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

细胞衰老和波形蛋白糖化：对细胞骨架和细胞力学的影响

• 批准号: 9134672
• 负责人: ROBERT D GOLDMAN
• 金额: $ 21.14万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9134672
• 关键词: Age; Aging; Amino Acid Substitution; Antibodies; Biochemical; Biological Assay; Cell Aging; Cells; Complementary DNA; Computing Methodologies; Cytoskeletal Proteins; Cytoskeleton; Cytoskeleton Alteration; Dermal; Diabetes Mellitus; Embryo; Fibroblasts; Fluorescence Recovery After Photobleaching; Genomic Instability; Glucose; Glyoxal; Goals; Health; Human; Imagery; Immunoblotting; In Vitro; Individual; Intermediate Filament Proteins; Intermediate Filaments; Life; Lysine; Mass Spectrum Analysis; Measurement; Mechanics; Methods; Microspheres; Mitochondria; Mus; Organizational Change; Oryctolagus cuniculus; Oxidative Stress; Peptides; Phase; Phased Innovation Awards; Post-Translational Protein Processing; Preparation; Process; Property; Proteins; Reagent; Rheology; Role; Site; Skin; Structure; System; Telomere Shortening; Time; Tissues; Vimentin; age related; aged; base; cell age; glucose metabolism; glycation; insight; laser tweezer; normal aging; particle; polyclonal antibody; protein structure function; response; senescence; sugar; synthetic peptide; vector

 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.

 描述（由申请人提供）：衰老涉及细胞水平上随时间变化的损伤积累。虽然人们对端粒缩短、基因组不稳定、氧化应激和线粒体功能障碍了解很多，但实际上对细胞骨架的年龄依赖性变化一无所知。蛋白质结构和功能。与衰老相关的一个重要的蛋白质修饰是糖残基的累积、非酶促添加（即糖化）也是糖尿病的一个重要因素。最近发现波形蛋白（一种细胞骨架中间丝 (IF) 蛋白）是糖化的主要目标。该提案的首要目标是确定糖化如何改变波形蛋白 IF 的正常结构和功能。 VIF)。这对于理解衰老的细胞基础非常重要，因为众所周知，IF 是决定细胞和组织机械性能的主要因素。还知道细胞机械性能的变化伴随着衰老。 .在该项目的探索 (R21) 阶段，我们将检查人类皮肤成纤维细胞，以确定供体年龄的增长是否伴随着 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。此外，在 R21 阶段，我们将制备针对波形蛋白中糖化区域的特异性抗体，以及表达波形蛋白并用相关赖氨酸残基进行氨基酸取代的 cDNA。对于 R33 阶段，该阶段将重点确定糖化对波形蛋白 IF 动力学、亚细胞组织及其亚细胞组织的影响。 这将涉及使用最先进的活细胞测定；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。最后，我们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联系起来。所提出的研究将为年龄如何变化提供重要的新见解。依赖蛋白糖化改变波形蛋白细胞骨架的组织和表达，以及这些改变如何影响细胞的微机械特性。