Development of Methods to Quantify Biological and Pathological Aging of Cartilage

描述（由申请方提供）：老化组织易发生自发性翻译后损伤。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，当蛋白质是组织的细胞外基质的组分时，蛋白质损伤不能被修复并且以时间依赖性方式累积。在这个项目中，我们计划专注于蛋白质损伤的一种形式，脱酰胺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，其中酰胺基团从天冬酰胺或谷氨酰胺中除去以形成天冬氨酸或谷氨酸。我们计划使用脱酰胺的时间依赖性发生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转换的区域结构。该方法依赖于通过质谱和表位的结构建模来定量蛋白质表位的脱酰胺和非脱酰胺形式，以确定其脱酰胺速率常数。该方法在理论上提供了一种估计无限数量的含有Asn或Gln的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，我们将以软骨的主要蛋白质之一聚集蛋白聚糖的脱酰胺作用为研究范例。通过质谱分析，我们已经鉴定了聚集蛋白聚糖内的9个表位，这些表位在体内进行自发脱酰胺，并将用作本项目的基础。该项目有望成为其他衰老系统研究的范例，并具体阐明聚集蛋白聚糖表位在正常和骨关节炎软骨组织中老化和翻转的速率。我们假设，蛋白脱酰胺将提供第一个全面的方法估计软骨基质蛋白半衰期，并将大大有助于在健康的老龄化和疾病的关节的分子理解。这里开发的信息和方法理论上可以应用于任何组织，用于监测改善组织再生的策略的效果。

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.