STRUCTURE/FUNCTION STUDIES OF VITAMIN D BINDING PROTEIN

描述（改编自申请人的摘要）：维生素D结合 蛋白（DBP）结合维生素D及其代谢产物以及G-肌动蛋白， 高亲和力，并用于将前者转运至靶组织， 抑制后者，以防止其在细胞损伤期间聚合。 DBP还结合脂肪酸，并增强中性粒细胞上的补体激活 通过与C5 a des Arg结合的趋化性。此外，已知DBP是 在体内转化为有效的巨噬细胞和破骨细胞活化因子 （DBP-MAF）。 本提案的目标是研究各种结构-功能 DBP和DBP-MAF方面使用多方法方法，包括 维生素D甾醇和脂肪酸结合结构域的化学修饰 DBP的亲和/光亲和标记，操纵DBP-基因， 表达DBP的突变体和片段及其功能表征， DBP及其与肌动蛋白复合物三维结构的X射线测定 晶体学、巨噬细胞-超氧化物生成和骨吸收 测定（用于DBP-MAF活性）。 DBP的多任务性质与特定的 DBP识别/结合各种配体。 这些过程强烈 受蛋白质结构的影响，特别是三维结构， 负责这种识别的蛋白质结构域， 约束力 因此，这些研究将提供有关重要性的信息 DBP的多结构域中的独立结构域，可能 各种内源性配体共享共同的结合口袋， 各种域之间的串扰，各种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）2D 3代谢活化中的作用至关重要， 促钙激素及其组织特异性递送，因此后者 可以负责钙和磷的体内平衡，调节 细胞的生长和成熟，恶性细胞的抗增殖，和 免疫调节 DBP与G-肌动蛋白的相互作用在细胞内是重要的。 防止肌动蛋白聚合，以及细胞增殖期间动脉阻塞 损伤 另一方面，通过DBP-MAF激活巨噬细胞强调了 DBP可能的免疫调节特性。 此外，委员会认为， 破骨细胞活化（DBP-MAF）提出了这种可能性， 细胞因子可能参与炎性关节疾病， 骨关节炎、类风湿性关节炎、牙周病等。 建议的结构-功能研究在评估 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.