Molecular Mechanisms Underlying Endothelial Weibel-Palade Body Biogenesis and Exocytosis

内皮 Weibel-Palade 体生物发生和胞吐作用的分子机制

• 批准号: 10407600
• 负责人: Anish Vaibhav Sharda
• 金额: $ 16.5万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2022-06-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10407600
• 关键词: Adaptor Signaling Protein; Advisory Committees; Affect; Albinism; Binding; Biogenesis; Blood; Blood Cells; Blood Coagulation Disorders; Blood Platelets; Cardiovascular system; Cell membrane; Cells; Clinical Research; Coagulation Process; Complex; Cytoplasmic Granules; Data; Defect; Dependence; Disease; Docking; Endosomes; Endothelial Cells; Endothelium; Environment; Exocytosis; Fellowship; Five-Year Plans; Genes; Goals; Growth; Hematology; Hemorrhage; Hemostatic Agents; Hemostatic function; Hermanski-Pudlak Syndrome; Human; Immunoprecipitation; Impairment; Individual; Israel; Lysosomes; Mediating; Medical center; Melanosomes; Membrane; Membrane Protein Traffic; Mentors; Mentorship; Molecular; Molecular Weight; Morbidity - disease rate; Neoplasm Metastasis; Organelles; Pathway interactions; Plasma; Play; Postdoctoral Fellow; Process; Proteins; Regulation; Reporting; Research Personnel; Role; SNAP receptor; SNAP23 gene; Scientist; Secondary to; Secretory Vesicles; Skin Pigmentation; Thrombosis; Training; Vesicle; Weibel-Palade Bodies; Yeasts; angiogenesis; cancer cell; cardiovascular risk factor; career; cellubrevin; experimental study; fluidity; in vivo; innovation; live cell microscopy; mouse model; platelet function; protein complex; protein transport; rab GTP-Binding Proteins; receptor; release factor; syntaxin 3; trafficking; trans-Golgi Network; von Willebrand Disease; von Willebrand Factor

Project Summary/Abstract Endothelium plays a vital role in hemostasis and thrombosis. It is essential to maintain blood fluidity, but it also expresses and releases numerous “factors” that regulate blood cell activation and coagulation. Among these, von Willebrand factor (vWF) is an essential plasma hemostatic factor released by the endothelium. Deficiency of mature vWF is the most common bleeding disorder in humans. On the other hand, elevated plasma levels of vWF, or abnormal concentrations of its high-molecular weight multimers, is associated with increased risk of cardiovascular morbidity. As such, understanding of the molecular mechanisms that underlie regulated release of vWF have broad implications in hemostasis and thrombosis. One factor that plays a crucial role in normal vWF exocytosis from endothelial cells is unimpaired biogenesis of its storage granules, the Weibel-Palade bodies (WPBs), which is a highly complex process that remains poorly characterized. We have previously shown that deficiency of biogenesis of lysosome-related organelle complex 2 (BLOC-2) results in impaired vWF exocytosis. Since, BLOC-2, and other related trafficking proteins, are essential for biogenesis of lysosomal-related organelles, a group of specialized granules that includes platelet dense granules and melanosomes, it seems plausible that this protein complex is also required for biogenesis of WPB. We hypothesize that BLOC-2-mediated endosomal trafficking is critical for biogenesis of WPB. We further postulate that the exocyst complex plays an essential role in WPB trafficking by 1) interacting with BLOC-2 in its role in endosomal trafficking 2) and regulating soluble NSF adaptor protein receptor (SNARE)-mediated fusion of WPBs at the plasma membrane. In Aim 1, we will characterize cargo trafficking from the endosomes to the maturing WPBs and the dependence of this pathway on BLOC-2. In Aim 2, we will evaluate the role of the exocyst complex in BLOC-2-dependent endosomal trafficking and characterize the core trafficking machinery involved in this pathway. And, finally, in Aim 3, we will evaluate the regulatory function of the exocyst complex in SNARE-mediated fusion of WPBs at the plasma membrane. The applicant, Dr. Anish Sharda, previously completed clinical and research fellowship in hematology and is currently pursuing a post-doctoral fellowship under the mentorship of Drs. Robert Flaumenhaft and Bruce Furie, who will serve as primary mentor and co-mentor, respectively. The Division of Hemostasis and Thrombosis at Beth Israel Deaconess Medical Center has a distinguished track record of scientific innovation and mentorship, and will provide an excellent environment for the applicant in pursuit of his career goals. The advisory committee of exceptional scientists that Dr. Sharda has established will bring diverse intellectual expertise to his training and scientific growth. Dr. Sharda is well-qualified to execute the proposed experiments and has presented a comprehensive five-year plan to meet his goal of becoming an independent researcher.

项目总结/摘要 内皮素在止血和血栓形成中起着至关重要的作用。维持血液流动性是必不可少的，但它也 表达并释放许多调节血细胞活化和凝结的“因子”。在这些国家中， 血管性血友病因子（vWF）是由内皮释放的必需血浆止血因子。缺乏 成熟vWF是人类最常见的出血性疾病。另一方面，血浆中的 vWF或其高分子量多聚体的异常浓度与以下风险增加相关： 心血管疾病发病率。因此，了解调控释放的分子机制 vWF在止血和血栓形成中具有广泛的意义。在正常情况下发挥关键作用的一个因素 内皮细胞的vWF胞吐作用是其储存颗粒未受损的生物合成， 体（WPB），这是一个高度复杂的过程，其特征仍然很差。我们先前已经 显示溶酶体相关细胞器复合物2（BLOC-2）的生物合成缺陷导致受损的 vWF胞吐作用。由于BLOC-2和其他相关的运输蛋白质是生物合成所必需的， 溶酶体相关的细胞器，一组专门的颗粒，包括血小板致密颗粒和 黑素体，这似乎是合理的，这种蛋白质复合物也需要WPB的生物合成。我们 假设BLOC-2介导的内体运输对于WPB的生物发生是关键的。我们进一步 假设外囊复合物通过1）与BLOC-2相互作用在WPB运输中发挥重要作用， 其在内体运输中的作用2）和调节可溶性NSF衔接蛋白受体（SNARE）介导的 WPB在质膜上融合。在目标1中，我们将描述从内体运输货物的特征 以及该途径对BLOC-2的依赖性。在目标2中，我们将评估 BLOC-2依赖的内体运输中的外囊复合物，并表征核心运输 参与这条道路的机器。最后，在目标3中，我们将评估 SNARE介导的WPB在质膜上的融合中的外囊复合物。 申请人Anish Sharda博士以前完成了血液学临床和研究奖学金 目前正在罗伯特·弗劳门哈夫特博士的指导下攻读博士后研究金， 布鲁斯福瑞，谁将担任主要导师和共同导师，分别。止血与止血科 贝丝以色列女执事医疗中心的血栓有着杰出的科学创新记录 和导师，并将提供一个良好的环境，为申请人在追求自己的职业目标。的 Sharda博士建立的杰出科学家顾问委员会将带来不同的知识分子， 专业知识，他的培训和科学的成长。Sharda博士完全有资格执行提议的实验 并提出了一个全面的五年计划，以实现他成为一名独立研究员的目标。