surrounded by investing membranes has been firmly established. Our observations favoring the involvement of labile cross- interactions in the formation of these structures, however, stands in contrast to what has become the prevailing view in the field. Numerous groups have argued that LC sequences adopt no molecular structure upon phase transition. The McKnight lab has provided multiple lines of evidence showing that formation of labile cross- interactions is at the heart of phase separation into liquid-like droplets, hydrogel formation, and LC domain function in living cells. Evidence supportive of these conclusions includes chemistry (14,16), pharmacology (12,16), correlative mutagenesis (10, 13, 14), human genetics (12, 15), and – most recently - solid state NMR spectroscopy (11). The narrative of this application avoids this controversy and instead focuses on several objectives important to the advancement of our research. An assessment of differences between the McKnight perspective (LC domain function as driven by defined structural organization) and that of most others who have joined the field (LC domain function sans molecular structure) can be found in our review chapter recently published the Annual Review of Biochemistry (17). The two projects described in this application represent extensions of our work on the LC domain of FUS. We are also interested in other settings wherein cells employ labile cross- interactions to abet normal or abnormal biology. We have found that the LC domains of intermediate filament proteins utilize labile, cross- interactions to mediate filament maturation. In the context of assembled filaments, we have shown that coalesced LC domains represent binding sites for RNA granules (12). We also deduced the basis of C9orf72 pathogenesis via the toxic GRn and PRn poly-dipeptides produced by RAN translation of repeat expansion transcripts – thus resolving how neurons die in the most prevalent form of ALS (12,15,16). Aside from continuing our collaborative SS-NMR studies with Dr. Robert Tycko, and NAI footprinting studies with Dr. Yonghao Yu to probe LC domain conformation in living cells, we have established a collaborative partnership with Dr. Ralf Langen of the University of Southern California to use electron paramagnetic resonance (EPR) methods for studies probing the earliest events wherein LC domains coalesce.

项目摘要/摘要：自2012年以来，McKnight实验室研究了一类神秘的蛋白质结构域 由低复杂度（LC）序列指定。某些LC序列组装成淀粉样聚合物， 导致形成液体状液滴，其依次成熟为水凝胶。虽然在形态上 与致病性朊病毒样淀粉样蛋白难以区分，由LC序列形成的聚合物在 稀释近年来，FUS聚合物结构的原子分辨率揭示了LC畴的基础 聚合物不稳定性（11）。自我们2012年在Cell上连续发表论文以来的六年中（9，10）， RNA颗粒、无膜细胞器和LC结构域已经爆炸。LC结构域参与了 已经牢固地建立了不被包埋膜包围的中尺度点的形成。我们 观察结果支持在这些结构的形成中涉及不稳定的交叉相互作用， 然而，这与实地的普遍看法形成对照。许多团体认为， LC序列在相变时不采用分子结构。麦克奈特实验室提供了 多条证据表明，不稳定的交叉相互作用的形成是阶段的核心， 分离成液体样液滴、水凝胶形成和LC结构域在活细胞中的功能。证据 支持这些结论的包括化学（14，16）、药理学（12，16）、相关诱变 (10，13，14），人类遗传学（12，15），和-最近-固态NMR光谱（11）。叙事 本申请的第一部分避免了这种争议，而是集中在对本申请重要的几个目标上。 推进我们的研究。McKnight观点（LC领域）之间差异的评估 由已定义的结构组织驱动的职能）和已加入该领域的大多数其他人的职能（LC 结构域功能sans分子结构）可以在我们最近出版的年度评论章节中找到。 生物化学（17）。本申请中描述的两个项目代表了我们在以下方面工作的扩展： FUS的LC结构域。我们也对其他的环境感兴趣，在这些环境中，细胞采用不稳定的交叉作用。 来教唆正常或异常的生物学我们发现中间丝蛋白的LC结构域 利用不稳定的，交叉的相互作用来调节花丝的成熟。在组装细丝的背景下，我们 已经表明，合并的LC结构域代表RNA颗粒的结合位点（12）。我们还推断出 C9orf72致病的基础是通过由RAN翻译产生的毒性GRn和PRn聚二肽， 重复扩增转录本-从而解决了神经元如何在最普遍的ALS形式中死亡（12，15，16）。 除了继续我们与Robert Tycko博士合作的SS-NMR研究和NAI足迹研究之外， 与Yonghao Yu博士一起探索活细胞中的LC结构域构象，我们建立了一个合作伙伴关系， 与南加州大学的Ralf Langen博士合作， 共振（EPR）方法用于研究探测其中LC域聚结的最早事件。