Project 3: RAGE/DIAPH1 interactions and cellular stress

项目摘要：项目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(RAGE)在糖尿病并发症发病机制中的作用，如心血管加速(CVD) 外周动脉疾病(PAD)及其常见并发症缺血再灌注(IR)损伤。由于 它在各种疾病状态中的中心作用，RAGE信号通路的拮抗被认为是一种 前景看好的治疗方法。我们计划项目组的研究发现DIAPH1是一种细胞内 RAGE信号的效应者，并证明了细胞内结构域(“尾”)之间的直接作用 RAGE或ctRAGE，以及DIAPH1的FH1(福尔曼同源1)结构域，在与RAGE相关的细胞类型中- 使用体外和体内方法的依赖病理学。巨噬细胞、心肌细胞和 内皮细胞、内质网和线粒体应激，这些都是RAGE依赖的一部分 病理学与DIAPH1和Mitofusin2(Mfn2)的相互作用有关。来自我们的初步结果 实验室强烈支持这样的观点，即小分子可以抑制RAGE-DIAPH1-Mfn2的相互作用。这个 关键的问题是这种抑制如何影响RAGE的生理上重要的相互作用。这样做的目的是 项目是建立RAGE-DIAPH1信号转导的生物物理和结构生物学基础，并 确定抑制的模式。我们假设，RAGE-DIAPH1抑制的体外模式，如一个 生物物理和结构生物学工具的结合，如核磁共振光谱学，将为 了解RAGE信号抑制的细胞机制，从而可以有效地用于优化 RAGE-DIAPH1-Mfn2相互作用的小分子抑制剂。核磁共振光谱学将作为主要的 结构工具，因为ctRAGE和DIAPH1的FH1结构域，以及Mfn2的大片段是灵活的 并且可能不适用于结晶或电子冷冻显微镜。我们将探索其结构生物学 新型RAGE-DIAPH1小分子拮抗剂及其细胞内荧光技术 描述RAGE-DIAPH1-Mfn2的相互作用。我们的项目将与项目1和项目2密切合作，探索 这种相互作用的机制基础，并找出新的和有效的治疗策略和药物 糖尿病、心血管疾病和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.