Complement-Induced Endothelial Cell Activation by a Novel Rab5-ZFYVE21-SMURF2 Signaling Axis

项目摘要 包括心肌梗死在内的缺血性并发症是老年人死亡的主要原因。 患有补体介导疾病的患者，包括移植动脉硬化（TA）和自身免疫性 结缔组织病补体是参与宿主防御的九种循环免疫蛋白 当在疾病条件下被激活时，引起内皮细胞（EC）功能障碍。一旦被Ab激活， 补体蛋白自组装成跨膜的孔状结构，称为膜攻击 复合物（MAC）。MAC插入EC表面，在那里它们启动炎症信号而不引起细胞凋亡。 死亡了解MAC驱动的炎症信号到EC可能会导致新的治疗方法， 补体介导疾病的缺血性后遗症。 为了研究这些过程，我使用了来自同种抗原致敏的 移植候选物在体外和体内诱导Ab介导的MAC组装在人EC上。在这个过程中， 我发现了一种新的MAC效应子途径，非经典NF-κB，其特征是快速和显著的 增加NF-κB诱导激酶（NIK）。NIK诱导炎性基因表达，增强EC对炎症反应的能力， 激活CD 4 + T细胞，并与TA有关，TA是一种血管闭塞性疾病， 心脏移植，在2个人源化小鼠模型中。 为了了解MAC是如何诱导NIK的，我进行了全基因组siRNA筛选，发现了一个新的靶基因。 新的，基于内体的机制，MAC稳定NIK。MAC被迅速内化， 转移至Rab 5+囊泡以形成MAC+ Rab 5+区室。以Rab 5依赖的方式， MAC+ Rab 5+隔室依次募集活化的Akt（pAkt）和NIK。在这里，Rab 5活性是 需要将活化的Akt募集到MAC+ Rab 5+内体以诱导NIK稳定化。定义机制 Rab 5可以募集pAkt，我对FACS分选的MAC+ Rab 5+内体进行了蛋白质组学分析， 并定义了Rab 5-ZFYVE 21-SMURF 2信号轴，其导致NIK稳定和EC功能障碍。 我假设MAC激活Rab 5-ZFYVE 21-SMURF 2轴，通过以下方式增强EC功能障碍： 稳定NIK在目标1中，我将定义调节Rab 5-ZFYVE 21-SMURF 2轴的三种机制。我会 检查1）脂质相关激酶和磷酸酶如何介导MAC+ Rab 5+的脂质重塑 2）MAPK如何调节Rab 5活性，以及3）pAkt如何被募集以 MAC+ Rab 5+内体。在Aim 2中，我将检查调节Rab 5-ZFYVE 21-E2的选择分子的相关性。 患者组织中、体内EC信号传导的人源化模型中和人源化小鼠中的SMURF 2信号传导 TA模型该提案的长期目标是更好地了解MAC诱导的炎症信号。 通过这样做，可以出现改善MAC诱导的缺血性并发症的药物靶点。

PROJECT SUMMARY Ischemic complications including myocardial infarction are the leading causes of mortality among patients with complement-mediated diseases including transplant arteriosclerosis (TA) and autoimmune connective tissue disease. Complement are a set of nine circulating immune proteins involved in host defense that, when activated under disease conditions, cause endothelial cell (EC) dysfunction. Upon activation by Ab, complement proteins self-assemble into transmembranous, pore-like structures called membrane attack complexes (MAC). MAC insert into EC surfaces where they initiate inflammatory signaling without causing cell death. Understanding the inflammatory signals driven by MAC to EC may lead to new therapies blocking the ischemic sequelae of complement-mediated diseases. To study these processes, I used panel reactive antibody (PRA) sera from alloantigen-sensitized transplant candidates to induce Ab-mediated MAC assembly on human EC in vitro and in vivo. In this process, I discovered a novel MAC effector pathway, non-canonical NF-κB, characterized by rapid and dramatically increased NF-κB inducing kinase (NIK). NIK induced inflammatory genes, enhanced the ability of EC to activate CD4+ T cells, and was implicated in TA, a vaso-occlusive condition occurring as a complication of heart transplantation, in 2 humanized mouse models. To understand how MAC could induce NIK, I performed a genome-wide siRNA screen and uncovered a novel, endosome-based mechanism by which MAC stabilized NIK. MAC was rapidly internalized and transferred to Rab5+ vesicles to form a MAC+Rab5+ compartment. In a Rab5-dependent manner, the MAC+Rab5+ compartment sequentially recruited activated Akt (pAkt) and NIK. Here, Rab5 activity was required to recruit activated Akt to MAC+Rab5+ endosome to induce NIK stabilization. To define mechanism(s) by which Rab5 could recruit pAkt, I performed proteomic analyses of FACS-sorted MAC+Rab5+ endosomes and defined a Rab5-ZFYVE21-SMURF2 signaling axis causing NIK stabilization and EC dysfunction. I hypothesize that MAC activates a Rab5-ZFYVE21-SMURF2 axis that elicits EC dysfunction by stabilizing NIK. In Aim 1, I will define three mechanism(s) regulating the Rab5-ZFYVE21-SMURF2 axis. I will examine 1) how lipid-associated kinases and phosphatases mediate lipid remodeling of MAC+Rab5+ endosomes to stabilize NIK, 2) how MAPK(s) regulate Rab5 activity, and 3) how pAkt is recruited to MAC+Rab5+ endosomes. In Aim2 I will examine the relevance of select molecules regulating Rab5-ZFYVE21- SMURF2 signaling in patient tissues, in humanized models of EC signaling in vivo, and in a humanized mouse model of TA. The long-term aims of this proposal are to better understand MAC-induced inflammatory signals. By doing this, druggable targets ameliorating MAC-induced ischemic complications may emerge.