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）：Hermansky-Pudlak综合征（HPS）是一组相关的遗传疾病，导致眼镜白化病（OCA），出血性疾病和通常致命的肺纤维纤维。这些症状归因于受影响细胞类型的细胞类型特异性相关细胞器（LROS）的生物发生和功能缺陷，例如黑色素体（皮肤和眼部色素细胞中的细胞器中的黑色素色素和储存黑色素色素的细胞器），骨骼浓密的脂质片和肺部浓密的细胞和lung lung lung Ephelial lamellial lamellial lamellal holdies。值得注意的是，OCA视力不良，由于黑色素和视网膜变性的紫外线保护丧失，患有皮肤癌的风险显着增加。 HPS中八个亚型中的五个是由于两个蛋白质复合物中的突变，与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，对它们在LRO生物发生中的机械作用的理解可能会导致HPS的新型治疗方法。该建议将使用黑色素体作为模型LROS以及来自野生型和HPS模型小鼠的永生化黑素细胞系在蛋白质转运中的BLOC-1和BLOC-2在蛋白质转运中的特定作用。我假设BLOC-1和BLOC-2与内体SNARE蛋白，Syntaxin13（STX13）和其他合作伙伴SNARE SNARE蛋白结合起作用，以调节回收内体衍生的运输中间体的动力学，并指定其与Melanosomes的对接和融合。 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.为了实现这些目的，我将通过以下方式比较黑色素体和内体货物在野生型，bloc-1-和Bloc-2缺陷的黑色素细胞中：定量活细胞显微镜研究研究与梅拉氏菌的相互作用，以调查与梅拉氏菌的相互作用，以评估梅拉氏液和流式细胞囊的相互作用。免疫荧光和免疫电子显微镜评估黑色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以识别STX13结合伴侣。这些研究将提供有关LRO生物发生的分子基础的见解，并阐明将LRO货物与内体系统隔离的特定事件，并允许专门LRO的成熟。这项工作将洞悉HPS的分子机制，确定治疗HP和可能其他类型白化病的潜在治疗靶标，并增加我们对影响LROS疾病的理解。 公共卫生相关性：该提案将研究Hermansky-Pudlak综合征（HPS）的几种亚型中的蛋白质运输缺陷，该疾病影响了波多黎各的〜1：1800个人，在世界其他人群中很常见。 HPS是由多种细胞类型中专门的亚细胞结构的畸形引起的，并导致眼态白化病，出血性疾病和致命的肺纤维纤维引起。该建议将使用黑色素体（在黑素细胞中发现的一种专业结构）作为一种模型系统，用于解剖HPS的分子机制，其长期目的是鉴定治疗HPS的治疗靶标。