Noncollagenous Protein Interaction in Biomineralization

生物矿化中的非胶原蛋白相互作用

• 批准号: 8317807
• 负责人: ADELE L BOSKEY
• 金额: $ 27.1万
• 依托单位: HOSPITAL FOR SPECIAL SURGERY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8317807
• 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

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.

描述（由申请人提供）：胶原蛋白组织（骨、牙本质、牙骨质、钙化软骨等）中生理性羟基磷灰石晶体沉积（矿化）的机制是一个知之甚少的复杂过程。我们相信，在医管局期间 （羟基磷灰石）的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，因此被归类为“本质无序蛋白质”(IDP)。 IDPs 富含脊椎动物和非脊椎动物的矿化系统，人们认为它们灵活的结构使它们能够与各自的“伙伴”相互作用。纤维状胶原蛋白是矿化组织 IDP 或其亚基的“伙伴”之一 已被证明可以绑定。该反应的驱动力以及发生这种结合的原因尚不清楚。矿化组织 IDP 的另一个“伙伴”是 HA。几种 NCP（例如富含亮氨酸的小蛋白聚糖 (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，小而灵活的 IDP 与胶原蛋白结合，然后与 HA 核或新生 HA 结合，结果变得更加有序。 IDP 中构象变化的能量被认为比更有序的 NCP 与胶原蛋白和 HA 的结合能量更有利，因为相互作用过程中对能量的需求较少。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 生长所需的相互作用能量，支持或反驳这一假设。存在三个具体目标： 目标 1)：使用 FTIR 光谱法展示乳骨桥蛋白 (mOPN) 和核心蛋白聚糖 (DCN)（在本文中均称为“研究蛋白”）与 HA 结合时发生的构象变化。除了这些数据之外，我们还将测量“研究蛋白”与 HA 和胶原蛋白的结合亲和力，并测量在 (a) 单独的胶原蛋白、(b) 涂有“研究蛋白”的胶原蛋白存在的情况下 HA 生长和复制的动力学。 ”，（c）在每种“研究蛋白质”单独存在的情况下形成胶原蛋白，以及（d）单独的“研究蛋白质”。这些研究将确定用于目标 2 和 3 的最佳“研究蛋白”与胶原蛋白比例。目标 2)：开发表面等离振子共振，以使用“研究蛋白”研究胶原蛋白-NCP-HA 相互作用。目标 3)：开发一组荧光方法来研究胶原蛋白-NCP-HA 相互作用，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 与胶原蛋白和 HA 相互作用提供新技术，并为胶原蛋白介导的生物矿化机制提供新的见解。 公共健康相关性：本研究的目的是开发分析方法来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有关许多以前在该领域未使用过的技术的信息，生成有可能改变我们对生物矿化过程的理解的数据。此外，这些研究可以促进骨软化症和骨质疏松症等常见骨和牙齿疾病以及成骨和牙本质发育不全和骨骼发育不良等罕见疾病的治疗方法的开发，以及新的组织工程策略。