BLOC-1 and BLOC-2 function in melanosome maturation

描述(申请人提供)：Hermansky-Pudlak综合征(HPS)是一组相关的遗传性疾病，会导致眼皮肤白化病(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特定类型的溶酶体相关细胞器(LRO)的生物发生和功能缺陷所致，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小体。值得注意的是，OCA患者视力不佳，由于黑色素失去紫外线保护和视网膜退化，患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体的突变引起的，这两种蛋白质复合体是溶酶体相关细胞器复合体(BLOC)-1和BLOC-2的生物发生。这些复合体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。本研究将以黑素小体为LRO模型，以野生型黑素细胞系和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与内体SNARE蛋白、合成素13(STX13)和其他伙伴SNARE蛋白一起发挥作用，以调节内小体来源的再循环运输中间产物的动态，并指定它们与黑素体的对接和融合。这项建议的具体目的是：(1)测试BLOC-2是否调节黑素小体的顺行货物运输~(2)测试BLOC-1和BLOC-2是否通过引导内膜转运中间体与黑素小体接触来调节它们的动态；(3)测试BLOC-1和BLOC-2是否影响黑素细胞中含有STX13的SNARE复合体的组成。为了实现这些目标，我将比较野生型、BLOC-1和BLOC-2缺陷的黑素细胞中黑素小体和内体货物的行为：定量活细胞显微镜观察含有STX13的内体载体与黑素小体的相互作用；流式细胞术内吞分析以评估黑素小体运输~以及免疫荧光和免疫电子显微镜来评估黑素小体货物的稳态分布。我将利用体外和体外技术来评估蛋白质之间的相互作用，并利用质谱学来确定STX13结合伙伴。这些研究将提供对LRO生物发生的分子基础的见解，并澄清将LRO货物从内体系统中分离出来并允许专门的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的潜在靶点，并可能治疗其他类型的白化病，并增加我们对影响LROS的疾病的了解。

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.