Structure and Mechanism: Hsp90 proteostasis, cilia biogenesis and the jumbo phage “nucleus”

结构和机制：Hsp90 蛋白质稳态、纤毛生物发生和巨型噬菌体 – 细胞核 –

• 批准号: 10407008
• 负责人: DAVID A. AGARD
• 金额: $ 84.51万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10407008
• 关键词: Animals; Bacteriophages; Biochemical; Biochemistry; Biological; Biophysics; Birth; Cell Nucleus; Cell physiology; Cells; Cellular biology; Centrioles; Centrosome; Cilia; Client; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Cryoelectron Microscopy; Cytoskeleton; DNA; DNA biosynthesis; Disease; Docking; Epithelial Cells; Excision; Freezing; Genetic Transcription; Goals; Immunity; In Situ; In Vitro; Infection; Interphase Cell; Knock-out; Life; Liquid substance; Maintenance; Malignant Neoplasms; Mature Centriole; Membrane; Microtubules; Molecular; Molecular Chaperones; Mus; Organelles; Organism; Process; Proteins; Proteome; Research; Resistance; Resolution; Sensory; Signal Transduction; Structure; System; Technology; Thinness; Tracheal Epithelium; Triage; Tubulin; Visualization; Work; ciliopathy; cilium biogenesis; cilium motility; human disease; kinetosome; misfolded protein; monolayer; pressure; protein aggregation; proteostasis; reconstitution; therapeutic target; tomography; ubiquitin-protein ligase; virtual

ABSTRACT My previous MIRA period focused on mechanisms of microtubule nucleation, centrosome structure and the phage-encoded cytoskeleton and “nucleus”. Now that my HHMI has ended, our strong efforts on protein ho- meostasis are included in this MIRA proposal. Throughout, our work seeks to understand fundamental molecu- lar mechanisms that underly cellular function. Where possible, complex systems are reconstituted in vitro and analyzed in atomic detail with the implications explored at a cellular level. The research has three parts. I. Birth, life and destruction: mechanisms of Hsp90/Hsp70-driven proteostasis: Maintenance of the cellular proteome is one of the most fundamental aspects of all organisms. Molecular chaperones facilitate folding and activation, sequester or recover aggregated proteins, participate in the removal of irreversibly mis- folded proteins, and help regulate folding capacity according to cellular need. While critical players have been identified, the molecular mechanisms by which most of these tasks are accomplished remain unknown. We focus on the cytosolic Hsp90 chaperones that facilitate the folding and activation of ~10% of the proteome. Hsp90's “clients” are enriched in proteins important for cellular signaling, proliferation, and survival making Hsp90 a valuable therapeutic target for multiple diseases. Despite the biological importance, the underlying mechanism of client remodeling is unknown, as is how the chaperones facilitate folding vs degradation triage decisions by presenting clients to E3 ligases. Through in vitro reconstitution, extensive biochemical, biophysi- cal and cryoEM structural analyses our goal is to elucidate the molecular mechanisms of these processes. II. Structure of the basal body transition zone, tomography technology: In non-dividing cells, centrioles mature into basal bodies that dock at the membrane leading to the formation of a primary cilium which serves as a sensory organelle on virtually all animal cells, or motile cilia to move fluid. These structures are important in numerous human diseases, including cancer and a broad array of ciliopathies. Unfortunately, there is only limited understanding of centriole or basal body structure, how the basal body docks at the membrane, transi- tions to an axoneme, or provides a distinct cellular compartment. We will use cultured mouse tracheal epithelial cells which can be grown and differentiated on grids to produce arrays of motile cilia. Cells will be high pres- sure frozen and FIB-milled to create thin lamella for high-resolution in situ cryoEM. Importantly, key proteins can be knocked out by CRISPR or tagged with Ferri-tag for simultaneous like/cryoEM visualization. Phage “nucleus” and host immunity evasion: The cell biology being revealed by Phi-KZ jumbo phages is simply extraordinary (collaboration Pogliano, UCSD), demonstrating what appears to be an entirely new concept in compartment formation. Upon infection, these phage form a “nucleus” from a self-assembling pro- tein monolayer shell that is centered by a dynamically unstable tubulin cytoskeleton. The shell grows as the phage DNA replicates, selectively imports DNA replication and transcription machinery, yet excludes cytosolic proteins and GFP. Collaborating with (Bondy-Denomy, UCSF) has shown that the shell confers resistance to all known host immunity factors (CRISPRs, restriction endoncleases). The molecular basis for these processes is unknown. We focus on the determining shell assembly principles and the mechanism of selective transport.

