Noncollagenous Protein Interaction in Biomineralization

描述（由申请人提供）：生理羟基磷灰石晶体（矿化）在胶原基组织（骨、牙本质、牙骨质、钙化软骨等）中沉积的机制是一个很难理解的复杂过程。我们相信，在HA期间， 胶原和非胶原蛋白（NCPs）调节（促进或抑制）HA的生长和增殖。许多NCP具有主要是无规卷曲的结构，因此被分类为“内分泌紊乱蛋白”（IDP）。境内流离失所者在脊椎动物和非脊椎动物的矿化系统中数量众多，据认为，他们灵活的结构使他们能够与各自的“伙伴”互动。原纤维胶原是矿化组织IDP或其亚基与之结合的“伙伴”之一， 显示绑定。这种反应的驱动力和这种结合发生的原因尚不清楚。矿化组织IDP的另一个“伙伴”是HA。几种NCP（例如富含小亮氨酸的蛋白聚糖（SLRPS）和其他糖蛋白）具有明确的非柔性结构，并且还与胶原蛋白和HA相互作用。我们的假设是，小的、柔性的IDP与胶原结合，然后与HA核或新生HA结合，并且因此变得更加有序。在IDP中的构象变化的能量被假定为比更有序的NCP与胶原和HA的结合能量更有利，由于在相互作用过程中对能量的需求较少。本R21申请的目的是开发和优化分析方法，以确定IDP的构象，以及调节HA生长所需的相互作用能量学，支持或反驳这一假设。有三个具体目标：目的1）：使用FTIR光谱证明在乳骨桥蛋白（mOPN）和核心蛋白聚糖（DCN）（在本文中均被称为“研究蛋白质”）与HA的结合中发生的构象变化。沿着这些数据，我们将测量“研究蛋白”与HA和胶原的结合亲和力，并且还测量在（a）单独的胶原、（B）涂覆有任一“研究蛋白”的胶原、（c）在每种“研究蛋白”单独存在下形成的胶原和（d）单独的“研究蛋白”存在下HA生长和复制的动力学。这些研究将确定用于目标2和3的最佳“研究蛋白”与胶原蛋白的比例。目的2）：开发表面等离子体共振用于使用“研究蛋白”研究胶原-NCP-HA相互作用。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”。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，并为胶原介导的生物矿化机制提供新的见解。 公共卫生相关性：本研究的目的是开发分析方法，研究胶原蛋白和胶原蛋白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技术的信息，以前未使用的，在该领域，产生的数据，有可能改变我们对生物矿化过程的理解。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，以及罕见的疾病，如骨生成和牙本质生成-骨发育不良和骨骼发育不良，沿着新的组织工程策略。

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.