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

• 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
• 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

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.

项目总结 分泌途径负责可溶性和膜蛋白的生物发生，涉及到 通讯、能量转导、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。 这种转运通过UDP-葡萄糖醛酸基等内质网驻留蛋白的意外外流导致内质网应激 转移酶(UGT)。这些是I型膜蛋白(T1MP)，负责脂质的修饰 激素和镇痛剂扑热息痛和吗啡。这些驻留在ER中的T1MP显示一个二进制 它们胞浆尾部的序列(Lys-x-Lys-x-x-x或Lys-Lys-x-x；x=任何氨基酸)，用于由Cotom检索内质网 蛋白I复合体(COPI)。该杂七聚体复合体的α和β‘亚基结合该二碱基序列 启动T1MP包装成来自ER后隔室(如顺式高尔基体)的囊泡。这些COPI 包被的囊泡运输并将T1MP蛋白送回内质网，以恢复分泌平衡。然而， T1MP与α和β的COPI亚单位的结合、释放和选择性相互作用的原子原理如下 不是很清楚。这是一个关键的知识缺口，因为T1MP的释放和摆脱COPI调节了T1MP 贩运、翻译后修饰和T1MP功能。Copi功能障碍被认为与 与发育、自身免疫和癌症有关的各种疾病。我们的长期目标是获得 对依赖于COPI的T1MP逆行贩运及其潜在原子水平的基本见解 疾病中导致COPI功能障碍的因素。在这项资助中，我们将阐明对 利用冠状病毒(CoV)尖峰的Copi招募、释放和T1MP翻译后修饰 蛋白质，一个具有二碱性Lys-x-His-x-x序列的T1MP，作为一种新的模型系统。这种双基序列确保了 依赖拷贝的从高尔基体到病毒后代组装部位的逆行递送 中间隔室(ERGIC)。在目标1中，我们将阐明构象调制的原子细节 COPI-SPEK相互作用的结果。在目标2中，我们将确定控制COPI和 随后对扣球进行翻译后的修改。在目标3中，我们将阐明COPI的原子基础 S蛋白的亚基选择性。这些研究将扩展尖峰突变体的工具包， 修改的COPI交互作用，如我们小组最近发布的。我们将在X射线中整合结构方法 使用生物物理工具的结晶学、核磁共振和Rosetta建模以及分泌转运的细胞分析 获得对COPI-SPEK相互作用的精细调制和构象调控的前所未有的见解。这个 创新地使用Spike蛋白作为T1MP模型系统将对基本分泌产生新的见解 贩卖人口。这些数据将同时为开发靶向疗法开辟道路 为更深入地了解冠状病毒组装和加工冠状病毒疫苗。