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，麦克奈特实验室研究了一类神秘的蛋白质结构域 由低复杂性 (LC) 序列指定，某些 LC 序列组装成淀粉样蛋白聚合物， 导致形成液体状液滴，随后成熟为水凝胶。 与致病性朊病毒样淀粉样蛋白无法区分，由 LC 序列分解形成的聚合物 FUS 聚合物结构的原子分辨率最近揭示了 LC 结构域的基础。 聚合物不稳定性 (11) 自我们于 2012 年在 Cell (9,10) 上连续发表论文以来的六年中，研究了 RNA 颗粒、无膜细胞器和 LC 结构域都参与其中。 没有被包埋膜包围的中尺度斑点的形成已经牢固确立。 观察结果支持不稳定的交叉相互作用参与这些结构的形成， 然而，这与该领域的普遍观点相反。 麦克奈特实验室提供了LC序列在相变时不采用分子结构。 多种证据表明，不稳定的交叉相互作用的形成是阶段的核心 活细胞中液体状液滴的分离、水凝胶的形成和 LC 结构域功能的证据。 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)，以及最近的固态核磁共振波谱 (11)。 该应用程序避免了这种争议，而是集中于对 我们研究的进展。麦克奈特观点（LC 领域）之间差异的评估。 由定义的结构组织驱动的功能）以及大多数其他加入该领域的人的功能（LC 领域功能无分子结构）可以在我们最近发表的年度评论章节中找到 生物化学综述 (17)。本申请中描述的两个项目代表了我们的工作的扩展。 我们也对其他设置感兴趣，因此细胞采用不稳定的交叉  相互作用。 我们发现中间丝蛋白的 LC 结构域会促进正常或异常的生物学。 在组装丝的背景下，我们利用不稳定的交叉相互作用来介导丝的成熟。 已经表明，合并的 LC 结构域代表 RNA 颗粒的结合位点 (12)。 C9orf72 发病机制的基础是通过 RAN 翻译产生的有毒 GRn 和 PRn 多二肽 重复扩增转录本——从而解决了最常见的 ALS 形式中神经元如何死亡的问题 (12,15,16)。 除了继续与 Robert Tycko 博士合作进行 SS-NMR 研究和 NAI 足迹研究之外 与于永浩博士探讨活细胞中的LC结构域构象，我们建立了合作 与南加州大学 Ralf Langen 博士合作利用电子顺磁 共振 (EPR) 方法，用于研究探测 LC 结构域合并的最早事件。