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

摘要 我之前的Mira时期主要关注微管成核、中心体结构和 噬菌体编码的细胞骨架和“核”。现在我的人机界面已经结束了，我们在蛋白质方面的巨大努力- 米拉的这项提案中包括了动态平衡。在整个过程中，我们的工作试图了解基本的分子- 破坏细胞功能的更大的机制。在可能的情况下，复杂的系统在体外重组并 对原子细节进行了分析，并在细胞水平上探索了其含义。这项研究分为三个部分。 I.出生、生命和破坏：Hsp90/Hsp70驱动的蛋白平衡的机制：维持 细胞蛋白质组是所有生物体最基本的方面之一。分子伴侣有助于 折叠和激活，隔离或回收聚集的蛋白质，参与不可逆转的错误的清除- 折叠蛋白质，并帮助根据细胞需要调节折叠能力。虽然关键人物一直是 尽管已经确定，但大多数这些任务完成的分子机制仍不清楚。我们 重点放在胞质中的Hsp90伴侣蛋白，它促进~10%的蛋白质组的折叠和激活。 热休克蛋白90的“客户”S富含对细胞信号、增殖和生存至关重要的蛋白质 HSP90是治疗多种疾病的有价值的靶点。尽管生物上很重要，但潜在的 客户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 通过向客户提供E3连接来做出决定。通过体外重组，广泛的生化、生物物理- CAL和低温电子显微镜结构分析我们的目标是阐明这些过程的分子机制。 基底体过渡区的结构，断层扫描技术：在未分裂的细胞中，中心粒 成熟到停靠在细胞膜上的基底体，导致形成初级纤毛， 作为几乎所有动物细胞上的感觉器，或移动的纤毛来移动液体。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，只有 对中心粒或基底体结构的了解有限，基底体如何停靠在膜上，传递 连接到轴丝，或提供一个明显的细胞室。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可移动的纤毛阵列的细胞。细胞将处于高压状态- 当然，冷冻和FIB-球磨可以为高分辨率的原位冷冻EM创造薄层。重要的是，关键蛋白质 可以被CRISPR剔除或用Ferri-Tag标记，以便同时进行Like/CryoEM可视化。 噬菌体“核”与宿主免疫逃避：Phi-KZ巨噬菌体揭示的细胞生物学 简直是非同寻常(Collaboration Pogliano，UCSD)，展示了一种似乎是全新的 车厢编队中的概念。一旦感染，这些噬菌体就会形成一个自组装的“核”-- 以动态不稳定的微管蛋白细胞骨架为中心的Tein单层外壳。外壳随着贝壳生长 噬菌体DNA复制，选择性地导入DNA复制和转录机器，但不包括胞浆 蛋白质和绿色荧光蛋白。与加州大学伯克利分校(Bondy-Denomy，UCSF)的合作表明，贝壳对 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 是未知的。重点介绍了壳层组装原理的确定和选择性传输的机理。

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.