Cell aging and vimentin glycation: Effects on the cytoskeleton and cell mechanics

 描述（由申请人提供）：衰老涉及细胞水平损伤的时间依赖性累积。虽然我们对端粒缩短、基因组不稳定性、氧化应激和线粒体功能障碍了解很多，但对细胞骨架蛋白结构和功能的年龄依赖性变化几乎一无所知。与衰老相关的一种重要蛋白质修饰是糖残基向蛋白质的累积性非酶促添加（即糖化）。由于葡萄糖水平升高，糖化也是糖尿病的重要因素。最近发现，波形蛋白，一种细胞骨架中间丝（IF）蛋白，是糖基化的主要靶点。本研究的主要目的是确定糖基化如何改变波形蛋白IF（VIF）的正常结构和功能，这对于理解衰老的细胞基础具有重要意义，因为众所周知，IF是决定细胞和组织力学性质的主要因素。还已知细胞的机械性质的变化伴随着老化。在本项目的探索（R21）阶段，我们将检查人皮肤成纤维细胞，以确定供体年龄的增加是否伴随着VIF网络的组织变化。还将进行研究，以确定实验诱导的糖化如何改变VIF网络组织。同样在R21阶段，我们将制备特异于波形蛋白中糖化区域的抗体和表达波形蛋白的cDNA，其中相关赖氨酸残基被氨基酸取代。这些试剂将用于R33阶段，该阶段将集中于确定糖化对波形蛋白IF的动力学、对其亚细胞组织以及对其亚细胞结构的影响。 在改变细胞的微观力学特性中的特殊作用。这将涉及使用最先进的活细胞测定法;以及糖化对波形蛋白组装、结构和机械特性的影响的体外分析。其中一些研究将与我们为这个项目招募的一组合作者一起进行。最后，将尝试将波形蛋白糖化的变化与从年轻和年老小鼠获得的组织的年龄相关参数相关联。提出的研究将提供重要的新的见解，如何年龄依赖性蛋白糖化改变组织和表达的波形蛋白细胞骨架，以及这些改变如何影响细胞的微观力学性能。

 DESCRIPTION (provided by applicant): 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.