Mechanisms regulating ion transport across the melanosomal membrane in health and disease

在健康和疾病中调节离子跨黑素体膜运输的机制

• 批准号: 10164721
• 负责人: Michael S Marks
• 金额: $ 35.94万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10164721
• 关键词: Affect; Albinism; Amino Acid Transporter; Anabolism; Biochemical; Biological Assay; Calcium; Cations; Cells; Characteristics; Charge; Chloride Channels; Ciliary Body; Complex; Data; Defect; Disease; Environment; Enzymes; Equilibrium; Eye; Fluorescence Microscopy; Gene Mutation; Genes; Genetic Variation; Goals; Hair; Health; Human; Hypopigmentation; Immunoelectron Microscopy; Impairment; Integral Membrane Protein; Ion Transport; Ions; Iris; Lead; Link; Lysosomes; Malignant Neoplasms; Mammals; Mediating; Melanins; Melanogenesis; Melanosomes; Membrane; Membrane Microdomains; Microscopy; Molecular; Monophenol Monooxygenase; Multiprotein Complexes; Mus; Mutate; Mutation; Oculocutaneous Albinism; Organelles; Pathway interactions; Patients; Permeability; Physiological; Physiology; Pigmentation Disorders; Pigmentation physiologic function; Pigments; Predisposition; Production; Property; Proteins; Protons; Public Health; Reporter; Resolution; Site; Skin; Skin Cancer; Skin Substitutes; Sodium; Solid; Structure of retinal pigment epithelium; Sucrose; System; Techniques; Testing; Tyrosinase related protein-1; Tyrosine; Variant; Vision; Visual impairment; Yeasts; base; gene product; genetic variant; in vivo; malignant neoplasm of eye; melanocyte; mutant; patch clamp; protein protein interaction; trafficking; vision development

PROJECT SUMMARY Melanosomes are unique lysosome-related organelles in skin, hair, and eye melanocytes and pigment epithelia of the retina, iris and ciliary body of the eye, in which melanins - the main pigments in mammals - are synthesized and stored. Genetic defects in melanosome components or biogenetic machinery result in albinism, characterized by hypopigmentation, impairments in vision, and increased susceptibility to skin and eye cancers. Some of the genes that are defective in various forms of oculocutaneous albinism (OCA), including OCA2 and SLC45A2, encode transmembrane proteins that regulate the ionic environment of melanosomes or melanosome precursors. For example, we recently showed that OCA2 functions as a chloride channel that neutralizes melanosome pH, thereby activating melanin synthesis, whereas two-pore channel 2 (TPC2) – the first identified melanosomal cation channel – negatively regulates pigmentation. Despite these advances, our understanding of ion transport across the melanosome membrane and how ion flux regulates pigmentation is rudimentary. In particular, it is not known how SLC45A2 regulates pigmentation, or how genetic variation in SLC45A2 interferes with pigment production. While melanocytes lacking SLC45A2 or OCA2 share some characteristics such as impaired in vivo activity of a key melanogenic enzyme, tyrosinase, how these two proteins influence the tyrosinase activity cooperatively or separately remains elusive. Finally, how TPC2 – a nonselective, sodium and calcium permeable channel – influences melanogenesis is completely unknown. The goal of this proposal is to answer critical questions regarding SLC45A2, OCA2 and TPC2 function and to dissect the molecular pathways that allow these proteins, directly or indirectly, to control the ionic milieu within melanosomes and melanin synthesis. Based on solid preliminary data, we will test that: (1) OCA2 and SLC45A2 each function to increase the luminal pH of melanosomes, but at distinct stages of maturation; (2) SLC45A2 functions directly on melanosomes from a specific microdomain and that assembly into the microdomain is disrupted in hypopigmentation-associated SLC45A2 variants; and (3) TPC2 functions as part of a multi-protein complex that mediates tyrosine transport across the melanosome membrane. Broader impact: These studies will have a broad impact on understanding the mechanisms that regulate skin and eye pigmentation, will advance our understanding of how ion transport across melanosomal membranes is critical for melanosomal function, will uncover mechanisms underlying pigmentation disorders, and will set a precedent for understanding ion transport control in other lysosome-related organelles. Relevance to public health: Mutations in the genes encoding several proteins involved in ion transport across melanosomal membranes cause albinism with pigmentation defects, impaired vision, and increased susceptibility of the skin and eye to cancer. Our studies will elucidate the molecular mechanisms by which these proteins affect melanogenesis and how patient mutations result in pigmentation and vision defects.

项目摘要 黑素体是皮肤、毛发、眼睛黑素细胞和色素中独特的溶酶体相关细胞器 视网膜、虹膜和眼睛睫状体的上皮细胞，其中黑色素-哺乳动物中的主要色素- 合成并存储。黑素体成分或生物遗传机制的遗传缺陷导致 白化病，特征是色素减退、视力受损和对皮肤和 眼癌一些基因是有缺陷的各种形式的眼皮肤白化病（OCA）， 包括OCA 2和SLC 45 A2，编码跨膜蛋白，调节细胞的离子环境。 黑素体或黑素体前体。例如，我们最近发现OCA 2的功能是氯化物， 中和黑素体pH的通道，从而激活黑素合成，而双孔通道2 （TPC 2）-第一个确定的黑素体阳离子通道-负调节色素沉着。尽管有这些 我们对黑素体膜离子转运的理解以及离子通量如何调节 色素沉着是基本的。特别是，尚不清楚SLC 45 A2如何调节色素沉着，或如何遗传， SLC 45 A2的变异干扰色素的产生。而缺乏SLC 45 A2或OCA 2的黑素细胞 一些特征，如受损的体内活性的一个关键的黑素生成酶，酪氨酸酶，如何这两个 蛋白质协同地或单独地影响酪氨酸酶活性仍然是难以捉摸的。最后，TPC 2- a 非选择性，钠和钙渗透通道-影响黑素生成是完全未知的。 本提案的目标是回答有关SLC 45 A2、OCA 2和TPC 2功能的关键问题， 剖析允许这些蛋白质直接或间接控制内部离子环境的分子途径。 黑素体和黑素合成。基于可靠的初步数据，我们将测试：（1）OCA 2和 SLC 45 A2各自作用于增加黑素体的内腔pH，但在不同的成熟阶段;（2） SLC 45 A2直接作用于来自特定微域的黑素体，并组装成 在色素减退相关的SLC 45 A2变体中，微结构域被破坏;以及（3）TPC 2作为 一种介导酪氨酸转运穿过黑素体膜的多蛋白复合物。 更广泛的影响：这些研究将对理解调节皮肤的机制产生广泛的影响。 和眼睛色素沉着，将推进我们对离子如何穿过黑色素体膜的理解， 对黑素体功能至关重要，将揭示色素沉着疾病的潜在机制，并将建立一个 这是理解其他溶酶体相关细胞器中离子转运控制的先例。 与公共卫生的相关性：编码几种参与离子转运的蛋白质的基因突变 黑色素体膜引起白化病，伴有色素沉着缺陷、视力受损和 皮肤和眼睛对癌症的易感性。我们的研究将阐明 这些蛋白质影响黑素生成以及患者突变如何导致色素沉着和视力缺陷。