CMP-Neu5Ac: A Central Molecule in Bleeding Diseases and Mediator of a Novel Platelet Effector Function

CMP-Neu5Ac：出血性疾病的中心分子和新型血小板效应功能的介质

• 批准号: 10001341
• 负责人: Marie Hollenhorst
• 金额: $ 5.48万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-29 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001341
• 关键词: Alkynes; Anabolism; Autoimmune Diseases; Biological; Biological Assay; Biology; Biotin; Blood Coagulation Disorders; Blood Platelets; Carbohydrates; Cell Surface Proteins; Cell surface; Cells; Chemicals; Chemistry; Coagulation Process; Coupled; Data; Development; Diagnosis; Disease; Enzyme Kinetics; Enzyme Tests; Enzymes; Flow Cytometry; Glycobiology; Glycocalyx; Goals; Hematologic Neoplasms; Hematological Disease; Hematology; Hematopoietic stem cells; Hemorrhage; Hemostatic function; Human; Human body; In Vitro; Incubated; Inherited; Learning; Light; Liquid substance; Malignant Neoplasms; Mediating; Mediator of activation protein; Megakaryocytes; Membrane Proteins; Molecular; Morbidity - disease rate; Mutation; Myocardial Infarction; N-Acetylneuraminic Acid; Optics; Outcome; Pathogenesis; Patients; Phosphotransferases; Physicians; Plasma; Platelet Count measurement; Play; Production; Proteins; Proteomics; Radiolabeled; Recombinants; Reporter; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Scientist; Sialyltransferases; Stroke; Surface; Syndrome; Testing; Therapeutic; Thrombocytopenia; Thrombosis; Training; Universities; Variant; Western Blotting; Work; base; effective therapy; epimerase; extracellular; glycosyltransferase; in vivo; liquid chromatography mass spectrometry; mortality; novel; overexpression; platelet function; precursor cell; professor; sialylation; small molecule; sugar; sugar nucleotide; tool; transfusion medicine; uptake

PROJECT SUMMARY/ABSTRACT There is a fundamental gap in understanding how platelet number and function are regulated. Until we gain a more detailed understanding of this, we will lack effective therapies for patients with bleeding and clotting diseases. CMP-Neu5Ac is a sugar nucleotide that plays an important role in the biology of platelets. After release from platelets, this molecule is used as a substrate for extracellular sialylation of platelet surface proteins, resulting in changes in platelet in vivo clearance and activity. The cellular origin of the CMP-Neu5Ac released from platelets and the platelet surface protein substrates for extracellular sialylation remain unknown. The long-term goal is to capitalize on a molecular understanding of the role of platelets as mediators of extracellular sialylation to develop platelet-based cellular therapeutics for hematologic diseases and cancer. The overall objective of this application is to determine the origin of CMP-Neu5Ac in platelets and to identify its biologically relevant sialylation cosubstrates. The central hypothesis is that platelets regulate the extracellular sialylation of functionally important platelet proteins via release of CMP-Neu5Ac that is biosynthesized in platelet precursors. This will be accomplished via the following specific aims: 1) Mechanistically define the functional effects of GNE enzyme variants found in patients with inherited thrombocytopenia. This will be accomplished by overexpression and purification of the recombinant enzyme followed by in vitro enzymatic characterization. 2) Determine the cellular origin of platelet CMP-Neu5Ac. Lysates from platelets, megakaryocytes, and hematopoietic stem cells will be analyzed for expression of CMP-Neu5Ac biosynthetic enzymes, and tested for their ability to enzymatically transform UDP-GlcNAc to CMP-Neu5Ac. 3) Identify the biologically relevant platelet surface protein substrates for extracellular sialylation. Click chemistry will be used to attach alkyne-modified chemical reporters to facilitate identification of novel extracellular sialylation substrates by liquid chromatography/mass spectrometry proteomics. The outcome will be an understanding of the cellular origin of CMP-Neu5Ac in the platelet lineage and an identification of platelet surface protein substrates for extracellular sialylation. This will have implications not only for the diagnosis and treatment of disorders of hemostasis and thrombosis, but also for a broader range of diseases involving alterations of the glycome, including cancer and autoimmune diseases. The training plan includes learning a new chemical glycobiology skillset from Professor Carolyn Bertozzi at Stanford University, and applying these tools to platelet biology with guidance from consultant experts. This training will be critical for me to develop a unique niche as an independent physician-scientist investigator at the interface of chemical biology and hematology/transfusion medicine.

项目总结/摘要 在理解血小板数量和功能如何调节方面存在根本性的差距。直到我们 如果对此有更详细的了解，我们将缺乏针对出血和凝血患者的有效治疗方法 疾病CMP-Neu 5Ac是一种糖核苷酸，在血小板生物学中起重要作用。释放后 来自血小板的该分子用作血小板表面蛋白细胞外唾液酸化的底物， 导致体内血小板清除率和活性的变化。释放的CMP-Neu 5Ac的细胞来源 来自血小板和用于细胞外唾液酸化的血小板表面蛋白质底物仍然未知。 长期目标是利用对血小板作为介导剂的作用的分子理解， 细胞外唾液酸化以开发用于血液病和癌症的基于血小板的细胞治疗剂。的 本申请的总体目的是确定血小板中CMP-Neu 5Ac的来源并鉴定其在血小板中的表达。 生物学相关的唾液酸化共底物。中心假设是血小板调节细胞外 通过释放血小板中生物合成CMP-Neu 5Ac使功能重要的血小板蛋白唾液酸化 前体 这将通过以下具体目标来实现：1）机械地定义功能效果 在遗传性血小板减少症患者中发现的GNE酶变体。这将通过 重组酶的过表达和纯化，然后进行体外酶表征。（二） 确定血小板CMP-Neu 5Ac的细胞来源。来自血小板、巨核细胞和 将分析造血干细胞的CMP-Neu 5Ac生物合成酶的表达，并测试 它们将UDP-GlcNAc酶促转化为CMP-Neu 5Ac的能力。3)识别生物学相关血小板 用于细胞外唾液酸化的表面蛋白质底物。点击化学将用于连接炔改性的 通过液相色谱法鉴定新型细胞外唾液酸化底物的化学报道分子 色谱/质谱蛋白质组学。其结果将是对细胞起源的理解， 血小板谱系中的CMP-Neu 5Ac和细胞外基质的血小板表面蛋白底物的鉴定 唾液酸化这不仅对止血障碍的诊断和治疗有意义， 血栓形成，但也适用于涉及糖组改变的更广泛的疾病，包括癌症和 自身免疫性疾病 培训计划包括从Carolyn Bertozzi教授那里学习新的化学糖生物学技能 在斯坦福大学，并在顾问专家的指导下将这些工具应用于血小板生物学。这 培训将是至关重要的，我发展一个独特的利基作为一个独立的医生，科学家调查员在 化学生物学与血液学/输血医学接口