Aging involves the time-dependent accumulation of damage at the cellular level. While much is known about telomere shortening, genomic instability, oxidative stress, and mitochondrial malfunction, virtually nothing is known about age-dependent changes in cytoskeletal protein structure and function. An important protein modification associated with aging is the cumulative, non-enzymatic addition of sugar residues to proteins (i.e. glycation). Glycation is also a significant factor in diabetes, due to elevated glucose levels. It has recently been discovered that vimentin, a cytoskeletal intermediate filament (IF) protein, is a major target of glycation. The overarching goal of this proposal is to determine how glycation alters the normal structure and function of vimentin IF (VIF).This is of great importance to understanding the cellular basis of aging, as it is well known that IF are major factors in determining the mechanical properties of cells and tissues. It is also known that changes in the mechanical properties of cells accompany aging. In the exploratory (R21) phase of this project we will examine human skin fibroblasts to determine if the increasing age of the donor is accompanied by organizational changes in VIF networks. Studies will also be carried out to determine how experimentally induced glycation alters VIF network organization. Also during the R21 phase we will prepare antibodies specific for the regions that are glycated in vimentin and cDNAs expressing vimentin with amino acid substitutions for the relevant lysine residues. These reagents will be used for the R33 phase which will focus on determining the effects of glycation on the dynamics of vimentin IF, on their subcellular organization and on their specific roles in altering the micromechanical properties of cells. This will involve using both state of the art live cell assays; and in vitro analyses of the effects of glycation on vimentin assembly, structure, and mechanical properties. Some of these studies will be carried out with a group of collaborators that we have enlisted for this project. Finally, attempts will be made to correlate the changes in vimentin glycation with age related parameters of tissues obtained from young and old mice. The studies proposed will provide important new insights into how age-dependent protein glycation alters the organization and expression of the vimentin cytoskeleton and how these alterations impact the micromechanical properties of the cell.
During normal aging, by-products of glucose metabolism are permanently attached to proteins in a process known as glycation. One of the major proteins modified in this manner is vimentin, a protein which assembles to form the cytoskeletal intermediate filaments that are important for maintaining the cell's mechanical properties. Understanding how this age-dependent glycation alters the structure and function of the vimentin intermediate filament system will advance our understanding of the mechanical changes that take place during cell and tissue aging.
