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: 10561709
• 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/10561709
• 关键词: Adopted; Amyloid; Applications Grants; Back; Binding Sites; Biochemistry; Biology; C9ORF72; California; Cells; Chemistry; Cytoplasmic Granules; Dipeptides; Electron Spin Resonance Spectroscopy; Eukaryotic Cell; Event; Explosion; Filament; Gene Transfer; Genes; Heart; Human Genetics; Hydrogels; Intermediate Filament Proteins; Investments; 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; 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 Lab已研究了一个神秘的蛋白质领域 由低复杂性（LC）序列指定。某些LC序列组装成淀粉样蛋白样聚合物， 导致形成液样液滴，这些液滴顺序成熟成水凝胶。虽然在形态上 与致病性，类似的淀粉样蛋白，由LC序列形成的聚合物无法区分 稀释。 FUS聚合物结构的原子分辨率最近揭示了LC结构域的基础 聚合物衰老（11）。自从我们的《牢房背靠背论文》发表以来的六年以来（9,10）的研究 已经探索了RNA颗粒，无膜细胞器和LC结构域。 LC域参与 在投资膜周围并没有牢固地建立中量表的刺激点。我们的 观察结果有利于不稳定的交叉相互作用参与这些结构的形成， 但是，与已成为现场的普遍观点相反。许多团体争辩 该LC序列在相变时不采用分子结构。 McKnight Lab提供了 多种证据表明，不稳定的跨-相互作用的形成是相位的核心 分离成液样液滴，水凝胶形成和LC结构域在活细胞中的功能。证据 这些结论的支持包括化学（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 - 固态NMR光谱法（11）。叙述 本应用程序避免了这一争议，而是专注于对 我们的研究进步。评估McKnight观点（LC域）之间的差异 由定义的结构组织和大多数加入该领域的其他结构组织驱动的功能（LC 域功能无分子结构）可以在我们的评论章节中找到，最近发布了年度 生物化学评论（17）。本应用程序中描述的两个项目代表了我们的工作的扩展 FUS的LC域。我们还对其他环境感兴趣，其中细胞员工交叉互动 为正常生物学或异常生物学。我们发现中间丝蛋白的LC结构域 利用不稳定的交叉相互作用来介导细丝成熟。在组装细丝的背景下，我们 已表明合并的LC结构域代表RNA颗粒的结合位点（12）。我们还推导了 C9ORF72通过RAN翻译产生的毒性GRN和PRN多二肽的发病机理的基础 重复扩展转录本 - 从而解决了神经元如何以最普遍的ALS形式死亡（12,15,16）。 除了继续与Robert Tycko博士和NAI足迹研究继续我们的协作SS-NMR研究。 与Yonghao Yu博士一起探测活细胞中的LC域构象，我们建立了一个协作 与南加州大学拉尔夫·兰根（Ralf Langen）博士的合作伙伴关系使用电子顺磁性 共振（EPR）方法用于探测LC域结合的最早事件的研究。