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

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 由低复杂度(LC)序列指定。某些LC序列组装成类淀粉聚合物， 导致形成类似液体的液滴，这些液滴随后成熟为水凝胶。虽然在形态上 由LC序列形成的聚合物与致病的普里恩样淀粉样蛋白难以区分，在 稀释。FUS聚合物结构的原子分辨最近揭示了LC结构域的基础 聚合物不稳定性(11)。自我们连续2012年的论文发表在《细胞》杂志(9，10)以来的六年里，研究 RNA颗粒、无膜细胞器和LC结构域发生爆炸。涉及的LC域 没有包被膜包围的中尺度斑点的形成已经被牢固地建立起来。我们的 支持不稳定的跨相互作用参与这些结构的形成的观察结果， 然而，这与该领域的主流观点形成了鲜明对比。许多团体争辩说 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，不稳定的跨相互作用的形成是阶段的核心 分离成液滴，形成水凝胶，并在活细胞中发挥LC结构域的功能。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)，以及最近的固体核磁共振光谱学(11)。叙事学 避免了这一争议，而是将重点放在了对 我们研究的进展。评估McKnight观点(LC领域)之间的差异 由明确的结构组织驱动的职能)和大多数已加入该领域的其他人的职能(LC 结构域功能(无分子结构)可以在我们最近发表的年度 生物化学综述(17)本申请中描述的两个项目代表了我们在 FUS的LC结构域。我们还对细胞使用不稳定的跨交互作用的其他设置感兴趣 煽动，煽动煽动正常或不正常的生物我们发现中间丝蛋白的LC结构域 利用不稳定的交叉相互作用来调节细丝成熟。在组装细丝的背景下，我们 已经表明，结合的LC结构域代表RNA颗粒的结合位置(12)。我们还推导出了 C9orf72通过RAN翻译产生的毒性GRN和PRN多肽致病的基础 重复扩展转录--从而解决最常见的肌萎缩侧索硬化症(12，15，16)形式的神经元如何死亡。 除了继续我们与Robert Tycko博士合作的SS-NMR研究，以及NAI足迹研究 与余永好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的 与南加州大学的拉尔夫·兰根博士合作使用电子顺磁 共振(EPR)方法，用于研究LC结构域合并的最早事件。

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.