Endocytic Trafficking and Cell Signaling in Models of ARC Syndrome

关节紊乱-肾功能障碍-胆汁淤积综合征(ARC)是一种常染色体隐性遗传性疾病， 导致多个器官系统的发育缺陷。症状包括关节僵硬， 肾和肝脏发育缺陷、皮肤鳞片、血小板无颗粒、中枢神经系统异常和持续性 感染，通常导致出生后第一年内死亡。弧光是由一种基因突变引起的 两个基因，VPS33B或VPS16B/VIPAS39。这些基因编码的两种ARC连锁蛋白是 啤酒花复合体的六个亚基中的两个亚基的同源物，这对内体与 溶酶体。这表明ARC主要是内吞途径膜融合事件的缺陷。至 剖析将这些内吞转运缺陷与其生理后果联系起来的机制 通过突变Vps33B和Vps16B，建立ARC的果蝇模型。令人惊讶的是， 两个突变株系均为纯合、存活、可育，无明显发育缺陷。然而， 两种ARC综合征模型都显示了感染后的缺陷：无法清除细菌和严重的 尽管增加了抗微生物多肽的合成，但对感染过敏。这种超敏的感觉 ARC感染模型被先天免疫受体PGRP-LC功能丧失逆转，表明 致命性的原因是宿主先天免疫反应失控。重要的是，要求 调节先天性免疫信号的ARC蛋白在哺乳动物中是保守的，因为敲除 VPS33B在哺乳动物巨噬细胞中引起相同的缺陷，包括显著增强的PRO-1。 炎症反应。此外，我们发现，在苍蝇组织和 哺乳动物巨噬细胞滞留在内部小泡中，可能是升高的来源 我们观察到的免疫信号。综上所述，这些发现提出了一种可能性，即尽管 膜转运，ARC患者的许多症状可能反映了信号的夸大。这项建议 旨在研究VPS33B介导的内体成熟影响信号转导的机制 以及信号如何反过来改变内体贩运并引发寿命缩短。果蝇突变体 消除PGRP-LC受体下游IMD途径的不同元件将在 Vps33B和Vps16B零等位基因的背景。它们在缩短寿命、提高免疫信号和 ARC突变体中改变的内吞运输将被测试。这些基因实验将得到以下补充 阻断IMD通路中不同的激酶的药理学方法，目的是抑制 我们在ARC模型中观察到了超敏反应。总之，这些实验将提供更好的理解 导致ARC综合征至少部分症状的分子机制因此具有 有可能显著改变ARC患者的治疗选择思路。

Arthrogryposis-renal dysfunction-cholestasis syndrome (ARC) is an autosomal recessive inherited disorder that leads to developmental defects in multiple organs systems. Symptoms include stiffness of joints, developmental defects in kidney and liver, scaly skin, agranular platelets, CNS anomalies and persistent infections and typically result in fatality within the first year after birth. ARC is caused by mutations in one of two genes, VPS33B or VPS16B/VIPAS39. The two ARC-linked proteins encoded by these genes are homologs of two of the six subunits of the HOPS complex, which is critical for the fusion of endosomes with lysosomes. This suggests that ARC is primarily a defect in membrane fusion events of endocytic pathways. To dissect the mechanisms that link these endocytic trafficking defects with their physiological consequences in ARC syndrome, Drosophila models of ARC were generated by mutating Vps33B and Vps16B. Surprisingly, both mutant lines were homozygous viable and fertile without any obvious developmental defects. However, both models of ARC syndrome revealed defects after infections: an inability to clear bacteria and severe hypersensitivity to infections despite an increased synthesis of anti-microbial peptides. This hypersensitivity of ARC models to infections was reversed by loss of function of the innate immune receptor PGRP-LC, indicating that the reason of lethality is an out-of-control host innate immune response. Importantly, the requirement of ARC proteins for the regulation of innate immune signaling is conserved in mammals, as knockdown of VPS33B caused equivalent defects in mammalian macrophages, including dramatically enhanced pro- inflammatory responses. Furthermore, we find that activated immune receptors in both fly tissues and mammalian macrophages get stranded in internal vesicles and possibly serve as the source for the elevated immune signaling we observe. Together, these findings raise the possibility that despite a primary defect in membrane trafficking, many of the symptoms of ARC patients may reflect exaggerated signaling. This proposal aims to investigate the mechanisms by which VPS33B-mediated endosomal maturation influences signaling and how, in turn, signaling alters endosomal trafficking and triggers shortened life span. Drosophila mutants eliminating different elements of the IMD pathway downstream of the PGRP-LC receptor will be tested in the context of Vps33B and Vps16B null alleles. Their effect on reduced life span, elevated immune signaling and altered endocytic trafficking in ARC mutants will be tested. These genetic experiments will be supplemented by pharmacological approaches blocking different kinases in the IMD pathway with the goal to suppress the hypersensitivity we observe in ARC models. Together, these experiments will provide a better understanding of the molecular mechanisms causing at least some of the symptoms in ARC syndrome and thus have the potential to significantly change the thinking about treatment options for ARC patients.