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

项目总结 包括心肌梗死在内的缺血性并发症是 补体介导的疾病患者，包括移植动脉硬化(TA)和自身免疫 结缔组织病。补体是一组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，会导致内皮细胞(EC)功能障碍。在被AB激活后， 补体蛋白自组装成跨膜、孔状结构，称为膜攻击 复合体(MAC)。将MAC插入EC表面，在那里它们启动炎症信号而不引起细胞 死亡。了解MAC对EC的炎症信号可能导致新的治疗方法阻断 补体介导性疾病的缺血性后遗症。 为了研究这些过程，我使用了同种异体抗原致敏的群体反应性抗体(Pra)血清。 移植候选者在体外和体内诱导抗体介导的人内皮细胞MAC组装。在这个过程中， 我发现了一种新的MAC效应途径，非典型的NF-κB，其特征是快速和戏剧性的 升高的核因子-κB诱导激酶(NIK)。NIK诱导炎性基因，增强EC 激活CD4+T细胞，并与TA有关，TA是一种血管闭塞状态，发生在 2只人源化小鼠心脏移植模型。 为了了解MAC如何诱导NiK，我进行了全基因组siRNA筛选，发现了一种 新的，基于内体的机制，通过MAC稳定Nik。Mac被迅速内化，并 转移到Rab5+囊泡形成一个MAC+Rab5+隔室。以依赖于Rab5的方式， Mac+Rab5+隔室依次招募激活的Akt(Pakt)和Nik。在这里，Rab5活动是 需要将激活的Akt募集到MAC+Rab5+内吞体内，以诱导Nik稳定。定义机制(S) 通过Rab5募集pakt，我对FACS分选的MAC+Rab5+内体进行了蛋白质组学分析 并定义了一个Rab5-ZFYVE21-SMURF2信号轴，导致NIK稳定和EC功能障碍。 我假设MAC激活了Rab5-ZFYVE21-SMURF2轴，通过以下方式引起EC功能障碍 让尼克稳定下来。在目标1中，我将定义三个调节Rab5-ZFYVE21-SMURF2轴的机制(S)。这就做 研究1)脂质相关激酶和磷酸酶如何介导MAC+、Rab5+的脂质重塑 2)MAPK(S)如何调节Rab5的活性，以及3)pakt如何被招募到 Mac+Rab5+内涵体。在AIM2中，我将研究调节Rab5-ZFYVE21-的部分分子的相关性 SMURF2信号在患者组织、体内EC信号的人源化模型和人源化小鼠中的信号 TA的模型。这项提议的长期目标是更好地理解MAC诱导的炎症信号。 通过这样做，可能会出现改善MAC诱导的缺血并发症的可用药靶点。

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.