Structure and Function of the VWF Helical Tubule Required for Hemostasis

止血所需的 VWF 螺旋管的结构和功能

基本信息

批准号：
10705010
负责人：
Jacob R Anderson
金额：
$ 3.96万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-01 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10705010
关键词：
3-Dimensional Adopted Architecture Binding Blood Blood Coagulation Disorders Blood Platelets Blood Proteins Blood Vessels C-terminal Cells Coagulation Process Cryo-electron tomography Cryoelectron Microscopy Cysteine Data Data Collection Development Dimerization Disease Disulfide Linkage Disulfides Endoplasmic Reticulum Endothelial Cells Endothelium Engineering Environment Factor VIII Failure Fellowship Generations Golgi Apparatus Head Hematological Disease Hemorrhage Hemostatic Agents Hemostatic function Human In Situ In Vitro Inherited Injury Length Link Maps Mediating Missense Mutation Modeling Molecular Molecular Structure Morbidity - disease rate Mutation Negative Staining Peptide Hydrolases Physicians Play Positioning Attribute Process Proteins Research Resolution Role Scientist Series Site Structure Tertiary Protein Structure Testing Therapeutic Thinness Thrombosis Time Training Umbilical vein Visualization Weibel-Palade Bodies Work cleavage factor clinically relevant crosslink dimer disease-causing mutation disorder subtype disulfide bond experience insight light scattering model building monomer novel pre-doctoral reconstruction tomography von Willebrand Disease von Willebrand Factor

项目摘要

PROJECT SUMMARY/ABSTRACT Von Willebrand Disease (VWD), the most common bleeding disorder worldwide, is caused by mutations in von Willebrand Factor (VWF), a large multidomain protein. In the blood, VWF circulates as a long multimer of head- to-head disulfide linked dimers of mature VWF. These long multimers are critical for VWF function as they give circulating VWF polyvalency for activating and binding platelets at sites of endothelial injury, forming a hemostatic plug to staunch bleeding. Additionally, long VWF multimers stabilize coagulation factor VIII (FVIII) in the blood. To form these long multimers, crucial for normal hemostasis, VWF forms helical tubules in the low pH of the late- Golgi and Weibel-Palade bodies (WPB). The helical tubule templates the disulfide bond formation needed to form long multimers by positioning D3 domains in close proximity. At the same time, VWF’s prodomain is cleaved, generating the mature VWF that binds FVIII in the blood. Aberrancy in these maturation steps due to VWF mutations causes several VWD subtypes. Despite the importance of the helical tubule for VWF multimerization, the high-resolution structure of the helical tubule is not known. This fellowship proposal aims to determine structures of VWF helical tubules at three stages of maturation, test Type 2A VWD mutations for causing short tubules, and interrogate the implications of prodomain cleavage for FVIII-VWF tubule association. In Aim 1, using a C-terminally truncated VWF construct, a high-resolution structure of the VWF tubule before and after head-to-head disulfide bonds form will be determined using cryo-electron microscopy (cryo-EM) and helical reconstruction. Using cryo-electron tomography (cryo-ET) and subtomogram averaging, a three- dimensional reconstruction of the in situ VWF tubule will be determined to test if the close packing of VWF helical tubules inside the native WPB environment has consequences for the molecular structure of VWF in the tubule. Guided by this structural insight, a subset of VWD mutations will be tested for their effect on robust tubule formation and normal VWF multimer length. Aim 2 will determine the structural rearrangements in VWF upon prodomain cleavage and test if the cleaved tubule can bind FVIII. This research will elucidate the molecular mechanism of VWF head-to-head disulfide bond formation, necessary for VWF multimerization and normal hemostasis. Structural characterization of the VWF tubule will lead to a molecular understanding of VWD caused by inefficient multimerization. Identification of FVIII-VWF tubule binding will provide a novel context to understand their association and inform therapeutic efforts to modulate FVIII-VWF binding before secretion into the blood. A preliminary VWF tubule reconstruction indicates that additional data collection will allow atomic model building. This research will be carried out under the sponsorship of Dr. Timothy Springer, experienced in structural characterization of VWF, and Dr. Alan Brown, an expert in cryo-EM, creating a strong training environment for predoctoral physician-scientist training.

项目概要/摘要血管性血友病 (VWD) 是世界上最常见的出血性疾病，由血管性血友病突变引起血友病因子 (VWF) 是一种大型多域蛋白，在血液中，VWF 以头多聚体的形式被破坏。成熟 VWF 的头二硫键连接二聚体这些长多聚体对于 VWF 功能至关重要，因为它们赋予 VWF 功能。循环 VWF 多价激活并结合内皮损伤部位的血小板，形成止血剂此外，长 VWF 多聚体可稳定血液中的凝血因子 VIII (FVIII)。为了形成这些对正常止血至关重要的长多聚体，VWF 在晚期的低 pH 条件下形成螺旋小管。高尔基体和 Weibel-Palade 小体 (WPB) 形成了二硫键形成所需的模板。通过将 D3 结构域紧密定位来形成长多聚体。同时，VWF 的前结构域是。裂解，产生与血液中的 FVIII 结合的成熟 VWF。尽管螺旋小管对于 VWF 很重要，但 VWF 突变会导致多种 VWD 亚型。多聚化，螺旋管的高分辨率结构尚不清楚，该奖学金提案的目的是。确定 VWF 螺旋管在三个成熟阶段的结构，测试 2A 型 VWD 突变导致短小管，并探讨前结构域裂解对 FVIII-VWF 小管关联的影响。在目标 1 中，使用 C 端截短的 VWF 构建体，这是之前 VWF 小管的高分辨率结构头对头二硫键形成后，将使用冷冻电子显微镜 (cryo-EM) 确定使用冷冻电子断层扫描（cryo-ET）和次断层扫描平均，进行螺旋重建。将确定原位 VWF 小管的尺寸重建，以测试 VWF 螺旋是否紧密堆积天然 WPB 环境内的肾小管对肾小管中 VWF 的分子结构有影响。在这种结构见解的指导下，将测试 VWD 突变的子集对坚固肾小管的影响目标 2 将决定 VWF 的结构重排。前结构域裂解并测试裂解的小管是否可以结合 FVIII。这项研究将阐明该分子。 VWF头对头二硫键形成的机制，是VWF多聚化和正常情况所必需的 VWF 小管的结构特征将导致对 VWD 引起的分子理解。通过低效多聚化鉴定 FVIII-VWF 小管结合将为理解提供新的背景。它们的关联并为在分泌到血液 A 之前调节 FVIII-VWF 结合的治疗工作提供信息。初步的 VWF 小管重建表明额外的数据收集将允许原子模型的构建。这项研究将在结构方面经验丰富的 Timothy Springer 博士的赞助下进行 VWF 的表征和冷冻电镜专家 Alan Brown 博士为 VWF 创造了强大的培训环境博士前医师科学家培训。