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

项目总结 黑素小体是皮肤、头发和眼睛黑素细胞和色素中独特的溶酶体相关细胞器。 眼睛的视网膜、虹膜和睫状体的上皮，其中黑色素是哺乳动物的主要色素. 合成并存储。黑素小体成分或生物遗传机制的遗传缺陷会导致 白化病，特征是色素减少，视力受损，对皮肤和 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 黑素小体或黑素小体前体。例如，我们最近展示了OCA2作为氯化物的功能 中和黑素小体pH从而激活黑色素合成的通道，而两孔通道2 (TPC2)--第一个被发现的黑素体阳离子通道--负向调节色素沉着。尽管如此 进展，我们对黑素体膜上离子传输的理解以及离子通量是如何调节的 色素沉着是基本的。特别是，目前尚不清楚SLC45A2是如何调节色素沉着的，或者是如何遗传的 SLC45A2的变异会干扰色素的产生。而缺乏SLC45A2或OCA2的黑素细胞 一些特征如体内一种关键的黑色素生成酶酪氨酸酶活性受损，这两种酶是如何 蛋白质共同或单独影响酪氨酸酶活性仍然难以捉摸。最后，TPC2-a如何 非选择性的钠和钙渗透通道对黑素生成的影响是完全未知的。 本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体内离子环境的分子途径。 黑素小体和黑色素合成。基于坚实的初步数据，我们将测试：(1)OCA2和 SLC45A2都能提高黑素小体的管腔pH值，但在不同的成熟阶段；(2) SLC45A2直接作用于来自特定微域的黑素小体，并组装成 在色素减退相关的SLC45A2变异体中微域被破坏；以及(3)TPC2作为 一种多蛋白质复合体，介导酪氨酸在黑素体膜上的运输。 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，将促进我们对离子如何通过黑素体膜运输的理解 对黑素小体功能至关重要，将揭示色素沉着障碍的潜在机制，并将设置 了解其他溶酶体相关细胞器中离子传输控制的先例。 与公共健康相关：编码几种参与离子转运的蛋白质的基因突变 黑素小体膜会导致白化病，并伴有色素沉着缺陷、视力受损和增加。 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的生成，以及患者突变如何导致色素沉着和视力缺陷。

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.