Noncollagenous Protein Interaction in Biomineralization

描述(申请人提供)：生理性羟基磷灰石晶体(矿化)在胶原基组织(骨、牙本质、牙骨质、钙化软骨等)中的沉积机制。是一个知之甚少的复杂过程。我们相信，在医管局 (羟基磷灰石)形成胶原蛋白和非胶原蛋白(NCP)调节(促进或抑制)HA的生长和增殖。许多NCP的结构主要是随机卷曲的，因此被归类为“内在无序蛋白质”(IDPs)。IDPs丰富地存在于脊椎动物和非脊椎动物的矿化系统中，人们认为它们灵活的结构使它们能够与各自的“伴侣”相互作用。纤维状胶原蛋白是矿化组织IDPs或其亚单位所具有的“伙伴”之一 被证明是有约束力的。这种反应的驱动力和发生这种结合的原因尚不清楚。矿化组织IDPs的另一个“伙伴”是HA。一些NCP(例如富含亮氨酸的小分子蛋白多糖和其他糖蛋白)具有明确的非柔性结构，并且还与胶原和透明质酸相互作用。我们的假设是，小而灵活的IDPs与胶原结合，然后与HA核或新生HA结合，从而变得更加有序。由于在相互作用过程中对能量的需求较少，因此构象变化的能量学被认为比更有序的NCPs与胶原和HA的结合能量学更有利。这种R21应用的目的是开发和优化分析方法来确定IDPs的构象，以及调节HA生长所需的相互作用能量学，支持或驳斥这一假说。目标1)：利用傅里叶变换红外光谱研究牛奶骨桥蛋白(MOPN)和核心蛋白聚糖(DCN)与透明质酸结合的构象变化。与这些数据一起，我们将测量“研究蛋白”与HA和胶原的结合亲和力，并在(A)单独存在胶原、(B)包被“研究蛋白”的胶原、(C)在每个“研究蛋白”单独存在下形成的胶原和(D)单独存在“研究蛋白”的情况下，还测量HA生长和复制的动力学。这些研究将确定用于AIMS 2和AIMS 3的最佳“研究蛋白”与胶原的比例。目标2)：利用“研究蛋白”研究胶原-NCP-HA相互作用的表面等离子体共振。目的3)：建立一组研究胶原-NCP-HA相互作用的荧光方法，并将其应用于“研究蛋白质”。这些方法的发展将使我们能够开始验证我们的假设，同时为研究IDP与胶原和HA的相互作用提供新的技术，并对胶原介导的生物矿化机制提供新的见解。 公共卫生相关性：这项研究的目标是开发分析方法，以研究胶原和胶原相关蛋白在矿化过程中的重要性。这项探索性研究的成功完成将提供许多以前没有在现场使用的技术的信息，产生有可能改变我们对生物矿化过程的理解的数据。此外，这些研究可以导致开发治疗常见的骨骼和牙齿疾病，如骨软化症和骨质疏松症，以及更罕见的疾病，如成骨和牙本质形成不全和骨骼发育不良，以及新的组织工程策略。

DESCRIPTION (provided by applicant): The mechanism of deposition of physiologic hydroxyapatite crystals (mineralization) in collagen-based tissues (bone, dentin, cementum, calcified cartilage, etc.) is a poorly understood complex process. It is our belief, that during HA (hydroxyapatite) formation both collagen and noncollagenous proteins (NCPs) regulate (promote or inhibit) the growth and proliferation of HA. Many of the NCPs have structures that are predominately random coils, and are thus classified as "Intrinsically Disordered Proteins" (IDPs). IDPs are abundant in mineralizing systems of vertebrates and non-vertebrates, and it is thought that their flexible structures allow them to interact with their respective "partners". Fibrillar collagen is one of the "partners" to which mineralized tissue IDPs or their subunits have been shown to bind. The driving force for this reaction and the reasons that this binding takes place are not known. Another "partner" for the mineralized tissue IDPs is HA. Several NCPs (e.g. small leucine rich proteoglycans (SLRPS) and other glycoproteins) have well defined, non-flexible structures, and also interact with collagen and HA. It is our hypothesis that the small, flexible IDPs, bind to collagen, then to HA nuclei or nascent HA, and become more ordered in consequence. The energetics of conformational change in the IDPs are postulated to be more favorable than the binding energetics of more ordered NCPs to collagen and HA, due to a lesser need for energy in the interaction process. The purpose of this R21 application is to develop and optimize analytical methods to determine the conformation of IDPs, and the interaction energetics needed for the regulation of HA growth, supporting or refuting this hypothesis. There are three specific aims: Aim 1):Demonstrate the conformational changes that occur in the binding of milk osteopontin (mOPN) and decorin (DCN), both referred to herein as "study proteins", to HA using FTIR spectroscopy. Along with these data we will measure the binding affinities of the "study proteins" to HA and collagen and also measure the kinetics of HA growth and replication in the presence of (a) collagen alone, (b) collagen coated with either "study protein", (c) collagen formed in the presence of each "study protein" separately, and (d) "study protein" alone. These studies will determine the optimal "study protein" to collagen ratio for use in aims 2 & 3. Aim 2): Develop surface plasmon resonance for studies of collagen-NCP-HA interactions using "study proteins". Aim 3): Develop a group of Fluorescence methods to study collagen-NCP- HA interaction and apply these to the "study proteins". The development of these methods will enable us to begin to validate our hypotheses, while providing both new techniques for the study of IDP interaction with collagen and HA and new insight into the mechanisms of collagen-mediated biomineralization. PUBLIC HEALTH RELEVANCE: The goal of this study is to develop analytical methods for investigating the importance of collagen and collagen-associated proteins in the process of mineralization. Successful completion of this exploratory study will provide information on many techniques, previously unused, in the field, generating data that has the potential to change our understanding of the biomineralization process. Furthermore, these studies can lead to the development of treatments for common bone and tooth diseases like osteomalacia and osteoporosis as well as rarer diseases such as osteogenesis- and dentinogenesis- imperfecta and skeletal dysplasias, along with new tissue engineering strategies.