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.

项目摘要 该秘密途径是导致与固体和膜蛋白的生物发生 沟通，能源转移，养分吸收和防御。这些蛋白质在 内质网（ER），然后被贩运到高尔基体和其他细胞器，例如质膜。 这种贩运会导致ER的ER压力，因为ER居民蛋白（例如UDP-葡萄糖隆）的意外出现 转移（UGT）。这些是I型膜蛋白（T1MP），负责修饰脂质 激素以及镇痛药对乙酰氨基酚和吗啡。这些ER居民T1MP的展示 序列（lys-x-lys-x-x或lys-lys-x-x; x = x =任何氨基酸）在其胞质尾部，用于通过coater进行er-retrieval 蛋白I配合物（COPI）。该异hepatomeric络合物的α和β''units结合了此二元序列 将T1MP包装启动到源自CIS-Golgi等后隔室的车辆中。这些copi 涂层的蔬菜交通并将T1MP蛋白交付回ER，以恢复秘密平衡。但是， T1MP结合，释放和选择性相互作用与α和β'COPI亚基的原子原理是 不太了解。这是一个关键的知识差距，因为T1MP释放并逃脱了COPI调制T1MP 贩运，翻译后修改和T1MP功能。 COPI功能障碍与 与发展，自动免疫和癌症有关的多种疾病。我们的长期目标是获得 对T1MP和基础原子级的COPI依赖逆行贩运的基本见解 导致疾病中COPI功能障碍的因素。在这笔赠款中，我们将阐明机械洞察力 使用冠状病毒（COV）SPIKE的COPI招募，释放和T1MP翻译后修饰 蛋白质，具有Dibasic Lys-X-HIS-X-X序列的T1MP，作为新的模型系统。此二元序列可确保 尖峰从高尔基体到ER-Golgi中的病毒后代装配部位的尖峰逆行递送 中间室（ERGIC）。在AIM 1中，我们将阐明构象调节的原子细节 Copi尖峰相互作用。在AIM 2中，我们将确定管辖从COPI释放的原则和 随后对尖峰的翻译后修饰。在AIM 3中，我们将阐明COPI的原子基础 峰值蛋白的亚基选择性。这些调查将扩展到具有尖峰突变体工具包 我们小组最近发布的修改了COPI互动。我们将在X射线中整合结构方法 晶体学，NMR和Rosetta建模，使用生物物理工具和秘书贩运的细胞测定 获得对COPI尖峰相互作用的精细调制和构象调节的前所未有的见解。这 峰值蛋白作为T1MP模型系统的创新使用将产生对基本分泌的新见解 贩运。这些数据将简单地为开发有针对性疗法的途径开放 复型选择性疾病，并更深入地了解COV组装和COV疫苗的处理。