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

摘要 我以前的MIRA期间集中在微管成核机制，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，我们对蛋白质的强烈努力- 在MIRA提案中包含了meostasis。在整个过程中，我们的工作旨在了解基本的分子- 更大的细胞功能机制。在可能的情况下，在体外重建复杂的系统， 在细胞水平探索的影响，在原子细节分析。研究分为三个部分。 I.出生、生命和毁灭：Hsp 90/Hsp 70驱动的蛋白稳态机制： 细胞蛋白质组是所有生物体最基本的方面之一。分子伴侣促进 折叠和激活，螯合或恢复聚集的蛋白质，参与不可逆的错误- 折叠蛋白质，并帮助调节折叠能力，根据细胞的需要。虽然关键球员一直在 虽然已经确定，但大多数这些任务完成的分子机制仍然未知。我们 集中在胞质Hsp 90分子伴侣，促进折叠和激活约10%的蛋白质组。 Hsp 90的“客户”富含对细胞信号传导、增殖和生存至关重要的蛋白质 热休克蛋白90是多种疾病治疗的重要靶点。尽管在生物学上很重要， 客户端重塑的机制尚不清楚，伴侣蛋白如何促进折叠与降解分流 通过将客户端呈现给E3连接酶来做出决定。通过体外重组，广泛的生化，生物物理- 我们的目标是阐明这些过程的分子机制。 二.基体过渡区的结构，断层摄影技术：在非分裂细胞中，中心粒 成熟为基底体，停靠在膜上，导致形成初级纤毛， 作为几乎所有动物细胞上的感觉器，或移动流体的运动纤毛。这些结构很重要 在许多人类疾病中，包括癌症和广泛的纤毛病。不幸的是， 对中心粒或基体结构的了解有限，基体如何停靠在膜上，transi- 连接到轴丝，或提供不同的细胞隔室。我们将使用培养的小鼠气管上皮细胞 这些细胞可以在网格上生长和分化以产生运动纤毛阵列。细胞将处于高压状态- 确保冷冻和FIB研磨以产生用于高分辨率原位cryoEM薄层。重要的是， 可以通过CRISPR敲除或用Ferri标签标记，用于同时进行类似/cryoEM可视化。 噬菌体“核”和宿主免疫逃避：Phi-KZ巨型细胞揭示的细胞生物学 简直是非凡的（合作Pogliano，UCSD），展示了一个全新的 概念形成。感染后，这些噬菌体从一个自组装的前体形成一个“核”， 以动态不稳定的微管蛋白细胞骨架为中心的单层壳。贝壳随着 噬菌体DNA复制，选择性地输入DNA复制和转录机制，但排除胞质 蛋白质和GFP。与（Bondy-Denomy，UCSF）的合作表明，该壳赋予对 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 不明我们专注于决定壳组装原则和选择性运输的机制。

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.