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

使用冠状病毒工具包对 COPI 依赖性逆行贩运进行生物物理、结构和细胞解剖

基本信息

批准号：
10646999
负责人：
Syed Saif Hasan
金额：
$ 41.33万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-30 至 2028-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10646999
关键词：
2019-nCoV Accidents Acetaminophen Affect Amino Acids Analgesics Applications Grants Autoimmunity Back Binding Binding Proteins Binding Sites Biochemical Biogenesis Biological Biological Models Biophysics C-terminal COVID-19 COVID-19 pandemic Cell membrane Cell physiology Cellular Assay Chimera organism Coated vesicle Coatomer Protein Communication Complex Coronavirus Coronavirus spike protein Data Destinations Development Devices Disease Disease Outbreaks Dissection Drug Metabolic Detoxication Electrostatics Endoplasmic Reticulum Ensure Environment Epidemic Equilibrium Functional disorder Future Glucuronosyltransferase Goals Golgi Apparatus Grant Health Heart Homeostasis Hormones Human Immune system Immunologics Interferometry Investigation Knowledge Lipids Malignant Neoplasms Mass Spectrum Analysis Membrane Proteins Middle East Respiratory Syndrome Coronavirus Modeling Modification Molecular Conformation Morphine Mutation Names Nutrient Organelles Pathway interactions Polysaccharides Post-Translational Protein Processing Process Protein Biosynthesis Proteins Publications Publishing Reagent Regulation Retrieval Role SARS-CoV-2 spike protein Site Stress Tail Testing Therapeutic Therapeutic Intervention Vesicle Viral Viral Proteins X-Ray Crystallography Xenobiotics biophysical tools coronavirus vaccine developmental disease endoplasmic reticulum stress experimental study gene product improved in silico innovation insight mimicry mutant novel novel coronavirus posters protein function protein transport receptor recruit secretory protein targeted treatment trafficking uptake

项目摘要

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.

项目概要分泌途径负责可溶性蛋白和膜蛋白的生物发生这些蛋白质是在细胞中合成的。内质网（ER），然后转运到高尔基体和其他细胞器，例如质膜。这种贩运通过内质网驻留蛋白（例如 UDP-葡萄糖醛酸基）的意外外流而导致内质网应激转移酶 (UGT) 这些是负责脂质修饰的 I 型膜蛋白 (T1MP)。激素以及止痛药对乙酰氨基酚和吗啡的这些 ER 驻留 T1MP 显示出二元性。其胞浆尾部的序列（Lys-x-Lys-x-x 或 Lys-Lys-x-x；x=任何氨基酸），用于通过涂层异构体进行 ER 修复该异源七聚体复合物的 α 和 β’ 亚基与该二碱基序列结合。启动 T1MP 包装到源自 ER 后区室（例如 cis-Golgi）的囊泡中。包被的囊泡运输并将 T1MP 蛋白输送回 ER 以恢复分泌平衡。 T1MP 结合、释放以及与 α 和 β’COPI 亚基选择性相互作用的原子原理是这是一个关键的知识差距，因为 T1MP 的释放和逃离 COPI 会调节 T1MP。转运、翻译后修饰和 T1MP 功能障碍与 T1MP 功能障碍有关。我们的长期目标是获得与发育、自身免疫和癌症相关的各种疾病的治疗。对 T1MP 的 COPI 依赖性逆行运输和潜在原子水平的基本见解在这项资助中，我们将阐明导致疾病中 COPI 功能障碍的因素。利用冠状病毒 (CoV) 尖峰进行 COPI 招募、释放和 T1MP 翻译后修饰蛋白质，具有二碱基 Lys-x-His-x-x 序列的 T1MP，作为新的模型系统，这种二碱基序列确保了。依赖于 COPI 的刺突从高尔基体逆行递送至 ER-高尔基体中的病毒后代组装位点在目标 1 中，我们将阐明构象调制的原子细节。在目标 2 中，我们将确定 COPI 释放的原则和在目标 3 中，我们将阐明 COPI 的原子基础。这些研究将扩展刺突突变体的工具包。修改后的 COPI 相互作用，正如我们小组最近发表的那样，我们将在 X 射线中集成结构方法。使用生物物理工具进行晶体学、NMR 和 Rosetta 建模以及分泌物运输的细胞测定获得对 COPI-spike 相互作用的精细调节和构象调节的前所未有的见解。刺突蛋白作为 T1MP 模型系统的创新使用将为基础分泌带来新的见解这些数据将同时为开发针对性疗法开辟途径。 COPI 选择性障碍，并更深入地了解 CoV 组装和 CoV 疫苗的加工。