A humidity-controlled system for reprodicable vitrification of macromolecules

用于大分子可重复玻璃化的湿度控制系统

• 批准号: 10581231
• 负责人: Gino Cingolani
• 金额: $ 10.29万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10581231
• 关键词: Bacteria; Bacteriophages; Biological; Biological Process; Biology; Biotechnology; Cell Nucleus; Cells; Complex; Cryoelectron Microscopy; DNA; Development; Eukaryotic Cell; Funding Mechanisms; Genome; Gram-Negative Bacteria; Guanosine Triphosphate Phosphohydrolases; Herpesviridae; Human; Humidity; Immune system; Importins; Injections; Laboratories; Literature; Mediating; Medicine; Membrane; Membrane Proteins; Molecular; Molecular Machines; Nuclear Import; Nuclear Pore Complex; Organelles; Principal Investigator; Process; Protein Biochemistry; Publications; Regulation; Research; Science; Signal Transduction; Structure; System; Viral; Viral Genome; Virulence; Virus; Work; X-Ray Crystallography; cell envelope; genetic information; interest; macromolecule; nanomachine; novel therapeutic intervention; nucleocytoplasmic transport; pressure; programs; receptor; structural biology

Project Summary Nearly 120 years since the discovery of the first virus, our understanding of how viruses deliver genomes into cells overcoming the complexity of biological membranes remains limited. While a vast scientific literature exists on viral surface proteins and their interaction with host receptors, and the immune system, little emphasis has been devoted to studying the delivery of entire viral genomes into cells. For instance, how do bacteriophages eject DNA through the cell envelope of Gram-negative bacteria? Or, in humans, how do herpesviruses deliver ~200 kb genome through the Nuclear Pore Complex (NPC) into the cell nucleus? For a quarter of a century, first as a trainee (1995-2003), and since 2004 as a principal investigator, I have investigated the mechanisms of nucleocytoplasmic transport and viral genome packaging. My work has resulted in close to 85 publications that contributed to elucidating the atomic structure and regulation of crucial factors implicated in nuclear import, and viral genome packaging. In this R35, I propose to combine the study of these two seemingly distinct biological processes by focusing on the mechanisms of viral genome delivery into living cells. Specifically, I will ask two biological questions that seek to compare and contrast how simple bacterial viruses (or bacteriophages) eject their DNA into bacteria with how Herpesviruses deliver their complex genomes into the nucleus of eukaryotic cells. The first question explores how bacteriophages eject ~45 kb genomes through the cell envelope of gram-negative bacteria. Long-thought to be a simple pressure-driven injection, this process uses a virus-encoded nanomachine, which we have begun to study in my laboratory. The second question explores how Herpesviruses deliver their large genome through the Nuclear Pore Complex (NPC) of human cells into the cell nucleus. This is a signal- and energy-mediated process that uses host importins and the GTPase Ran, exploiting the cellular transport machinery to promote entry of an exogenous genome into the nucleus. Overall, understanding how viruses transfer genetic information through biological membranes into cells and organelles is vital for deciphering the molecular mechanisms of virulence as well as the development of novel therapeutic approaches. The common denominator of this R35 lies in our interest in the structure and transport mechanisms of biological macromolecules. Our research approach marries established sciences like protein biochemistry and X-ray crystallography with the power of cryo-electron microscopy (cryo-EM) to visualize biological macromolecules in near-native conditions. We believe that this R35 MIRA funding mechanism will fuel the creative and diligent pursuit of answers to the questions we pose, permitting our research program to achieve significant advancements in structural biology.

项目概要 自发现第一种病毒以来近 120 年，我们对病毒如何将基因组传递到体内的了解 细胞克服生物膜复杂性的能力仍然有限。虽然有大量的科学文献 存在于病毒表面蛋白及其与宿主受体和免疫系统的相互作用，很少 重点致力于研究将整个病毒基因组传递到细胞中。例如，如何 噬菌体通过革兰氏阴性菌的细胞膜喷射DNA？或者说，对于人类来说，如何 疱疹病毒通过核孔复合体 (NPC) 将约 200 kb 的基因组传递到细胞核中？ 四分之一个世纪以来，首先作为实习生（1995-2003），自 2004 年作为首席研究员以来，我 研究了核细胞质运输和病毒基因组包装的机制。我的工作有 发表了近 85 篇出版物，有助于阐明关键的原子结构和调控 与核输入和病毒基因组包装有关的因素。在这个R35中，我建议结合研究 通过关注病毒基因组传递机制来研究这两个看似不同的生物过程 进入活细胞。具体来说，我会问两个生物学问题，旨在比较和对比如何简单 细菌病毒（或噬菌体）将其 DNA 喷射到细菌中，疱疹病毒如何传递其 DNA 复杂的基因组进入真核细胞的细胞核。第一个问题探讨噬菌体如何 通过革兰氏阴性细菌的细胞膜喷射约 45 kb 的基因组。一直以为很简单 压力驱动注射，这个过程使用病毒编码的纳米机器，我们已经开始研究它 我的实验室。第二个问题探讨疱疹病毒如何通过 核孔复合体（NPC）进入人体细胞的细胞核。这是一种信号和能量介导的 使用宿主导入蛋白和 GTPase Ran 的过程，利用细胞运输机制来促进 外源基因组进入细胞核。总的来说，了解病毒如何转移遗传 通过生物膜进入细胞和细胞器的信息对于破译分子至关重要 毒力机制以及新治疗方法的开发。常见的 这个 R35 的分母在于我们对生物的结构和运输机制的兴趣 大分子。我们的研究方法与蛋白质生物化学和 X 射线等成熟科学相结合 借助冷冻电子显微镜 (cryo-EM) 的晶体学技术，可以可视化生物大分子 接近原生条件。我们相信，R35 MIRA 资助机制将激发创意和勤奋的人们 追求我们提出的问题的答案，使我们的研究计划取得重大成果 结构生物学的进展。