INTERMEDIATE FILAMENTS AND THEIR ASSOCIATED PROTEINS

中间丝及其相关蛋白质

• 批准号: 3286445
• 负责人: MICHAEL William KLYMKOWSKY
• 金额: $ 8.59万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1988-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286445
• 关键词: affinity chromatography; antibody formation; autoradiography; cell cell interaction; chemical binding; cytoskeleton; electron microscopy; fluorescence microscopy; gel electrophoresis; immunoelectron microscopy; ion exchange chromatography; monoclonal antibody; morphology; phosphorylation

In each eukaryotic cell, there is a "cytoskeleton" of protein filaments that determine cell shape, morphology, movement and cytoplasmic organization. The most insoluble component of the cytoskeleton are intermediate filaments (IFs). Disruption of normal IF organization has been shown to occur in a number of neuropathies, including Alzheimer's disease. IFs are encoded by a complex, multigene family whose expression is closely linked to cellular differentiation. To study IF function, I will use the purified IF subunit protein vimentin adsorbed to nitrocellulose paper and purified vimentin filaments attached to chromatography resins to identify and isolate vimentin-binding proteins (VBPs). AntiVBP antibodies will be used to localize VBPs within the cell, and through intracellular injection studies, to study their function. Where antibodies against a VBP would not be useful for the study of function, we will subject vimentin to proteolytic or chemical treatment, in order to cleave it into its structural domains. I have used this approach to identify the phosphorylated portion of vimentin; antibodies directed against the "phospho-domain" will be used to study the role of IF phosphorylation within the cell. The binding of VBPs to vimentin domains will be studies, and antibodies directed against these domains will be generated and tested for their ability to block the interaction between vimentin and VBPs. These antibodies will then be used to study the role of vimentin/VBP interaction in vivo. I have identified proteins present in cell residues that may be related to IF; using nitrocellulose-immobilized antigen presentation, we are now generating antibodies against these proteins, which we will use to study their cellular distribution and function. Together these studies examine the function of a major cytoskeletal system, they should provide new information which may prove useful in understanding cellular behavior and its aberrations.

在每个真核细胞中，都有一个由蛋白质细丝组成的“细胞骨架” 决定细胞形状、形态、运动和细胞质 organization. 细胞骨架中最难溶解的成分是 中间丝（IF）。 国际单项体育联合会正常组织的中断， 已经被证明会发生在一些神经病中，包括阿尔茨海默氏症 疾病 IFs由一个复杂的多基因家族编码， 与细胞分化密切相关。 为了研究IF函数， 将使用纯化的IF亚基蛋白波形蛋白吸附到 硝酸纤维素纸和纯化的波形蛋白长丝附着在 用于鉴定和分离波形蛋白结合蛋白的层析树脂 （VBPs）。 抗VBP抗体将用于定位细胞内的VBP， 并通过细胞内注射研究，来研究它们的功能。 如果针对VBP的抗体对研究没有用处， 功能，我们将波形蛋白进行蛋白水解或化学处理， 以便将其切割成结构域。 我用过这种方法 以鉴定波形蛋白的磷酸化部分; 针对“磷酸结构域”将被用来研究IF的作用 细胞内的磷酸化。 VBP与波形蛋白结构域的结合 将进行研究，针对这些结构域的抗体将被 生成并测试了它们阻止 波形蛋白和VBP。 这些抗体将用于研究 波形蛋白/VBP相互作用。 我已经鉴定出了 可能与IF相关的细胞残留物;使用硝化纤维素固定化 抗原呈递，我们现在正在产生针对这些 蛋白质，我们将用它来研究它们的细胞分布， 功能 这些研究共同探讨了一个主要的功能， 细胞骨架系统，他们应该提供新的信息，可以证明 有助于理解细胞行为及其畸变。