Project 3: RAGE/DIAPH1 interactions and cellular stress

项目简介：Project 3 广泛的研究为多配体受体在晚期糖基化中的作用提供了强有力的支持 终末产物（endproducts，EDP）在糖尿病并发症的发病机制中的作用，例如加速的心血管疾病（CVD） 外周动脉疾病（peripheral arterial disease，PAD）及其常见并发症缺血再灌注（ischemia-reperfusion，IR）损伤。由于 由于它在各种疾病状态中的中心作用，因此认为拮抗TNF信号通路是一种重要的治疗方法。 有前途的治疗方法。我们的项目团队的研究确定DIAPH 1是一个细胞内的 的细胞内结构域（“尾”）之间的直接相互作用， 在细胞类型中，DIAPH 1的FH 1（同源性1）结构域与α-淀粉样蛋白相关， 使用体外和体内方法的依赖性病理学。在巨噬细胞、心肌细胞和 内皮细胞、内质网（ER）和线粒体应激，它们是RAGE依赖性的一部分 已经将DIAPH 1与线粒体融合蛋白2（MFN 2）的相互作用联系起来。初步结果显示， 实验室强烈支持小分子可以抑制RAGE-DIAPH 1-MFN 2相互作用的观点。的 关键的问题是这种抑制如何影响生理上重要的相互作用。这个目标 本项目旨在建立RAGE-DIAPH 1信号转导的生物物理学和结构生物学基础， 识别抑制模式。我们假设RAGE-DIAPH 1抑制的体外模式，如通过一个实验所揭示的， 生物物理和结构生物学工具的组合，如NMR光谱，将为 了解细胞内信号传导抑制的机制，因此可以有效地用于优化 RAGE-DIAPH 1-MFN 2相互作用的小分子抑制剂。NMR光谱将被用作主要的 由于CTR 4和DIAPH 1的FH 1结构域以及MFN 2的大片段是柔性的， 并且可能不适于结晶或电子低温显微镜检查。我们将探索 RAGE-DIAPH 1的新型小分子拮抗剂，并使用细胞内荧光技术， 表征RAGE-DIAPH 1-MFN 2相互作用。我们的项目将与项目1和项目2密切合作， 这种相互作用的机制基础，并确定新的和有效的治疗策略和药物， 糖尿病、CVD和PAD。

Project Summary: Project 3 Extensive research has provided strong support for the role of the multi-ligand receptor for advanced glycation endproducts (RAGE) in the pathogenesis of diabetes complications, such as accelerated cardiovascular (CVD) and peripheral arterial disease (PAD) and their common complication, ischemia-reperfusion (IR) injury. Due to its central role in various disease states, antagonism of RAGE signaling pathways is considered to be a promising therapeutic approach. Our Program Project team's research identified DIAPH1 as an intracellular effector of RAGE signaling and demonstrated a direct interaction between the intracellular domain (“tail”) of RAGE, or ctRAGE, and the FH1 (formin homology 1) domain of DIAPH1, in cell types that are relevant to RAGE- dependent pathologies using both in vitro and in vivo approaches. In macrophages, cardiomyocytes and endothelial cells, endoplasmic reticulum (ER) and mitochondrial stress, which are part of RAGE-dependent pathologies, have been linked to the interaction of DIAPH1 with Mitofusin2 (MFN2). Preliminary results from our labs strongly support the notion that small molecules can inhibit the RAGE-DIAPH1-MFN2 interaction. The critical question is how this inhibition affects physiologically important interactions of RAGE. The goal of this Project is to establish the biophysical and structural biology basis for RAGE-DIAPH1 signal transduction and identify modes of inhibition. We hypothesize that the in vitro mode of RAGE-DIAPH1 inhibition, as revealed by a combination of biophysical and structural biology tools, such as NMR spectroscopy, will provide a base for understanding cellular mechanisms of RAGE signaling inhibition and thus can be effectively used to optimize small molecule inhibitors of RAGE-DIAPH1-MFN2 interactions. NMR spectroscopy will be used as a main structural tool since ctRAGE and the FH1 domain of DIAPH1, as well as large segments of MFN2, are flexible and may not be amenable to crystallization or electron cryomicroscopy. We will probe the structural biology of novel small molecule antagonists of RAGE-DIAPH1 and use in-cell fluorescence techniques to meticulously characterize the RAGE-DIAPH1-MFN2 interaction. Our Project will work closely with Projects 1 and 2 to explore the mechanistic basis of this interaction and to identify novel and potent therapeutic strategies and agents for diabetes, CVD and PAD.