抽象的 我之前的 MIRA 时期重点关注微管成核机制、中心体结构和 噬菌体编码的细胞骨架和“细胞核”现在我的 HHMI 已经结束了，我们对蛋白质的大力努力将继续下去。 自始至终，我们的工作都旨在了解基本分子。 在可能的情况下，在体外和体外重建复杂的系统。 该研究分为三个部分，以原子细节进行分析，并在细胞水平上探讨其影响。 I. 诞生、生命和毁灭：Hsp90/Hsp70 驱动的蛋白质稳态机制：维持 细胞蛋白质组是所有生物体最基本的方面之一。 折叠和激活，隔离或恢复聚集的蛋白质，参与不可逆错误的去除 折叠蛋白质，并根据细胞需要帮助调节折叠能力，而关键参与者一直是。 尽管已经确定，但完成大多数这些任务的分子机制仍然未知。 重点关注促进约 10% 蛋白质组折叠和激活的胞质 Hsp90 分子伴侣。 Hsp90 的“客户”富含细胞信号传导、增殖和生存的重要蛋白质 Hsp90 是多种疾病的有价值的治疗靶点，尽管其具有重要的生物学意义，但其根本原因是。 客户端重塑的机制尚不清楚，伴侣如何促进折叠与降解分类也是未知的 通过体外重组、广泛的生物化学、生物物理学向客户展示E3连接酶来做出决定。 cal 和cryoEM 结构分析我们的目标是阐明这些过程的分子机制。 II. 基底体过渡区的结构，断层扫描技术：在非分裂细胞中，中心粒 成熟为基体，基体停靠在膜上，导致初级纤毛的形成，初级纤毛的作用 作为几乎所有动物细胞上的感觉细胞器，或移动液体的活动纤毛，这些结构都很重要。 不幸的是，在许多人类疾病中，包括癌症和多种纤毛病，都存在这种现象。 对中心粒或基底体结构、基底体如何与膜对接、转运的了解有限 我们将使用培养的小鼠气管上皮。 可以在网格上生长和分化以产生活动纤毛细胞阵列的细胞将是高浓度的。 确保冷冻和 FIB 研磨以形成用于高分辨率原位冷冻电镜的薄层，重要的是，关键蛋白质。 可以通过 CRISPR 敲除或用 Ferri 标签标记，以同时进行样/冷冻电镜可视化。 噬菌体“细胞核”和宿主免疫逃避：Phi-KZ 巨型噬菌体揭示的细胞生物学 简直是非凡的（与加州大学圣地亚哥分校 Pogliano 合作），展示了一个全新的东西 感染后，这些噬菌体从自组装亲形成“细胞核”。 蛋白质单层外壳，以动态不稳定的微管蛋白细胞骨架为中心，外壳随着蛋白质的生长而生长。 噬菌体 DNA 复制，选择性导入 DNA 复制和转录机制，但排除胞质 与（Bondy-Denomy，UCSF）合作表明，外壳具有抗性。 所有已知的宿主免疫因子（CRISPR、限制性内切酶）这些过程的分子基础。 我们的重点是确定壳组装原理和选择性运输机制。

项目成果

Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

HP1α 的液滴形成表明异染色质中相分离的作用。

• 发表时间: 2017
• 影响因子: 64.8
• 作者: Larson, Adam G;Elnatan, Daniel;Keenen, Madeline M;Trnka, Michael J;Johnston, Jonathan B;Burlingame, Alma L;Agard, David A;Redding, Sy;Narlikar, Geeta J
• 通讯作者: Narlikar, Geeta J

Competing protein-protein interactions regulate binding of Hsp27 to its client protein tau.

竞争性蛋白质-蛋白质相互作用调节 Hsp27 与其客户蛋白 tau 的结合。

• 发表时间: 2018
• 影响因子: 16.6
• 作者: Freilich, Rebecca;Betegon, Miguel;Tse, Eric;Mok, Sue;Julien, Olivier;Agard, David A;Southworth, Daniel R;Takeuchi, Koh;Gestwicki, Jason E
• 通讯作者: Gestwicki, Jason E

Electron cryo-tomography provides insight into procentriole architecture and assembly mechanism.

电子冷冻断层扫描提供了对原中心粒结构和组装机制的深入了解。

• 发表时间: 2019
• 影响因子: 7.7
• 作者: Li, Sam;Fernandez, Jose;Marshall, Wallace F;Agard, David A
• 通讯作者: Agard, David A

Mind the gap: Micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae.

注意间隙：微膨胀节大大减少了 FIB 铣削冷冻片层的弯曲。

• 发表时间: 2019
• 影响因子: 3
• 作者: Wolff, Georg;Limpens, Ronald W A L;Zheng, Shawn;Snijder, Eric J;Agard, David A;Koster, Abraham J;Bárcena, Montserrat
• 通讯作者: Bárcena, Montserrat

Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping.

用于定量巨噬细胞-细菌相互作用图谱的下一代相互作用蛋白质组学。

• 发表时间: 2023-02-23
• 作者: Fossati, Andrea;Mozumdar, Deepto;Kokontis, Claire;Mèndez;Nieweglowska, Eliza;Pelin, Adrian;Li, Yuping;Guo, Baron;Krogan, Nevan J;Agard, David A;Bondy;Swaney, Danielle L
• 通讯作者: Swaney, Danielle L