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.

项目概要 黑素体是皮肤、头发和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，其特征是色素沉着不足、视力受损以及对皮肤和皮肤的敏感性增加 眼癌。一些在各种形式的眼皮肤白化病（OCA）中存在缺陷的基因， 包括 OCA2 和 SLC45A2，编码调节离子环境的跨膜蛋白 黑素体或黑素体前体。例如，我们最近证明 OCA2 具有氯化物的功能 中和黑素体 pH 值的通道，从而激活黑色素合成，而双孔通道 2 (TPC2)——第一个被识别的黑素体阳离子通道——负向调节色素沉着。尽管有这些 进步，我们对跨黑素体膜的离子传输以及离子通量如何调节的理解 色素沉着是初级的。特别是，尚不清楚 SLC45A2 如何调节色素沉着，或者遗传如何调节色素沉着。 SLC45A2 的变化会干扰颜料的产生。而缺乏 SLC45A2 或 OCA2 的黑素细胞则共享 一些特征，例如关键黑色素生成酶酪氨酸酶的体内活性受损，这两种酶如何 蛋白质协同或单独影响酪氨酸酶活性仍然难以捉摸。最后，TPC2 是如何 非选择性、钠和钙可渗透通道——对黑素生成的影响完全未知。 该提案的目标是回答有关 SLC45A2、OCA2 和 TPC2 功能的关键问题，并 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 黑素体和黑色素合成。基于可靠的初步数据，我们将测试：(1) OCA2 和 SLC45A2 各自的功能是增加黑素体的管腔 pH 值，但处于不同的成熟阶段； (2) SLC45A2 直接作用于来自特定微域的黑素体，并将其组装成 与色素沉着不足相关的 SLC45A2 变体中的微结构域被破坏； (3) TPC2 的功能是 介导酪氨酸跨黑素体膜转运的多蛋白复合物。 更广泛的影响：这些研究将对了解调节皮肤的机制产生广泛的影响 和眼睛色素沉着，将增进我们对离子穿过黑素体膜的运输方式的理解 对黑素体功能至关重要，将揭示色素沉着疾病的潜在机制，并将设定一个 了解其他溶酶体相关细胞器中离子运输控制的先例。 与公共卫生的相关性：编码参与离子转运的几种蛋白质的基因突变 黑素体膜导致白化病，伴有色素沉着缺陷、视力受损和视力增加 皮肤和眼睛对癌症的易感性。我们的研究将阐明其分子机制 这些蛋白质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。