Biophysical, Structural, and Cellular Dissection of COPI-Dependent Retrograde Trafficking Using a Coronavirus Toolkit

项目摘要 分泌途径负责参与细胞内可溶性和膜蛋白的生物合成， 通讯、能量转换、营养吸收和防御。这些蛋白质是在 内质网（ER），然后运输到高尔基体和其他细胞器，如质膜。 这种运输通过ER驻留蛋白如UDP-葡萄糖醛酸酯的意外流出而引起ER应激 转移酶（UGT）。这些是I型膜蛋白（T1 MP），负责脂质的修饰 激素和止痛药醋氨酚和吗啡。这些ER驻留T1 MP显示出二元的 在它们的胞质尾部中的序列（Lys-x-Lys-x-x或Lys-Lys-x-x; x=任何氨基酸），用于通过外被体进行ER-回收 蛋白I复合物（COPI）。这种异源七聚体复合物的α和β'亚基结合这种二元序列 以启动T1 MP包装到源自ER后区室如顺式高尔基体的囊泡中。这些COPI 包被的囊泡运输并将T1 MP蛋白递送回ER以恢复分泌平衡。但 T1 MP与α和β 'COPI亚基结合、释放和选择性相互作用的原子原理是 没有被很好地理解。这是一个关键的知识差距，因为T1 MP释放和COPI逃逸调节T1 MP 运输、翻译后修饰和T1 MP功能。COPI功能障碍与 与发育、自身免疫和癌症相关的各种疾病。我们的长期目标是 对T1 MP的COPI依赖的逆行运输和潜在的原子水平的基本见解 导致疾病中COPI功能障碍的因素。在这份补助金中，我们将阐明机械的见解， 利用冠状病毒（CoV）加标的COPI募集、释放和T1 MP翻译后修饰 蛋白，T1 MP与二元Lys-x-His-x-x序列，作为一个新的模型系统。这种二元序列确保了 COPI依赖的刺突从高尔基体到ER-高尔基体中病毒子代组装位点的逆行递送 中间室（ERGIC）。在目标1中，我们将阐明构象调制的原子细节 COPI-spike相互作用的影响。在目标2中，我们将确定管理COPI发布的原则， 随后的尖峰的翻译后修饰。在目标3中，我们将阐明COPI的原子基础 刺突蛋白的亚基选择性。这些研究将扩展穗突变体工具包， 修改的COPI相互作用，正如我们小组最近发表的那样。我们将在X射线中整合结构方法 晶体学，NMR和Rosetta建模与生物物理工具和细胞测定分泌贩运， 获得前所未有的深入了解精细调制和构象调控的COPI穗相互作用。的 作为T1 MP模型系统的刺突蛋白的创新使用将产生对基本分泌的新见解， 贩卖人口这些数据将同时为靶向治疗药物的开发开辟道路， COPI选择性疾病和更深入地了解CoV组装和CoV疫苗的加工。

PROJECT SUMMARY The secretory pathway is responsible for the biogenesis of soluble and membrane proteins involved in communication, energy transduction, nutrient uptake, and defense. These proteins are synthesized in the endoplasmic reticulum (ER) and then trafficked to Golgi and other organelles such as the plasma membrane. This trafficking causes ER stress by accidental exodus of ER-resident proteins such as UDP-glucuronyl transferases (UGT’s). These are type I membrane proteins (T1MP’s) responsible for modifications of lipid hormones and of analgesics acetaminophen and morphine. These ER-resident T1MP’s display a dibasic sequence (Lys-x-Lys-x-x or Lys-Lys-x-x; x=any amino acid) in their cytosolic tail for ER-retrieval by the coatomer protein I complex (COPI). The α and β’ subunits of this hetero-heptameric complex bind this dibasic sequence to initiate T1MP packaging into vesicles originating from post-ER compartments such as cis-Golgi. These COPI coated vesicles traffic and deliver the T1MP proteins back to ER to restore secretory balance. However, the atomic principles underlying T1MP binding, release, and selective interactions with α and β’COPI subunits are not well understood. This is a critical knowledge-gap as T1MP release and escape from COPI modulate T1MP trafficking, post-translational modifications, and T1MP functions. COPI dysfunction has been implicated in a variety of disorders related to development, auto-immunity, and cancers. Our long-term objective is to gain fundamental insights into COPI-dependent retrograde trafficking of T1MP’s and the underlying atomic-level factors responsible for COPI dysfunction in diseases. In this grant, we will elucidate mechanistic insights into COPI recruitment, release, and T1MP post-translational modifications utilizing the coronavirus (CoV) spike protein, a T1MP with a dibasic Lys-x-His-x-x sequence, as a new model system. This dibasic sequence ensures COPI-dependent retrograde delivery of the spike from Golgi to the viral progeny assembly site in ER-Golgi intermediate compartment (ERGIC). In Aim 1, we will elucidate the atomic details of conformational modulation of COPI-spike interactions. In Aim 2, we will determine the principles that govern release from COPI and subsequent post-translational modifications of the spike. In Aim 3, we will elucidate the atomic basis of COPI subunit selectivity for the spike protein. These investigations will expand on a toolkit of spike mutants with modified COPI interactions, as recently published by our group. We will integrate structural approaches in X-ray crystallography, NMR, and Rosetta modeling with biophysical tools and cellular assays of secretory trafficking to gain unprecedented insights into fine modulation and conformational regulation of COPI-spike interactions. The innovative use of the spike protein as a T1MP model system will yield novel insights into fundamental secretory trafficking. These data will simultaneously opening avenues for the development of targeted therapeutics for COPI-selective disorders and for a deeper understanding of CoV assembly and processing of CoV vaccines.