Structural and Functional Characterization of Noncollagenous Bone Matrix Proteins

骨骼和牙齿中的矿化涉及调节矿物质形成的非胶原基质蛋白（NCPs）。进化分析表明，这些NCPs的基因来自一个共同的祖先，并聚集在一起。胶原基钙化组织中的NCP形成小整联蛋白结合配体N-连接糖蛋白（SIBLING）家族，并具有许多共同的生化特性和功能。SIBLING的特征在于决定其与细胞、基质和矿物质相互作用的基序和结构域，因此理解这些基序和结构域的作用对于确定SIBLING如何调节矿化具有根本重要性。我们已经集中在一个兄弟姐妹-骨唾液酸蛋白（BSP）-因为它是与初始矿化事件。BSP具有增加成骨细胞相关基因表达和基质矿化的基序。它还含有两个酸性结构域，据信这两个酸性结构域部分负责成核矿物质并与其结合，尽管只有间接证据表明这一点。我们已经证明，突变这两个域对体外成骨没有影响。然而，出乎意料的是，去除第二个基序导致矿物形成的主要抑制，这是一个具有重要功能意义的新发现。与所有的同胞基因一样，BSP被认为含有一个赋予矿物质抑制和促进能力的基序，但对BSP中的这个基序知之甚少。我们的目标是通过利用综合实验方法对BSP分子的功能结构域进行全面的了解。我们将：1.进行细胞培养和动物研究，以测试功能域的活性。这些研究将独特地考虑到身体的生理学。2.检查BSP结构的相关修改引起的功能变化。3.研究正常和突变的BSP分子的结合效率-到目前为止只在非生理学测定中研究-使用我们小组在动物研究中开创的方法。利用BSP获得的知识，从长远来看，我们打算将我们的研究扩展到SIBLING的其他成员，以全面了解这个蛋白质家族如何调节生理和病理矿化。HQP在拟议的研究计划将暴露在一个独特的培训环境，只有在世界各地的实验室数量有限。他们的培训预计也将在高需求的工业和研究机构。我们要求每年支持2个硕士和1个博士生。硕士生将主要参与体外程序和研究。博士生将承担先进的分子分析和动物研究。拟议的研究将提供有关NCP功能的基本信息，这些信息对于理解硬组织形成的基本生物学以及蛋白质结构的变化如何导致疾病是必要的。

Mineralization in bones and teeth involves noncollagenous matrix proteins (NCPs) that regulate the formation of mineral. Evolutionary analysis has shown that genes for these NCPs derive from a common ancestor and are clustered together. NCPs in collagen-based calcified tissues form the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family and share a number of biochemical properties and functions. The SIBLINGs are characterized by motifs and domains that determine their interaction with cells, matrix and mineral. Understanding the role of these motifs and domains is thus of fundamental importance for defining how SIBLINGs regulate mineralization. We have focused on one of the SIBLINGs - bone sialoprotein (BSP) - because it is associated with initial mineralization events. BSP has a motif that increases osteoblast-related gene expression and matrix mineralization. It also contains two acidic domains that are believed to be partially responsible for the nucleating mineral and binding to it, although there is only circumstantial evidence for this in vivo. We have shown that mutating both domains has no effect on in vitro osteogenesis. However, unexpectedly, removal of the second motif results in a major inhibition of mineral formation, a novel finding that has important functional implications. As all SIBLINGs, BSP is believed to contain a motif that confers both mineral inhibition and promotion capacity, yet little is known about this motif in BSP. Our objective is to obtain a comprehensive understanding of the functional domains of the BSP molecule by exploiting integrative experimental approaches. We will: 1.carry out cell culture and animal studies to test the activity of the functional domains. These studies will uniquely take into account the physiology of the body. 2.examine functional changes induced by relevant modifications of the BSP structure. 3.investigate the binding efficiency of normal and mutated BSP molecules – so far only studied in non-physiological assays - using methods our group has pioneered for studies in animals. Taking advantage of the knowledge acquired for BSP, in the longer term we intend to extend our studies to other members of the SIBLINGs in order to obtain a comprehensive understanding of how this family of proteins regulates both physiologic and pathological mineralization. HQPs in the proposed research program will be exposed to a unique training environment that is only available in a limited number of laboratories throughout the world. Their training is also expected to be in high demand industry and research institutions. We are requesting annual support for 2 MSc and 1 PhD students. MSc students will be primarily implicated in vitro procedures and studies. The PhD student will assume advanced molecular analyses and studies in animals. The proposed research will provide fundamental information on the function of NCPs necessary for understanding the basic biology of hard tissue formation and how changes in protein structure can lead to disease.