ROLE OF MATRIX VESICLES IN CALCIFICATION

描述(申请人提供)：软骨内骨化是正常骨骼发育所必需的，与骨折修复有关。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化。为了设计有效的治疗钙化功能障碍的方法，必须了解矿物质沉积的机制。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，我们的目标是表征MV钙化所涉及的基本成分。我们的目标是阐明关键蛋白质、脂类和电解质是如何相互作用形成MV的，并具有诱导磷酸钙矿物形成的能力。第一个目的是确定MV Annexin V与其主要功能修饰物：CA2、Zn2、ATP、GTP和磷脂结合的三维结构和结合热力学。X射线结晶学将被用来阐明膜联蛋白V和这些重要的活性调节剂之间的结构-功能关系，并确定蛋白质中的磷脂结合部位(S)。用透射电子显微镜观察磷脂酰丝氨酸(PS)：钙(Ca)：无机磷(PI)：膜联蛋白V复合体的堆积排列。等温滴定量热法将被用来获得Annexin V与其配体结合的化学计量和热力学数值。在第二个目标中，将继续对构成MV核心的PS：CA：PI与Annexin V的络合物的合成、物化表征和分子结构的测定进行精细化。通过将膜联蛋白V和电解质包裹到含有与天然MV相似的脂谱的大的单层囊泡中，可以合成功能类似MV的结构。可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、用于Ca：P化学计量比的电子显微镜和EDAX分析，以及固态31P-核磁共振，以提供MV诱导的早期矿物相和成核复合体的详细信息。虽然大部分拟议工作将使用南卡罗来纳大学的设施进行，但我们也可以使用阿贡国家实验室先进光子源同步加速器和巴特尔太平洋西北国家实验室的高场核磁共振设备。这项研究的目的是进一步确定关键成分和事件的特征，在许多情况下是在原子水平上，它们对MV功能的机制至关重要。我们的长期目标是生产合成的MV和/或核材料，能够诱导矿化并促进骨折或其他顽固性骨损伤的愈合。

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.