Molecular Mechanisms of Pigmentation in Health and Disease

项目摘要/摘要 黑色素负责皮肤、头发和眼睛的色素沉积，并在一个名为 黑素细胞和视网膜色素上皮细胞中的黑素小体。就皮肤黑素细胞而言，黑色素是 然后转移到邻近的角质形成细胞。以黑素小体缺陷为特征的色素沉着障碍的例子 生物发生和功能包括Hermansky-Pudlak综合征、眼皮肤白化病和Griscelli综合征。此外， 黑素小体对细胞内细胞毒药物的隔离和分泌是多药联用发展的基础 黑色素瘤的耐药性。因此，抑制黑素小体功能是增强 黑色素瘤细胞的化疗敏感性。因此，了解调节色素沉着的分子机制 具有生理意义，并有可能导致临床应用。尽管色素沉着与人类 在健康方面，我们对黑素小体的生物发生和功能的了解存在着重大的知识差距。我们的目标是 以综合的方式和重点回答这一领域中的根本突出问题 分子机制。 目的1：酪氨酸酶、酪氨酸酶相关蛋白1(Tyrp1)和Tyrp2合成黑色素 黑素体管腔。黑素小体的生物发生需要将三种黑素生成酶输送到成熟的黑素小体 细胞器。新合成的酪氨酸酶和Tyrp1到黑素小体的运输是相对较好的理解。在……里面 相比之下，Tyrp2通向黑素小体的途径尚不清楚。我们有证据表明Tyrp2使用了不同的 酪氨酸酶和Tyrp1使用的运输路线。我们将利用我们最近开发的一种新的分析方法来确定 Tyrp2所遵循的途径，并与酪氨酸酶和Tyrp1所使用的途径进行比较。细菌的生物发生 黑素小体(和其他与溶酶小体相关的细胞器)依赖于几种蛋白质的功能，这些蛋白质由 基因在不同形式的Hermanky-Pudlak综合征(HPS)中发生突变。最常见和最严重的两种形式的 疾病是HPS1和HPS4，但对这些HPS蛋白在细胞器生物发生中的功能知之甚少。我们会 检验假设，HPS1和HPS4与小GTP酶Rab32一起定义了一条运输途径 由Tyrp2和其他黑素小体蛋白使用的黑素小体。目标2：一项全基因组关联研究确定 双孔通道2(TPC2)是色素沉着的遗传决定因素，但TPC2是如何调节色素沉着的仍是一个谜。 我们的数据表明，TPC2是调节黑素小体功能的黑素小体膜的一个组成部分。我们将测试 该假说认为，通过改变黑素小体的pH值，TPC2控制酪氨酸酶的活性和 合成的黑色素的数量和类型。此外，我们还将研究TPC2的功能和多态性的机制 与色素变化有关的。目的3：黑素小体生物合成和黑色素合成后，皮肤 黑素细胞将色素转移到角质形成细胞。细胞间黑色素转移的机制尚不清楚，但有四种 已经提出了一些模型。我们将测试哪一个模型是正确的，或者如果黑色素转移发生的时间超过 一种机制。此外，还将研究这一过程中涉及的分子机制。

PROJECT SUMMARY / ABSTRACT Melanin pigments are responsible for pigmentation of skin hair and eyes and are synthesized in an organelle called melanosome in melanocytes and retinal pigmented epithelial cells. In the case of skin melanocytes, melanin pigments are then transferred to neighboring keratinocytes. Examples of pigmentation disorders characterized by defective melanosome biogenesis and function include Hermansky-Pudlak syndrome, oculocutaneous albinism and Griscelli syndrome. Further, the sequestration and secretion of intracellular cytotoxic drugs by melanosomes underlies the development of multidrug resistance in melanomas. Consequently, inhibiting melanosome function constitutes an important approach to enhance the chemosensitivity of melanoma cells. Thus, understanding the molecular mechanisms regulating pigmentation has physiological significance and will likely lead to clinical applications. In spite of the relevance of pigmentation for human health, there are significant knowledge gaps in our understanding of melanosome biogenesis and function. Our goal is to answer fundamental outstanding questions in this area of the pigmentation field in an integrated manner and focusing on molecular mechanisms. Aim 1: The enzymes tyrosinase, tyrosinase-related protein 1 (Tyrp1) and Tyrp2 synthesize melanin in the melanosome lumen. Melanosome biogenesis requires the delivery of the three melanogenic enzymes to the maturing organelle. The transport of newly synthesized tyrosinase and Tyrp1 to melanosomes is relatively well understood. In contrast, the pathway followed by Tyrp2 to melanosomes is unknown. We have evidence that Tyrp2 uses a different transport route from the one used by tyrosinase and Tyrp1. We will utilize a novel assay we recently developed to define the pathway followed by Tyrp2 and compare it with the pathway used by tyrosinase and Tyrp1. The biogenesis of melanosomes (and other lysosome-related organelles) depends on the function of several proteins that are encoded by genes mutated in various forms of Hermanky-Pudlak syndrome (HPS). The two most common and severe forms of the disease are HPS1 and HPS4, yet little is known about the function of these HPS proteins in organelle biogenesis. We will test the hypothesis that HPS1 and HPS4 together with the small GTPase Rab32 define a transport pathway to melanosomes that is used by Tyrp2 and other melanosomal proteins. Aim 2: A genome-wide association study identified Two-Pore Channel 2 (TPC2) as a genetic determinant of pigmentation but how TPC2 regulates pigmentation is a mystery. Our data suggests TPC2 is a component of the melanosome membrane that regulates melanosome function. We will test the hypothesis that through changes in melanosome pH, TPC2 controls the activity of the tyrosinase enzymes and the amount and type of melanin synthesized. Moreover, we will study the mechanism of TPC2 function and polymorphisms associated with pigmentation variations. Aim 3: After melanosome biogenesis and melanin synthesis take place, skin melanocytes transfer the pigments to keratinocytes. The mechanism for intercellular melanin transfer is unclear but four models have been proposed. We will test which one of the models is correct or if melanin transfer occurs by more than one mechanism. Further, the molecular machinery involved in this process will be investigated.