ROLE OF MATRIX VESICLES IN CALCIFICATION

基质囊泡在钙化中的作用

• 批准号: 6913534
• 负责人: ROY E WUTHIER
• 金额: $ 31.54万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-01-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6913534
• 关键词: X ray crystallography; annexins; binding sites; calcification; calcium ion; calcium phosphate; calorimetry; chickens; chondrocytes; electrolytes; extracellular matrix; extracellular matrix proteins; interferometry; intermolecular interaction; lipids; molecular dynamics; normal ossification; phosphatidylserines; phospholipids; protein structure function; spectrometry; thermodynamics; vesicle /vacuole; zinc

DESCRIPTION (provided by applicant): Endochondral ossification is essential for normal skeletal development and is involved in fracture repair. On the other hand, pathological calcification occurs in osteoarthritis, atherosclerosis, and chondrocalcinosis. In order to design effective therapies for treatment of dysfunctional calcification the mechanisms involved in mineral deposition must be understood. Since matrix vesicles (MV) are intimately associated with mineral formation in many vertebrate calcifying tissues, our goal is to characterize the essential components involved in MV calcification. Our goal is to elucidate how key proteins, lipids, and electrolytes interact to form MV with the ability to induce calcium phosphate mineral formation. The first aim is to determine the 3-D structures and the thermodynamics of binding of MV annexin V with its major functional modifiers: Ca2+, Zn2+, ATP, GTP, and the phospholipids. X-ray crystallography will be used to elucidate structure-function relationships between annexin V and these important modulators of its activity and to determine the phospholipid bind site(s) in the protein. The packing arrangement of phosphatidylserine (PS): calcium (Ca):inorganic phosphate (Pi): annexin V complexes will be observed by transmission electron microscopy (TEM). Isothermal titration calorimetry will be used to obtain stoichiometric and thermodynamic values for annexin V binding to its ligands. In the second aim, refinement in the synthesis and physicochemical characterization and the molecular structure determination of the complex of PS:Ca:Pi and annexin V that constitutes the nucleational core of MV will continue. Functional MV-like structures will be synthesized by encapsulating annexin V and electrolytes into large unilamellar vesicles containing lipid profiles similar to those of native MV. Analytical techniques to be used include Fourier transform infrared spectroscopy, high resolution X-ray diffraction, TEM with EDAX analysis for Ca:P stoichiometry, and solid-state 31P-NMR to provide details of the early mineral phases induced by MV and the nucleational complex. While most of the proposed work will take place using the facilities at the University of South Carolina, we also have access to state-of-the-art X-ray and 31P-NMR facilities at the Advanced Photon Source synchrotron at Argonne National Laboratory and the high-field NMR at the Battelle Pacific Northwest National Laboratory. The objective of the proposed research is to further characterize key components and events, in many cases at the atomic level, which are critical to the mechanism of MV function. Our long-term goal is to produce synthetic MV and/or nucleational materials that can induce mineralization and promote healing of bone fractures or other recalcitrant bone injuries.

描述（由申请人提供）：软骨内骨化对于正常骨骼发育至关重要，并参与骨折修复。另一方面，病理性钙化发生在骨关节炎、动脉粥样硬化和软骨钙质沉着症中。为了设计治疗功能失调性钙化的有效疗法，必须了解矿物质沉积所涉及的机制。由于基质囊泡 (MV) 与许多脊椎动物钙化组织中的矿物质形成密切相关，因此我们的目标是表征 MV 钙化中涉及的基本成分。我们的目标是阐明关键蛋白质、脂质和电解质如何相互作用形成具有诱导磷酸钙矿物质形成能力的 MV。第一个目标是确定 MV 膜联蛋白 V 与其主要功能修饰剂（Ca2+、Zn2+、ATP、GTP 和磷脂）结合的 3-D 结构和热力学。 X 射线晶体学将用于阐明膜联蛋白 V 与其活性的这些重要调节剂之间的结构-功能关系，并确定蛋白质中的磷脂结合位点。通过透射电子显微镜（TEM）观察磷脂酰丝氨酸（PS）：钙（Ca）：无机磷酸盐（Pi）：膜联蛋白V复合物的堆积排列。等温滴定量热法将用于获得膜联蛋白 V 与其配体结合的化学计量值和热力学值。在第二个目标中，将继续完善构成 MV 核核心的 PS:Ca:Pi 和膜联蛋白 V 复合物的合成和物理化学表征以及分子结构测定。通过将膜联蛋白 V 和电解质封装到含有与天然 MV 相似的脂质谱的大单层囊泡中，可以合成功能性 MV 样结构。使用的分析技术包括傅里叶变换红外光谱、高分辨率 X 射线衍射、TEM 和 EDAX 分析 Ca:P 化学计量，以及固态 31P-NMR，以提供由 MV 和成核复合物诱导的早期矿物相的详细信息。虽然拟议的大部分工作将使用南卡罗来纳大学的设施进行，但我们还可以使用阿贡国家实验室先进光子源同步加速器的最先进的 X 射线和 31P-NMR 设施以及巴特尔太平洋西北国家实验室的高场 NMR 设施。本研究的目的是进一步表征关键成分和事件，在许多情况下是在原子水平上，这对 MV 功能机制至关重要。我们的长期目标是生产能够诱导矿化并促进骨折或其他顽固性骨损伤愈合的合成 MV 和/或成核材料。