Lamellar Body Biogenesis in Health and Disease

描述（由申请人提供）：Hermansky-Pudlak综合征（HPS）是一组罕见的遗传性疾病，其特征为皮肤白化病、过度出血和其他症状，反映了生成组织特异性溶酶体相关细胞器（LROs）所需的膜运输过程的缺陷。HPS 1、2或4型患者还会发生致死性肺纤维化，反映肺泡II型上皮细胞（AT2）的损伤。A2损伤与板层体（LBs）缺陷有关，板层体是富含脂质的LROs，表面活性剂在其中合成并包装以供分泌。尽管LBs对人类健康至关重要，但人们对其膜蛋白组成、内溶酶体的来源、导致其成熟的膜运输途径，以及HPS中有缺陷的蛋白复合物如何在这些途径中发挥作用知之甚少。在这个基本的细胞生物学提案中，我们将定义LB生物发生和成熟所需的膜运输过程，并将它们与HPS - AP-3和block -1， -2和-3中有缺陷的蛋白质复合物联系起来。我们将验证以下假设：(1)LB在AT2中的不同阶段成熟；(2)AP-3和BLOCs通过靶向LB特异性整体膜货物从常规内体到早期LB阶段促进LB成熟；(3)LB生物发生中涉及的运输途径对于lro生成细胞类型是“通用的”，并且独立于货物。这些假设直接源于黑素细胞LB生物发生与LRO成熟的比较，其中黑素小体通过AP-3和block依赖的黑色素生成酶从早期内体到非色素前体的递送，经过不同的形态阶段成熟。我们的研究结果将进一步加深我们对LBs作为关键LROs的理解，有助于开发新的HPS诊断工具和治疗方法，并为了解HPS患者肺纤维化的病因奠定基础。我们的具体目标是：1。测试不同密度的LB是否代表LB成熟的不同阶段，以及渐进式货物蛋白递送是否影响成熟LB的脂质谱。我们将分析成熟AT2中富集的、不同的LB组分的含量，并通过免疫荧光和免疫电镜验证我们的发现。2. 测试LB货物交付是否需要AP-3和/或AP-1。我们将测试假定货物的细胞质结构域与用于向黑素体分类的黑素体蛋白使用的细胞质接头的结合，并评估AT2中货物定位到lb时对这些接头的需求。3. 测试LRO成熟是否“普遍”由AT2和黑素细胞中hps相关蛋白复合物介导的货物递送途径定义。我们将测试异位表达的黑素小体和LB货物是否定位于AT2中的LB和黑素细胞中的黑素小体，以及两种细胞类型的定位是否同样需要BLOCs。

DESCRIPTION (provided by applicant): Hermansky-Pudlak syndrome (HPS) is a group of rare genetic diseases characterized by oculocutaneous albinism, excessive bleeding, and other symptoms that reflect defects in membrane transport processes required to generate tissue-specific lysosome-related organelles (LROs). Patients with HPS types 1, 2 or 4 additionally suffer from a lethal lung fibrosis, reflecting injury to alveolar type II epithelial cells (AT2). A2 injury correlates with defects in lamellar bodies (LBs), which are lipid-enriched LROs in which surfactant is synthesized and packaged for secretion. Despite the major importance of LBs to human health, little is known about their membrane protein composition, their derivation from endolysosomes, the membrane trafficking pathways that lead to their maturation, or how the protein complexes that are defective in HPS function within these pathways. In this basic cell biology proposal, we will define membrane transport processes required for LB biogenesis and maturation, and link them to the protein complexes that are defective in HPS - AP-3 and BLOC-1, -2 and -3. We will test the hypotheses that (1) LBs mature through distinct stages in AT2, (2) AP-3 and the BLOCs facilitate LB maturation by targeting LB-specific integral membrane cargoes from conventional endosomes to early LB stages, and (3) the trafficking pathways involved in LB biogenesis are "universal" to LRO-generating cell types and are independent of cargo. These hypotheses stem directly from comparisons of LB biogenesis to LRO maturation in melanocytes, in which melanosomes mature through distinct morphological stages through the AP-3- and BLOC-dependent delivery of melanogenic enzymes from early endosomes to non-pigmented precursors. Our results will further our understanding of LBs as critical LROs, aid in the development of new HPS diagnostic tools and therapies, and lay the foundation to understand the etiology of the lung fibrosis in HPS patients. Our specific aims are: 1. To test whether LBs of different densities represent distinct stages in LB maturation and if progressive cargo protein delivery shapes the lipid profile of maturing LBs. We will profile the contents of enriched, distinct LB fractions from maturing AT2, and validate our findings by immunofluorescence and immunoelectron microscopy. 2. To test whether LB cargo delivery requires AP-3 and/or AP-1. We will test for binding of the cytoplasmic domains of putative cargoes to cytoplasmic adaptors used by melanosomal proteins for sorting to melanosomes, and assess the requirement for these adaptors in cargo localization to LBs in AT2. 3. To test whether LRO maturation is "universally" defined by a cargo delivery pathway mediated by HPS-associated protein complexes in AT2 and melanocytes. We will test whether ectopically expressed melanosome and LB cargoes localize to LBs in AT2 and melanosomes in melanocytes, and whether localization in both cell types similarly requires BLOCs.