BLOC-1 and BLOC-2 function in melanosome maturation

BLOC-1 和 BLOC-2 在黑素体成熟中发挥作用

• 批准号: 8255978
• 负责人: Megan Kathleen Dennis
• 金额: $ 5.16万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8255978
• 关键词: Affect; Albinism; Anabolism; Behavior; Binding; Biogenesis; Biological Models; Blindness; Blood Platelets; Cell Line; Cell surface; Cells; Clinical; Complex; Cytoplasmic Granules; Data; Defect; Development; Disease; Docking; Early Endosome; Endocytosis; Endosomes; Epithelial Cells; Epithelium; Epitopes; Event; Eye; Fibroblasts; Fibrosis; Genes; Goals; Golgi Apparatus; Hemorrhage; Hereditary Disease; Hermanski-Pudlak Syndrome; Human; Immunoblot Analysis; Immunoelectron Microscopy; Immunofluorescence Microscopy; Immunoprecipitation; In Vitro; Individual; Integral Membrane Protein; Lead; Length; Life; Lung; Lysosomes; Mass Spectrum Analysis; Melanins; Melanosomes; Membrane; Microscopy; Modeling; Molecular; Mus; Mutation; Oculocutaneous Albinism; Organelles; Pathway interactions; Photobleaching; Pigments; Population; Process; Property; Proteins; Puerto Rico; Recycling; Retinal Degeneration; Risk; Role; SNAP receptor; Skin; Skin Cancer; Sorting - Cell Movement; Specificity; Structure; Subcellular structure; Symptoms; System; Techniques; Testing; Tissues; To specify; UV protection; Visual Acuity; Work; alveolar lamellar body; base; cell type; cellular imaging; cohort; insight; malformation; melanocyte; member; mouse model; novel therapeutic intervention; photoactivation; protein complex; protein protein interaction; protein transport; research study; therapeutic target; trafficking

DESCRIPTION (provided by applicant): Hermansky-Pudlak syndrome (HPS) is a group of related genetic disorders that result in oculocutaneous albinism (OCA), bleeding disorders and often lethal lung fibroses. These symptoms are due to defects in the biogenesis and function of cell type-specific lysosome related organelles (LROs) in affected cell types, such as melanosomes (the organelles in skin and eye pigment cells in which melanin pigments are synthesized and stored), platelet dense granules and lung epithelial cell lamellar bodies. Notably, individuals with OCA suffer from poor visual acuity and are at significantly increased risk of skin cancer due to loss of UV protection by melanin and to retinal degeneration. Five of the eight subtypes of HPS in humans result from mutations in two protein complexes, Biogenesis of Lysosome-related Organelles Complex (BLOC)-1 and BLOC-2. The molecular function of these complexes is not known, and understanding of their mechanistic role in LRO biogenesis will likely lead to novel therapeutic approaches for HPS. This proposal will investigate the specific roles of BLOC-1 and BLOC-2 in protein transport using melanosomes as model LROs and immortalized melanocyte cell lines from wild type and HPS model mice as an experimental system. I hypothesize that BLOC-1 and BLOC-2 function in conjunction with the endosomal SNARE protein, syntaxin13 (STX13), and other partner SNARE proteins to regulate the dynamics of recycling endosome-derived transport intermediates and to specify their docking and fusion with melanosomes. The specific aims of this proposal are: (1) to test whether BLOC-2 regulates anterograde cargo delivery specifically to melanosomes~ (2) to test whether BLOC-1 and BLOC-2 regulate the dynamics of endosomal transport intermediates by directing them to contact melanosomes~ and (3) to test whether BLOC-1 and BLOC-2 influence the composition of STX13-containing SNARE complexes in melanocytes. To achieve these aims, I will compare the behavior of melanosome and endosomal cargoes in wild-type, BLOC-1- and BLOC-2-deficient melanocytes by: quantitative live cell microscopy to investigate interactions of STX13-containing endosomal carriers with melanosomes~ flow cytometric analyses of endocytic dynamics to assess melanosome cargo trafficking~ and immunofluorescence and immunoelectron microscopy to assess steady state distribution of melanosome cargoes. I will exploit in vitro and ex vivo techniques for assessing protein-protein interactions and mass spectrometry to identify STX13 binding partners. These studies will provide insights into the molecular basis for LRO biogenesis and clarify the specific events which segregate LRO cargoes from the endosomal system and allow for maturation of specialized LROs. This work will provide insight into the molecular mechanisms of HPS, identify potential therapeutic targets for treatment of HPS and possibly other types of albinism and increase our understanding of diseases affecting LROs. PUBLIC HEALTH RELEVANCE: This proposal will investigate protein trafficking defects in several subtypes of Hermansky-Pudlak syndrome (HPS), a disease which affects ~ 1:1800 individuals in Puerto Rico and is common in other populations around the world. HPS is caused by malformation of specialized subcellular structures in a variety of cell types and results in oculocutaneous albinism, bleeding disorders and lethal lung fibroses. This proposal will use melanosomes, a specialized structure found in melanocytes, as a model system for dissecting the molecular mechanisms of HPS with the long term aim of identifying therapeutic targets for treatment of HPS.

