Proteomics and crystal structure analysis to elucidate vascular pathological mechanisms

本项目旨在通过蛋白质组学方法阐明血管疾病病理生理学中的重要因素。晶体结构分析也用于了解作用机制的理化基础。这些研究的最终目的是开发新的治疗和诊断方法。KLF 5是应激反应中调节心血管重构过程的因子，目前被认为是最重要的心血管病理因子之一。为了了解其调节，通过蛋白质组学方法鉴定相互作用蛋白，其中包括阻遏物SET。分析了SET在KLF 5上的功能。SET的晶体结构也被解决，这将允许“针点”药物设计。我们还表明，KLF 5是乙酰化的，而p300是乙酰化酶，而SET抑制这种乙酰化。p300和SET因子对KLF 5的正性和负性调节与相互作用和乙酰化作用有关。去乙酰化酶HDAC 1进一步负调控KLF 5，并与p300进行物理竞争。KLF 5还具有抗凋亡活性，通过分析发现KLF 5与PARP-1的促凋亡片段相互作用，并且这种相互作用也受到KLF 5乙酰化修饰的调控。通过我们的研究，我们发现了相互作用和修饰如何在功能上影响KLF 5的作用，特别是表观遗传调控。更好地理解血管病理学中的分子作用将有助于开发新的治疗方法。

This project was undertaken to elucidate factors important in vascular disease pathophysiology through proteomic methods. Crystal structure analysis was also used to understand the physiochemical basis of the mechanisms of action. These studies were done with the ultimate objective to make possible the development of new therapies and diagnostic methods.KLF5 is a factor which regulates the cardiovascular remodeling process in response to stress, and is presently considered one of the most important cardiovascular pathological factors. To understand its regulation, interacting proteins were identified by proteomic methods, which included the repressor SET. The functions of SET on KLF5 were analyzed. The crystal structure of SET was also solved which will allow for 'pin-point' drug design.We also showed that KLF5 is acetylated, and for p300 to be the acetylase while SET inhibits this acetylation. Positive and negative regulation of KLF5 by respective p300 and SET factors was coupled to interaction and acetylation. The deacetylase HDAC1 further negatively regulated KLF5, and to physically compete with p300. KLF5 was also shown to possess anti-apoptotic activity, through analysis of which, we showed that KLF5 interacts with a pro-apoptotic fragment of PARP-1, and for this interaction to also be regulated by acetylation of KLF5.Through our studies, we have shown how interaction and modifications functionally affect the actions of KLF5, with a particular focus on epigenetic regulation. This better understanding of molecular actions in vascular pathology will allow for development of new therapies.