Noncollagenous Protein Interaction in Biomineralization

生物矿化中的非胶原蛋白相互作用

基本信息

批准号：
8450737
负责人：
ADELE L BOSKEY
金额：
$ 19.99万
依托单位：
HOSPITAL FOR SPECIAL SURGERY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8450737
关键词：
Affinity Belief Binding Bone Diseases Bone Tissue Budgets Calcified Capsid Proteins Cartilage Cell Nucleus Collagen Collagen Fibril Complex Data Dental Cementum Dentin Dentinogenesis Imperfecta Deposition Development Disease Fibrillar Collagen Fluorescence Free Energy Glycoproteins Goals Gold Grant Growth Hydroxyapatites Kinetics Lead Leucine Ligaments Measurement Measures Mediating Methods Milk Modality Molecular Conformation Osteogenesis Osteomalacia Osteoporosis Physiological Process Protein Binding Proteins Proteoglycan Rare Diseases Reaction Regulation Spectroscopy, Fourier Transform Infrared Structure Study Section Surface Surface Plasmon Resonance System Techniques Tendon structure Time Tissue Engineering Tissues Tooth Diseases Tooth structure Tryptophan Vertebrates analytical method base biomineralization bone common treatment decorin driving force flexibility insight method development mineralization osteopontin skeletal dysplasia therapy development

项目摘要

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.

描述（由申请人提供）：基于胶原蛋白组织（骨，牙本质，牙骨质，钙化软骨等）生理羟基磷灰石晶体（矿化）的沉积机理是一个不知所知的复杂过程。我们的信念是在HA期间（羟基磷灰石）形成胶原蛋白和非胶原蛋白（NCP）都调节HA的生长和增殖。许多NCP的结构主要是随机线圈，因此被归类为“本质上无序的蛋白质”（IDPS）。 IDP在脊椎动物和非脊椎动物的矿化系统中很丰富，人们认为它们的柔性结构使他们能够与各自的“伴侣”互动。 Fibrillar胶原蛋白是矿化组织IDP或其亚基的“合作伙伴”之一被证明是结合的。该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“伴侣”是HA。几个NCP（例如，富含亮氨酸的小蛋白聚糖（SLRP）和其他糖蛋白）具有良好的定义，非纤维化结构，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与胶原蛋白结合，然后与HA核或新生HA结合，并因此变得更加有序。由于在交互过程中对能量的需求较小，因此IDP中构象变化的能量比更有序的NCPS与胶原蛋白和HA的结合能量更有利。该R21应用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增长，支持或反驳该假设所需的相互作用能量学。有三个特定的目的：目标1）：证明牛奶骨桥蛋白（MOPN）和Decorin（DCN）的结合中发生的构象变化，均称此处称为“研究蛋白”，用于使用FTIR光谱法进行HA。与这些数据一起，我们将测量“研究蛋白”对HA和胶原蛋白的结合亲和力，并在单独存在（a）胶原蛋白的存在下测量HA生长和复制的动力学，（（b）胶原蛋白涂有“研究蛋白”，（C）在每个“研究蛋白质”中形成的胶原蛋白，并单独使用（c）单独研究蛋白质。这些研究将确定在目标2和3中使用的最佳“研究蛋白”与胶原蛋白比。目标2）：开发表面等离子体共振，用于使用“研究蛋白”进行胶原-NCP-HA相互作用的研究。目标3）：开发一组荧光方法来研究胶原-NCP- HA相互作用，并将其应用于“研究蛋白”。这些方法的开发将使我们能够开始验证我们的假设，同时为IDP与胶原蛋白和HA的相互作用提供了两种新技术，以及对胶原蛋白介导的生物矿化机制的新见解。