REGULATION OF CELL PROLIFERATION

细胞增殖的调节

• 批准号: 2910585
• 负责人: MICHAEL J. GETZ
• 金额: $ 28.25万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910585
• 关键词: DNA binding protein; DNA footprinting; actins; atherosclerotic plaque; cell proliferation; developmental genetics; genetic enhancer element; genetic library; genetic transcription; human tissue; immunologic assay /test; protein biosynthesis; protein protein interaction; protein structure function; vascular smooth muscle

This research seeks to establish the molecular basis for the regulation of the vascular smooth muscle (VSM) alpha-actin gene during fibroproliferative responses characteristic of vascular injury and disease. Previous studies in our laboratories point to a model in which transcriptional repression of the mouse VSM alpha-actin gene in fibroblasts and undifferentiated myoblasts results from the interaction of sequence-specific single-stranded DNA (ssDNA) binding proteins with opposite strands of an essential TEF-1 enhancer element. Binding site screening of cDNA expression libraries has now resulted in the cloning of these proteins and their subsequent identification as MSY1, a member of the Y-box family of nucleic acid binding proteins, Puralpha, a retinoblastoma (Rb)-binding protein initially identified in Hela cells, and Purbeta, a related protein of unknown properties. Preliminary studies of human atherosclerotic coronary arteries suggest a functional role for these proteins in cardiovascular disease. Experiments proposed in this application will test a central hypothesis that transcriptional repression results from a ssDNA-binding protein-dependent disruption of base pairing within the TEF-1 enhancer and that regulation of this process depends, in part, upon highly specific protein-protein interactions. This model will be tested by delineating functional domains within the ssDNA-binding proteins essential for protein-protein interactions, by determining the effects of mutations within these domains on the ability of these proteins to modulate enhancer topology and function, and by in vivo footprinting of enhancer structure in VSM alpha-actin expressing cell types. Lastly, the involvement of these proteins in human atherogenesis will be studied using a repertoire of anti-Pur and anti-MSYl peptide-specific antibodies. These studies will extend our understanding of a novel mechanism for the regulation of smooth muscle actin synthesis which may be important to the pathogenesis of coronary artery disease.

本研究旨在为血管损伤和疾病的纤维增殖性反应中血管平滑肌(VSM)α-肌动蛋白基因的调控奠定分子基础。我们实验室以前的研究指出了一个模型，在该模型中，小鼠VSMα-肌动蛋白基因在成纤维细胞和未分化成肌细胞中的转录抑制是由序列特异性单链DNA(SsDNA)结合蛋白与基本TEF-1增强子元件的相反链相互作用的结果。通过对表达文库结合位点的筛选，克隆了这些蛋白，并将其鉴定为MSY1、Purpha和Purbeta。MSY1是Y-box核酸结合蛋白家族的成员，Purpha是最初在Hela细胞中发现的视网膜母细胞瘤(RB)结合蛋白，Purbeta是一种未知性质的相关蛋白。对人类动脉粥样硬化冠状动脉的初步研究表明，这些蛋白在心血管疾病中具有功能作用。本申请中提出的实验将检验一个中心假设，即转录抑制是由于SSDNA结合蛋白依赖于TEF-1增强子内碱基配对的破坏而导致的，并且这一过程的调节部分依赖于高度特异的蛋白质-蛋白质相互作用。这一模型将通过描绘对蛋白质-蛋白质相互作用至关重要的单链DNA结合蛋白中的功能结构域，通过确定这些结构域中的突变对这些蛋白质调节增强子拓扑和功能的能力的影响，以及通过在体内足迹VSMα-肌动蛋白表达细胞类型的增强子结构来测试。最后，将使用一系列抗PUR和抗MSY1多肽特异性抗体来研究这些蛋白在人类动脉粥样硬化形成中的作用。这些研究将扩大我们对调节平滑肌肌动蛋白合成的新机制的理解，这可能在冠状动脉疾病的发病机制中发挥重要作用。