Endocytic Trafficking and Cell Signaling in Models of ARC Syndrome

ARC 综合征模型中的内吞转运和细胞信号转导

• 批准号: 9895825
• 负责人: Helmut J Kramer
• 金额: $ 33.21万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895825
• 关键词: Affect; Alleles; Amino Acids; Arthrogryposis; Bacteria; Basic Science; Biological Assay; Birth; Blood Platelets; Cells; Cessation of life; Cholestasis; Clinical Sciences; Complex; Data; Defect; Development; Disease; Dissection; Drosophila genus; Elements; Endosomes; Event; Genes; Genetic; Goals; Hereditary Disease; Hypersensitivity; Immune signaling; Immunologic Receptors; Infection; Inflammatory; Inflammatory Response; Innate Immune Response; Interleukin-6; KRAS2 gene; Kidney; Ligands; Link; Liver; Longevity; Lysosomes; Mammalian Cell; Mammals; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Mutate; Mutation; Pathway interactions; Patients; Pattern recognition receptor; Perception; Phagosomes; Pharmacologic Substance; Pharmacological Treatment; Pharmacology; Phenotype; Phosphotransferases; Physiological; Proteins; Regulation; Role; Sepsis; Signal Pathway; Signal Transduction; Skin; Source; Symptoms; Syndrome; TLR4 gene; TNF gene; Testing; Thinking; Tissues; Vesicle; Virus; antimicrobial peptide; base; body system; chronic infection; cytokine; experimental study; fly; immunoregulation; infancy; innate immune function; joint stiffness; kidney dysfunction; knock-down; link protein; loss of function; lysosomal proteins; macrophage; mutant; neoplastic cell; paralogous gene; premature; protein function; rare genetic disorder; receptor; response; stressor; tool; trafficking; uptake

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.

关节弯曲-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， 导致多个器官系统发育缺陷。症状包括关节僵硬、 肾脏和肝脏发育缺陷、鳞状皮肤、无颗粒血小板、中枢神经系统异常和持续性 感染，通常会在出生后一年内导致死亡。 ARC 是由其中之一的突变引起的 两个基因，VPS33B 或 VPS16B/VIPAS39。这些基因编码的两个 ARC 连接蛋白是 HOPS 复合物六个亚基中两个亚基的同源物，这对于内体与内含体的融合至关重要 溶酶体。这表明 ARC 主要是内吞途径膜融合事件的缺陷。到 剖析将这些内吞运输缺陷与其生理后果联系起来的机制 ARC综合征，ARC的果蝇模型是通过突变Vps33B和Vps16B产生的。出奇， 两个突变系都是纯合的，可存活且可育，没有任何明显的发育缺陷。然而， 两种 ARC 综合征模型在感染后都显示出缺陷：无法清除细菌和严重的感染 尽管抗微生物肽的合成增加，但仍对感染过敏。这种超敏反应 ARC 感染模型因先天免疫受体 PGRP-LC 功能丧失而逆转，表明 致命的原因是失控的宿主先天免疫反应。重要的是，要求 用于调节先天免疫信号的 ARC 蛋白在哺乳动物中是保守的，因为 VPS33B 在哺乳动物巨噬细胞中引起相同的缺陷，包括显着增强的亲 炎症反应。此外，我们发现果蝇组织和 哺乳动物巨噬细胞滞留在内部囊泡中，可能是升高的来源 我们观察到的免疫信号。总之，这些发现提出了一种可能性，尽管存在主要缺陷 膜运输，ARC患者的许多症状可能反映了夸大的信号传导。这个提议 旨在研究 VPS33B 介导的内体成熟影响信号传导的机制 以及信号如何反过来改变内体运输并引发寿命缩短。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 无效等位基因的背景。它们会缩短寿命、增强免疫信号和 将测试 ARC 突变体中改变的内吞运输。这些基因实验将得到补充 阻断 IMD 通路中不同激酶的药理学方法，目的是抑制 我们在 ARC 模型中观察到的超敏反应。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 可能会显着改变 ARC 患者治疗选择的想法。