描述（由申请人提供）：赫曼斯基-普德拉克综合征（HPS）是一组相关的遗传性疾病，可导致眼皮肤白化病（OCA）、出血性疾病以及通常致命的肺纤维化。这些症状是由于受影响细胞类型中细胞类型特异性溶酶体相关细胞器 (LRO) 的生物发生和功能缺陷造成的，例如黑素体（皮肤和眼睛色素细胞中合成和储存黑色素的细胞器）、血小板致密颗粒和肺上皮细胞板层体。值得注意的是，患有 OCA 的人视力较差，并且由于黑色素失去紫外线防护和视网膜变性，患皮肤癌的风险显着增加。人类 HPS 的 8 种亚型中有 5 种是由两种蛋白复合物（溶酶体相关细胞器复合物的生物发生 (BLOC)-1 和 BLOC-2）的突变引起的。这些复合物的分子功能尚不清楚，了解它们在 LRO 生物发生中的机制作用可能会带来新的 HPS 治疗方法。该提案将使用黑素体作为模型 LRO，并使用来自野生型和 HPS 模型小鼠的永生化黑素细胞系作为实验系统，研究 BLOC-1 和 BLOC-2 在蛋白质运输中的具体作用。我假设 BLOC-1 和 BLOC-2 与内体 SNARE 蛋白、突触蛋白 13 (STX13) 和其他 SNARE 蛋白结合发挥作用，调节内体衍生转运中间体的再循环动力学，并指定它们与黑素体的对接和融合。该提案的具体目的是：（1）测试BLOC-2是否专门调节黑素体的顺行货物输送〜（2）测试BLOC-1和BLOC-2是否通过引导内体运输中间体接触来调节它们的动态黑素体〜和（3）测试BLOC-1和BLOC-2是否影响黑素细胞中含有STX13的SNARE复合物的组成。为了实现这些目标，我将通过以下方式比较野生型、BLOC-1 和 BLOC-2 缺陷黑素细胞中黑素体和内体货物的行为： 定量活细胞显微镜研究含有 STX13 的内体载体与黑素体的相互作用〜流内吞动力学的细胞计数分析可评估黑素体货物的运输，免疫荧光和免疫电子显微镜可评估黑素体货物的稳态分布。我将利用体外和离体技术来评估蛋白质-蛋白质相互作用和质谱法来识别 STX13 结合伴侣。这些研究将深入了解 LRO 生物发生的分子基础，并阐明将 LRO 货物与内体系统分离并允许专门的 LRO 成熟的特定事件。这项工作将深入了解 HPS 的分子机制，确定治疗 HPS 和可能的其他类型白化病的潜在治疗靶点，并增加我们对影响 LRO 的疾病的了解。 公共健康相关性：该提案将调查 Hermansky-Pudlak 综合征 (HPS) 几种亚型的蛋白质运输缺陷，这种疾病影响波多黎各约 1:1800 人，并且在世界其他人群中很常见。 HPS 是由多种细胞类型的特殊亚细胞结构畸形引起的，可导致眼皮肤白化病、出血性疾病和致命性肺纤维化。该提案将使用黑素体（黑素细胞中发现的一种特殊结构）作为模型系统来剖析 HPS 的分子机制，长期目标是确定 HPS 的治疗靶点。