BLOC-1 and BLOC-2 function in melanosome maturation

描述（由申请人提供）：Hermansky-Pudlak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）、出血性疾病和通常致命的肺纤维化。这些症状是由于受影响的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，所述细胞类型例如黑素体（皮肤和眼睛色素细胞中的细胞器，其中合成和储存黑色素）、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的个体患有视力差，并且由于黑色素和视网膜变性导致的紫外线保护的丧失而导致皮肤癌的风险显著增加。人类HPS的八种亚型中有五种是由两种蛋白质复合物的突变引起的，即溶酶体相关细胞器复合物（BLOC）-1和BLOC-2的生物发生。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导致HPS的新治疗方法。本研究以黑素体为模型LRO，以野生型和HPS模型小鼠的永生化黑素细胞系为实验系统，研究BLOC-1和BLOC-2在蛋白质转运中的具体作用。我假设BLOC-1和BLOC-2的功能与内体陷阱蛋白，syntaxin 13（STX 13），和其他伙伴陷阱蛋白调节循环内体衍生的运输中间体的动力学，并指定其对接和融合与黑素体。本提案的具体目的是：（1）测试BLOC-2是否特异性地调节黑素体的顺行货物递送~（2）测试BLOC-1和BLOC-2是否通过引导内体转运中间体接触黑素体来调节内体转运中间体的动力学~和 (3)以测试BLOC-1和BLOC-2是否影响黑素细胞中含有STX 13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：定量活细胞显微镜检查，以研究含STX 13的内体载体与黑素体的相互作用;内吞动力学的流式细胞术分析，以评估黑素体货物运输;免疫荧光和免疫电子显微镜检查，以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鉴定STX 13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货物从内体系统中分离出来并允许专门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的疾病的理解。 公共卫生相关性：该提案将调查Hermansky-Pudlak综合征（HPS）几种亚型的蛋白质运输缺陷，HPS是一种影响波多黎各约1：1800个体的疾病，在世界各地的其他人群中很常见。HPS是由各种细胞类型中的特化亚细胞结构的畸形引起的，并导致眼皮肤白化病、出血性疾病和致死性肺纤维化。该提案将使用黑素体（黑素细胞中发现的一种特殊结构）作为剖析HPS分子机制的模型系统，其长期目标是确定治疗HPS的治疗靶点。

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.