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: 10400351
• 负责人: Michael S Marks
• 金额: $ 5.76万
• 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/10400351
• 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 黑色素体是皮肤，头发和眼睛黑色素细胞和色素的独特与溶酶体相关的细胞器 视网膜上皮，虹膜和睫状体的眼睛，黑色素（哺乳动物的主要色素）为 合成和存储。黑色素体成分或生物遗传学机械的遗传缺陷导致 白化症，其特征在于异形，视力障碍以及对皮肤和皮肤的敏感性增加 眼睛癌。某些在各种形式的眼皮白化病（OCA）中有缺陷的基因， 包括OCA2和SLC45A2，编码调节离子环境的跨膜蛋白 黑素体或黑色素体前体。例如，我们最近表明OCA2充当氯化物 中和黑色素pH的通道，从而激活黑色素合成，而两孔通道2 （TPC2） - 第一个鉴定出的黑素阳离子通道 - 负调节色素沉着。尽管如此 进步，我们对跨黑色素体膜的离子传输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SLC45A2如何调节色素沉着或遗传如何调节 SLC45A2的变化会干扰色素的产生。而黑素细胞缺乏SLC45A2或OCA2共享 一些特征，例如关键黑色素生成酶的体内活性受损，酪氨酸酶，这两种 蛋白质合作或分别影响酪氨酸酶活性仍然难以捉摸。最后，如何TPC2 - 非选择性，钠和钙可渗透通道 - 影响黑色素发生是完全未知的。 该提案的目的是回答有关SLC45A2，OCA2和TPC2功能的关键问题，以及 剖析允许这些蛋白质直接或间接控制离子环境的分子途径 黑色素体和黑色素合成。基于可靠的初步数据，我们将测试：（1）OCA2和 SLC45A2每个功能都会增加黑色素酶体的腔pH，但在不同的成熟阶段。 （2） SLC45A2直接在特定微域的黑素体上起作用，该组装到 微域在与异形相关的SLC45A2变体中受到破坏。 （3）TPC2在 一种多蛋白质复合物，可介导诸如黑色素体膜的酪氨酸转运。 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，将促进我们对离子跨膜膜如何运输的理解 对于黑色素词功能至关重要，将发现色素沉着障碍的机制，并将设置一个 了解其他溶酶体相关细胞器中离子传输控制的先例。 与公共卫生的相关性：编码几种参与离子运输的基因突变 黑色素体膜引起色素沉着缺陷，视力受损和增加的白化病 皮肤和眼睛对癌症的敏感性。我们的研究将阐明分子机制 这些蛋白质会影响黑色素生成以及患者突变如何导致色素沉着和视力缺陷。