Development of Methods to Quantify Biological and Pathological Aging of Cartilage

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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. PUBLIC HEALTH RELEVANCE: As proteins age they can undergo spontaneous chemical modifications that can alter their structure and function. Cells contain processes for repairing much of this damage but these repair enzymes are not present outside cells so proteins, especially long-lived proteins that make up the tissues around cells, can accumulate damage over time. Although these modifications may be deleterious, they can be put to good use as tags of a protein's age - the older the protein, the greater the number of modifications accumulated. We will study one form of protein damage, called deamidation, to determine how quickly proteins regenerate in cartilage and map the areas of slow and fast regeneration in normal ageing versus and arthritic tissue. The information and method developed here could theoretically be applied to any tissue to gain insights for improving tissue regeneration of all kinds.