DNA based platform for visualization of scarce protein assemblies with electron cryo-microscopy

基于 DNA 的平台，用于通过电子冷冻显微镜可视化稀有蛋白质组装体

• 批准号: RGPIN-2018-06070
• 负责人: MazhabJafari, MohammadT
• 金额: $ 2.7万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=686915
• 关键词: DNA; based; platform; visualization; scarce

Background:***Many clinically relevant protein complexes form transiently in cells at low abundance in response to external stimuli. Additionally, growth signaling proteins undergo heterogeneous phosphorylation regulated via upstream signaling that strictly regulate their conformation, subunit composition, and intra-cellular localization within cells. Many of these regulatory class' of proteins are not amenable to over-expression that is necessary to achieve milligram protein quantities for traditional structure determination techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Therefore, it is critical to target native complexes expressed at endogenous levels. However, another level of complication arises due to chemical and conformational heterogeneity of endogenous proteins. To overcome this challenge, we sought to take advantage of post-translationally modified states and scaffolding subunits that define function, subunit composition and intra-cellular localization. Mammalian target of rapamycin (mTOR) protein is found in association with regulatory scaffold proteins RPTOR or RICTOR that target the kinase to mTORC1 or mTORC2, respectively, with define chemical composition. Indeed, these scaffolding proteins have been used to selectively pull-down endogenous mTOR complexes. On the other hand, tuberous sclerosis complex (TSC) function, complex formation, and sub-cellular localization is strictly controlled via phosphorylation by upstream kinases. In starving cells, TSC is found on the lysosomal surface where it down-regulates mTORC1 activity. Upon insulin stimulation, TSC is rapidly phosphorylated by Akt and translocates from lysosomes to the cytoplasm where it remains in a catalytically active state for up to 1h post insulin stimulation. ***Objectives:***We proposed a research program for strtuctural investigation of protein components of TOR pathway, with emphasis on TORC2 from yeast and human TSC.***1) Development and optimization of polymer-coated holey EM grids. ***2) cryoEM of genetically-tagged TORC2 from yeast Saccharomyces cerevisiae. ***3) Signal-integrated affinity cryoEM of TSC. ***Significance:***Atomic-resolution structural knowledge is acutely lacking for mTORCs and TSC holo-complexes and is instrumental in design of selective agonist and antagonist. A detailed structural knowledge of TORC2 is necessary for generation of selective inhibitors for basic biological research into mTORC2 dependent signaling pathways. Similarly TSC structure, will reveal the mechanistic basis of disease associated mutations and identify interfaces between different TSC subunits and their interaction with upstream kinases. Our cryoEM sample preparation scheme will be adoptable to other scarce complexes.

背景：* 许多临床相关的蛋白质复合物在细胞中以低丰度短暂形成，以响应外部刺激。此外，生长信号传导蛋白经历通过上游信号传导调节的异源磷酸化，所述上游信号传导严格调节它们的构象、亚基组成和细胞内定位。这些调节类蛋白质中的许多不适于过表达，而过表达对于传统的结构测定技术如X射线晶体学和核磁共振（NMR）光谱学来说是实现毫克蛋白质量所必需的。因此，靶向内源性水平表达的天然复合物至关重要。然而，由于内源性蛋白质的化学和构象异质性，出现了另一个水平的复杂性。为了克服这一挑战，我们试图利用后修饰状态和支架亚基，定义功能，亚基组成和细胞内定位。哺乳动物雷帕霉素靶蛋白（mTOR）与调节支架蛋白RPTOR或RICTOR相关，RPTOR或RICTOR分别将激酶靶向mTORC 1或mTORC 2，具有确定的化学组成。事实上，这些支架蛋白已被用于选择性地下拉内源性mTOR复合物。另一方面，结节性硬化症复合体（TSC）的功能、复合体形成和亚细胞定位通过上游激酶的磷酸化严格控制。在饥饿细胞中，TSC存在于溶酶体表面，在那里它下调mTORC 1活性。在胰岛素刺激后，TSC被Akt迅速磷酸化，并从溶酶体易位到细胞质，在胰岛素刺激后1小时内保持催化活性状态。* 目的：* 我们提出了一个研究计划，对TOR通路的蛋白质组分进行结构研究，重点是酵母和人TSC的TORC 2。* 1)聚合物涂层多孔EM网格的开发和优化。*2）来自酵母酿酒酵母的遗传标记的T0RC 2的cryoEM。 *3）TSC的信号整合亲和cryoEM。* 重要性：* 对于mTORC和TSC全复合物，原子分辨率结构知识严重缺乏，并且有助于设计选择性激动剂和拮抗剂。 TORC2的详细结构知识对于产生用于mTORC2依赖性信号通路的基础生物学研究的选择性抑制剂是必要的。类似地，TSC结构将揭示疾病相关突变的机制基础，并鉴定不同TSC亚基之间的界面及其与上游激酶的相互作用。我们的cryoEM样品制备方案将适用于其他稀缺的复合物。