Lamellar Body Biogenesis in Health and Disease

描述(申请人提供)：Hermansky-Pudlak综合征(HPS)是一组罕见的遗传性疾病，以眼皮肤白化、大量出血和其他症状为特征，这些症状反映了产生组织特异性溶酶体相关细胞器(LRO)所需的膜运输过程中的缺陷。HPS 1型、2型或4型患者还伴有致死性肺纤维化，这反映了肺泡II型上皮细胞(AT2)的损伤。A2损伤与板层小体缺陷有关，板层小体是一种富含脂质的LRO，表面活性物质在其中合成和包装以供分泌。尽管LBS对人类健康具有重要意义，但人们对LBS的膜蛋白组成、来自内溶体的来源、导致其成熟的膜转运途径以及HPS缺陷的蛋白质复合体在这些途径中的作用知之甚少。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转运过程，并将它们与HPS-AP-3和BLOC-1、-2和-3中存在缺陷的蛋白质复合体联系起来。我们将检验以下假设：(1)LB在AT2中通过不同的阶段成熟，(2)AP-3和这些区块通过靶向从传统内体到早期LB阶段的LB特异性整膜货物来促进LB成熟，以及(3)参与LB生物发生的运输途径对LRO产生细胞类型是“通用的”，并且不依赖于货物。这些假说直接源于对黑素细胞中的LB生物发生和LRO成熟的比较，在黑素细胞中，黑素小体通过不同的形态阶段成熟，通过AP-3和区块依赖的方式将黑素生成酶从早期的内吞体递送给无色素的前体。我们的结果将进一步加深我们对LBS作为关键LRO的认识，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Lbs是否代表Lb成熟的不同阶段，以及递进性货物蛋白输送是否影响成熟Lb的脂谱。我们将从成熟的AT2中提取丰富的、不同的LB组分的含量，并通过免疫荧光和免疫电子显微镜来验证我们的发现。2.测试陆路货运是否需要AP-3和/或AP-1。我们将测试推测货物的细胞质结构域与黑素体蛋白用于分选黑素小体的细胞质适配器的结合，并评估在货物定位到AT2中的LBS时对这些适配器的需求。3.测试LRO的成熟是否由AT2和黑素细胞中HPS相关蛋白复合体介导的货物运输途径“普遍”定义。我们将测试异位表达的黑素小体和LBCargo是否定位于AT2中的LBS和黑素细胞中的黑素小体，以及在这两种细胞类型中的定位是否同样需要区块。

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.