STRUCTURE/FUNCTION STUDIES OF VITAMIN D BINDING PROTEIN

描述(摘自申请者的摘要)：维生素D结合 蛋白质(DBP)与维生素D及其代谢产物以及G-肌动蛋白结合 高亲和力，并用于将前者输送到靶组织，并 清除后者，以防止其在细胞损伤过程中聚合。 DBP还与脂肪酸结合，并增强中性粒细胞上的补体激活 通过与C5a Des Arg结合而趋化。此外，众所周知，DBP是 体内转化为强大的巨噬细胞和破骨细胞激活因子 (DBP-MAF)。 这项建议的目标是研究各种结构-功能 使用多种方法的DBP和DBP-MAF的各个方面，包括 维生素D、甾醇和脂肪酸结合域的化学修饰 通过亲和/光亲和标记DBP，操纵DBP-基因以 表达DBP突变体和片段及其功能特性， X射线测定DBP及DBP-肌动蛋白复合体的三维结构 结晶学、巨噬细胞超氧化物生成和骨吸收 化验(DBP-MAF活性)。 DBP的多任务特性直接与特定的 DBP对各种配体的识别/结合。这样的过程是强烈的 受蛋白质结构，特别是三维结构的影响 负责这种识别和识别的蛋白质结构域 有约束力的。因此，这些研究将提供有关重要性的信息 在DBP的多域结构中的独立结构域，可能 各种内源配体共用一个共同的结合口袋，程度 不同领域之间的串扰，不同配体之间的相互依赖，以及 配体结合对DBP-MAF活性的影响。 DBP在1，25(OH)2D3的代谢活化中起着至关重要的作用。 促钙激素及其组织特异性递送，使后者 可负责钙磷的动态平衡、调节 细胞的生长和成熟，恶性细胞的抗增殖，以及 免疫调节。DBP与G-肌动蛋白的相互作用在 细胞过程中肌动蛋白聚合和动脉阻塞的预防 受伤。另一方面，DBP-MAF对巨噬细胞的激活也强调了 DBP可能的免疫调节作用。此外， 破骨细胞激活(通过DBP-MAF)增加了这一可能性 细胞因子可能参与炎症性关节疾病，如 骨关节炎、类风湿性关节炎、牙周病等 建议的结构-功能研究应该在评估方面有价值 DBP和DBP的多种功能及其可能的生理意义 DBP-MAF。

DESCRIPTION (Adapted from the Applicant's Abstract): Vitamin D-binding protein (DBP) binds vitamin D and its metabolites as well as G-actin with high affinity and serves to transport the former to target tissues, and to scavenge the latter so as to prevent its polymerization during cell-injury. DBP also binds fatty acids, and enhances complement activation on neutrophil chemotaxis by binding to C5a des Arg. Additionally, DBP is known to be converted in vivo to a potent macrophage and osteoclast activating factor (DBP-MAF). The objectives of this proposal are to study various structure-functional aspects of DBP and DBP-MAF using a multiple-methods approach including chemical modification of the vitamin D sterol and fatty acid-binding domains of DBP by affinity/photoaffinity labeling, manipulations of the DBP-gene to express mutants and segments of DBP and their functional characterization, determination of the 3-D structures of DBP and DBP-actin complex by X-ray crystallography, and macrophage-superoxide production and bone resorption assays (for DBP-MAF activities). Multi-tasking nature of DBP is directly related to specific recognition/binding of various ligands by DBP. Such processes are strongly influenced by the structure of the protein, particularly the 3-D structures of the domains of the protein that are responsible for such recognition and binding. Hence, these studies will provide information about the importance of independent domains in the multi-domained structure of DBP, possible sharing of a common binding pocket by various endogenous ligands, degree of cross-talk among various domains, interdependence among various ligands, and influence of ligand-binding on DBP-MAF activities. The role of DBP is crucial in the metabolic activation of 1,25(OH)2D3, the calciotropic hormone, and its tissue-specific delivery, so that the latter can be responsible in calcium and phosphorus homeostasis, regulation of growth and maturity of cells, antiproliferation of malignant cells, and immunoregulation. Interaction of DBP with G-actin is important in the prevention of actin-polymerization, and clogging of arteries during cellular injury. On the other hand, macrophage-activation by DBP-MAF has stressed the possible immunoregulatory property of DBP. Furthermore, osteoclast-activation (by DBP-MAF) has raised the possibility that this cytokine may be involved in inflammatory joint diseases such as osteoarthritis, rheumatoid arthritis, periodontal diseases, etc. The proposed structure-function studies should be valuable in evaluating multiple functions and their possible physiological implications of DBP and DBP-MAF.