Development of Methods to Quantify Biological and Pathological Aging of Cartilage

描述(申请人提供)：老化的组织容易受到自发性的翻译后损伤。当蛋白质在细胞内时，这些修饰可以被修复或替换。然而，当蛋白质是组织细胞外基质的组成部分时，蛋白质损伤无法修复，并以时间依赖的方式积累。在这个项目中，我们计划将重点放在一种形式的蛋白质损伤上，即被认为是限制蛋白质使用寿命的因素之一的脱酰胺化。蛋白质脱酰胺是从天冬酰胺或谷氨酰胺中去掉一个酰胺基团以形成天冬氨酸或谷氨酸的化学反应。我们计划使用与时间相关的脱酰胺化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周转的带状结构图。该方法依赖于通过质谱学和表位结构模型来量化蛋白质表位的去酰胺化和非去酰胺化形式，以确定其去酰胺化速率常数。从理论上讲，这种方法将提供一种估计含有天冬氨酸或谷氨酰胺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，我们将以软骨的主要蛋白质之一aggrecan的脱酰胺化为研究范式。通过质谱学方法，我们已经在聚集素中确定了9个在体内发生自发去酰胺化的表位，这些表位将作为本项目的基础。该项目有望成为研究其他衰老系统的范例，并具体阐明aggrecan表位在正常和骨关节炎软骨组织中的老化和翻转速度。我们假设，蛋白质脱酰胺法将提供第一个估计软骨基质蛋白质半衰期的全面方法，并将有助于从分子水平上理解关节在健康衰老和疾病中的作用。从理论上讲，这里开发的信息和方法可以应用于任何组织，以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.