Lamellar Body Biogenesis in Health and Disease

健康和疾病中的层状体生物发生

• 批准号: 8760573
• 负责人: Susan H. Guttentag
• 金额: $ 42.19万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8760573
• 关键词: AGTR2 gene; Adaptor Signaling Protein; Alveolar; Binding; Biogenesis; Biological Assay; Blood Platelets; Cell Fractionation; Cells; Cellular biology; Chimeric Proteins; Complex; Consensus; Cytoplasmic Granules; Cytoplasmic Tail; Data; Defect; Derivation procedure; Development; Diagnostic; Disease; Early Endosome; Endosomes; Enzymes; Epithelial Cells; Etiology; Fibrosis; Fluorescence Microscopy; Foundations; Functional disorder; Genes; Health; Hemorrhage; Hereditary Disease; Hermanski-Pudlak Syndrome; Human; Hybrids; Immunoelectron Microscopy; Immunofluorescence Microscopy; In Vitro; Injury; Integral Membrane Protein; Lead; Link; Lipids; Lung; Lung diseases; Lysosomes; Measures; Mediating; Melanosomes; Membrane; Membrane Protein Traffic; Membrane Proteins; Modeling; Molecular; Mutation; Oculocutaneous Albinism; Organelles; Pathway interactions; Patients; Pigments; Play; Population; Predisposition; Process; Proteins; Proteomics; Relative (related person); Role; Shapes; Signal Transduction; Site; Sorting - Cell Movement; Staging; Symptoms; TFF1 gene; Testing; Therapeutic; Tissues; Transcription Factor AP-1; Transmembrane Transport; Transport Process; Yeasts; alveolar lamellar body; base; cell type; density; family genetics; insight; malformation; melanocyte; mouse model; protein complex; public health relevance; segregation; stem; surfactant; tool; trafficking

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.

描述（由申请方提供）：Hermansky-Pudlak综合征（HPS）是一组罕见的遗传性疾病，其特征为眼皮肤白化病、过度出血和其他症状，这些症状反映了产生组织特异性溶酶体相关细胞器（LRO）所需的膜转运过程缺陷。患有HPS 1、2或4型的患者还患有致命的肺纤维化，反映了肺泡II型上皮细胞（AT 2）的损伤。A2损伤与板层体（LB）缺陷相关，板层体是富含脂质的LRO，其中合成并包装表面活性剂用于分泌。尽管LB对人类健康非常重要，但对其膜蛋白组成、其来自内溶酶体、导致其成熟的膜运输途径或HPS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建议中，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中有缺陷的蛋白质复合物联系起来。我们将测试以下假设：（1）LB通过AT 2中的不同阶段成熟，（2）AP-3和BLOC通过将LB特异性整合膜货物从常规内体靶向到早期LB阶段来促进LB成熟，以及（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LB生物发生与黑素细胞中LRO成熟的比较，其中黑素体通过不同的形态学阶段通过AP-3和BLOC依赖性的黑素生成酶从早期内体递送到非色素前体而成熟。我们的研究结果将进一步加深我们对LBs作为关键LRO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，以及渐进的货物蛋白递送是否塑造成熟LB的脂质谱。我们将配置文件的内容丰富，不同的LB馏分成熟AT 2，并通过免疫荧光和免疫电子显微镜验证我们的研究结果。2.测试LB货物交付是否需要AP-3和/或AP-1。我们将测试推定货物的细胞质结构域与黑素体蛋白用于分选黑素体的细胞质衔接子的结合，并评估在AT 2中货物定位于LB中对这些衔接子的需求。3.为了测试LRO成熟是否是“普遍的”由AT 2和黑素细胞中的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体和LB货物是否定位于AT 2中的LB和黑素细胞中的黑素体，以及在两种细胞类型中的定位是否同样需要BLOC。