Development of Methods to Quantify Biological and Pathological Aging of Cartilage

量化软骨生物和病理老化的方法的发展

• 批准号: 8321475
• 负责人: Virginia Kraus
• 金额: $ 12.87万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321475
• 关键词: Age; Aging; Amides; Amino Acids; Architecture; Area; Arthritis; Asparagine; Aspartate; Biological; Biological Aging; Biological Markers; Cartilage; Cells; Chemicals; Collagen; Degenerative polyarthritis; Disease; Doctor of Philosophy; Epitopes; Extracellular Matrix; Forensic Medicine; Fossils; Frozen Sections; Future; Generations; Glutamates; Glutamine; Half-Life; Hip region structure; Human; Individual; Investigation; Joints; Label; Lasers; Life; Maps; Mass Spectrum Analysis; Measurement; Methods; Microscopy; Modeling; Modification; Molecular; Monitor; Natural regeneration; Osteoarthrosis Deformans; Post-Translational Protein Processing; Principal Investigator; Process; Proteins; Research; Sampling; Serum; Specimen; Structural Models; Structure; System; Time; Tissues; Treatment Efficacy; Urine; Variant; Virginia; age related; aged; aggrecan; base; cartilage matrix protein; chemical reaction; deamidation; extracellular; glycation; improved; in vivo; insight; method development; nitration; normal aging; novel; oxidation; pathological aging; protein degradation; protein structure; racemization; regenerative; repair enzyme; repaired; research study; time use; tissue regeneration; tool

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.

描述（由申请人提供）：衰老组织容易受到自发翻译后损害。当蛋白质内细胞内时，可以修复这些修饰或更换蛋白质。但是，当蛋白质是组织细胞外基质的成分时，无法修复蛋白质损伤并以时间依赖的方式积累。在这个项目中，我们计划专注于一种蛋白质损伤形式，即脱氨化，这被认为是限制蛋白质有用寿命的因素之一。蛋白质脱氨酸是一种化学反应，其中将酰胺基从天冬酰胺或谷氨酰胺中去除以形成天冬氨酸或谷氨酸。我们计划将脱氨酸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周转率的区域结构。这种方法依赖于通过质谱和表位的脱膜和非打干形式的蛋白质表位形式的定量，以确定其脱氨速率常数。从理论上讲，该方法将提供一种估计包含ASN或GLN的蛋白质表位的半衰期的手段。为了调查衰老系统的这一新方面，我们将作为研究范式，研究软骨的主要蛋白质之一的研究。根据质谱，我们已经确定了Aggrecan中的9个表位，它们在体内进行了自发的脱氨酸，并且将被用作该项目的基础。预计该项目将作为研究其他衰老系统的范式，并专门阐明了Aggrecan表位年龄的速率，并在正常和骨关节炎软骨组织中移交。我们假设蛋白质脱氨酸将提供估计软骨基质蛋白半衰期的第一种综合方法，并将为健康衰老和疾病中关节的分子理解做出重大贡献。从理论上讲，这里开发的信息和方法可以应用于任何组织，以监视改善组织再生策略的影响。