Structure-Function of Nucleo-Cytoplasmic Communication

核-细胞质通讯的结构-功能

• 批准号: 10475615
• 负责人: Thomas Schwartz
• 金额: $ 37.99万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10475615
• 关键词: Address; Cardiac; Cell Nucleus; Cell physiology; Cells; Communication; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Cytoskeleton; Drug Design; Elements; Emery-Dreifuss Muscular Dystrophy; Eukaryotic Cell; Functional disorder; Genetic Materials; Genetic Transcription; Goals; Human; Individual; Malignant Neoplasms; Maps; Membrane; Methods; Molecular; Muscular dystrophy cardiomyopathy; Myopathy; Nuclear Envelope; Nuclear Pore Complex; Organelles; Positioning Attribute; Premature aging syndrome; Primary Dystonias; Process; Proteins; Resolution; SWP29; Structure; Technology; Translations; X-Ray Crystallography; grasp; human disease; innovation; mechanotransduction; protein complex; skeletal; success

PROJECT SUMMARY / ABSTRACT Eukaryotic cells are defined by their organelles, membrane-enclosed compartments in which specific cellular processes are carried out. The nucleus is the largest organelle, contains all genetic material, and enables separation of gene transcription from protein translation. As the nuclear envelope (NE) serves as a tight barrier enclosing the nucleus, the cell requires machinery to establish and control nucleo-cytoplasmic communication. There are two principally different components to this machinery. On one hand, nuclear pore complexes (NPCs) serve as the main conduit for molecular exchange across the NE. On the other hand, universally conserved linker of nucleo- and cytoskeleton (LINC) complexes serve as physical tethers across the NE, which are necessary for positioning the nucleus and for mechano-sensing in a diverse set of circumstances. Dysfunction of the machinery is at the core of important human diseases, including skeletal and cardiac myopathies, premature aging, and cancer. Our goal is to understand the structure of the protein complexes involved in nucleo-cytoplasmic communication at high (atomic) resolution. Such information helps to identify and separate the myriad functions this machinery carries out and that we are still only beginning to fully grasp. High resolution information further provides the basis for structure-guided drug design to interfere with the salient human diseases, such as Emery-Dreifuss Muscular Dystrophy (EDMD) and Primary Dystonia, which are still not cured. The structural characterization of the NPC and the LINC complex are challenging, because of the size and complexity of these multi-MDa assemblies. Over the past 15 years, we have made significant advances on both problems. For the NPC, we have chosen a highly productive bottom-up approach, in which we characterized multi-subunit complexes predominantly by X-ray crystallography, the building blocks of the massive, 40-100 MDa NPC. Those structures have now been used in combination with cryo-electron tomographic (cryo-ET) maps of assembled NPCs to generate composite structures that attempt to position the roughly 500 individual proteins within one NPC. For the LINC complex, we solved the universally conserved core component and have started to untangle the diverse network of its components, the Sad1/UNC-84 (SUN) and Klarsicht/ANC1/Syne-Homology (KASH) proteins. Going forward, the challenge is the structural characterization of large and dynamic assemblies, which is true for both, the NPC and the LINC complex, for the latter particularly when including the connection to the nucleo- and cytoskeletal components. The dramatic advances in cryo-electron microscopy (cryo-EM) over the recent past make this technology particularly important for our studies. We anticipate combining X-ray crystallography and cryo-EM for studying the most relevant structures going forward. The success of this will depend upon innovative, tailored methods to address the particular challenges that come with each project. We have repeatedly shown over the past decade how to successfully approach such challenges and have devised methods to meet them.

项目摘要/摘要 真核细胞由它们的细胞器定义，细胞器是膜封闭的隔室，其中特定的细胞 过程进行。细胞核是最大的细胞器，包含所有遗传物质， 基因转录与蛋白质翻译的分离。由于核被膜（NE）是一个紧密的屏障， 包围细胞核的细胞需要机制来建立和控制核质通讯。 这一机制有两个主要不同的组成部分。一方面，核孔复合体 细胞间质（NPC）作为分子交换穿过NE的主要管道。另一方面，普遍 核骨架和细胞骨架的保守连接体（LINC）复合物充当跨NE的物理系链， 对于在不同的环境中定位细胞核和机械感测是必要的。 该机制的功能障碍是重要的人类疾病的核心，包括骨骼和心脏疾病。 肌病、早衰和癌症。我们的目标是了解蛋白质复合物的结构 在高（原子）分辨率下参与核质通讯。这些信息有助于识别 并将这个机器执行的无数功能分开，我们仍然只是开始完全掌握。 高分辨率信息进一步为结构指导的药物设计提供了基础，以干扰药物的作用。 突出的人类疾病，如Emery-Dreifuss肌营养不良症（EDMD）和原发性肌张力障碍， 仍然没有治愈。NPC和LINC复合物的结构表征具有挑战性，因为 这些多MDa组件的大小和复杂性。在过去的15年里，我们取得了重大进展。 这两个问题的进展。对于全国人民代表大会，我们选择了一种富有成效的自下而上的方法， 我们主要通过X射线晶体学来表征多亚基复合物， 40-100 MDa的大型NPC。这些结构现在已经与低温电子结合使用， 组装NPC的断层扫描（cryo-ET）图，以生成复合结构， 一个NPC中大约有500个蛋白质对于LINC复合体，我们解决了普遍保守的 核心部件，并已开始解开其组成部分，萨德1/萨德-84（SUN）的多样化网络 和Klarsicht/ANC 1/Syne-Homology（KASH）蛋白。展望未来，挑战是结构性的 大型和动态组件的表征，这对于NPC和LINC复合体都是正确的， 后者特别是当包括与核骨架和细胞骨架组分的连接时。的戏剧性 近年来低温电子显微镜（cryo-EM）的发展使这项技术特别 对我们的学习很重要。我们预计结合X射线晶体学和冷冻电镜研究最 相关结构向前推进。这方面的成功将取决于创新的、量身定制的方法， 解决每个项目带来的特殊挑战。我们过去一再表明， 十年来，我们一直在研究如何成功地应对这些挑战，并制定了应对这些挑战的方法。