ROLE OF MATRIX VESICLES IN CALCIFICATION

描述（由申请人提供）：软骨内骨化对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化症中。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钙化中所涉及的基本成分。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MV，并具有诱导磷酸钙矿物质形成的能力。第一个目的是确定MV膜联蛋白V与其主要功能修饰剂：Ca 2+，Zn 2+，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和这些重要的调节剂的活性之间的结构-功能关系，并确定磷脂结合位点（S）的蛋白质。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结合其配体的化学计量和热力学值。在第二个目标，完善的合成和物理化学表征和PS：Ca：Pi和膜联蛋白V的复合物的分子结构的测定，构成了核MV的核心将继续。功能性MV样结构将通过将膜联蛋白V和电解质封装到含有与天然MV类似的脂质分布的大单层囊泡中来合成。要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与EDAX分析的Ca：P化学计量，和固态31 P-NMR提供的早期矿物相诱导MV和nucralational复杂的细节。虽然大多数拟议的工作将使用南卡罗来纳州大学的设施进行，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的最先进的X射线和31 P-NMR设施以及巴特尔太平洋西北国家实验室的高场NMR。拟议研究的目的是进一步表征关键组件和事件，在许多情况下在原子水平上，这对MV功能的机制至关重要。我们的长期目标是生产合成MV和/或成核材料，可以诱导矿化并促进骨折或其他骨损伤的愈合。

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.