Development of Methods to Quantify Biological and Pathological Aging of Cartilage

描述（由申请人提供）：老化组织易受自发翻译后损伤的影响。当蛋白质在细胞内时，这些修饰可以被修复或蛋白质被替换。然而，当蛋白质是组织细胞外基质的组成部分时，蛋白质损伤不能修复，并以时间依赖性的方式积累。在这个项目中，我们计划关注一种形式的蛋白质损伤，脱酰胺，这被认为是限制蛋白质使用寿命的因素之一。蛋白质脱酰胺是一种化学反应，从天冬酰胺或谷氨酰胺中去除酰胺基团，形成天冬氨酸或谷氨酸。我们计划利用脱酰胺的时间依赖性作为确定蛋白质表位半衰期的新方法的基础，并在空间上绘制组织周转的区域结构。该方法依赖于通过质谱和表位结构建模对蛋白质表位的脱酰胺和非脱酰胺形式进行定量，以确定其脱酰胺速率常数。理论上，这种方法将提供一种估计含有Asn或Gln的无限数量蛋白质表位的半衰期的方法。为了研究衰老系统的这一新方面，我们将以研究为范例，研究软骨的主要蛋白质之一的脱酰胺，聚集蛋白。通过质谱分析，我们已经确定了聚集蛋白中9个在体内发生自发脱酰胺的表位，这将作为本项目的基础。该项目有望成为研究其他老化系统的范例，并具体阐明正常和骨关节炎软骨组织中聚集蛋白表位老化和翻转的速率。我们假设蛋白质脱酰胺将提供第一种评估软骨基质蛋白质半衰期的综合方法，并将对关节在健康衰老和疾病中的分子理解做出重大贡献。在此开发的信息和方法理论上可以应用于任何组织，以监测改善组织再生策略的效果。

DESCRIPTION (provided by applicant): Ageing tissues are susceptible to spontaneous post-translational damage. When a protein is intracellular, these modifications can be repaired or the protein replaced. However, when a protein is a component of an extracellular matrix of a tissue, protein damage cannot be repaired and accumulates in a time-dependent manner. In this project we plan to focus on one form of protein damage, deamidation that is believed to be one of the factors that limit the useful lifetime of proteins. Protein deamidation is a chemical reaction in which an amide group is removed from asparagine or glutamine to form aspartate or glutamate. We plan to use the time- dependent occurrence of deamidation as the basis for a new method of determining the half-lives of protein epitopes and to spatially map the zonal architecture of tissue turnover. This method relies on the quantification of the deamidated and non-deamidated forms of a protein epitope by mass spectroscopy and structural modeling of the epitope to determine its deamidation rate constant. This method would theoretically provide a means of estimating half-lives of an unlimited number of protein epitopes containing Asn or Gln. To investigate this novel aspect of ageing systems, we will take as a paradigm for study, investigation of deamidation of one of the major proteins of cartilage, aggrecan. By mass spectroscopy, we have already identified 9 epitopes within aggrecan that undergo spontaneous deamidation in vivo and which will be used as the basis for this project. This project is expected to serve as a paradigm for the study of other ageing systems and to specifically elucidate the rates at which aggrecan epitopes age and are turned over in normal and osteoarthritic cartilage tissue. We hypothesize that protein deamidation will provide the first comprehensive method of estimating cartilage matrix protein half-lives, and will contribute substantially to a molecular understanding of the joint in healthful aging and disease. The information and method developed here could theoretically be applied to any tissue for monitoring the effects of strategies for improving tissue regeneration.