Molecular Mechanisms of Pigmentation in Health and Disease

项目总结/摘要 黑色素负责皮肤、毛发和眼睛的色素沉着， 黑素细胞和视网膜色素上皮细胞中的黑素小体。在皮肤黑素细胞的情况下，黑色素是 然后转移到邻近的角质形成细胞。以黑素体缺陷为特征的色素沉着疾病的实例 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，本发明还 黑素体对细胞内细胞毒性药物的隔离和分泌是多药治疗的基础。 黑色素瘤的抵抗力。因此，抑制黑素体功能构成了增强黑素体功能的重要途径。 黑素瘤细胞的化学敏感性。因此，了解调节色素沉着的分子机制， 生理意义，并可能导致临床应用。尽管色素沉着与人类的 然而，在我们对黑素体生物发生和功能的理解方面存在重大的知识差距。我们的目标是 以综合的方式回答色素沉着领域这一领域的基本突出问题，并专注于 分子机制 目的1：酪氨酸酶、酪氨酸酶相关蛋白1（Tyrp 1）和Tyrp 2在黑色素细胞中合成黑色素。 黑素小体腔黑素体的生物发生需要将三种黑素生成酶递送至成熟的黑素体。 细胞器新合成的酪氨酸酶和Tyrp 1向黑素体的转运相对较好地理解。在 相反，Tyrp 2到黑素体的途径是未知的。我们有证据表明Tyrp 2使用不同的 酪氨酸酶和Tyrp 1所使用的转运途径。我们将利用我们最近开发的一种新的测定方法来确定 Tyrp 2所遵循的途径，并将其与酪氨酸酶和Tyrp 1所使用的途径进行比较。的生物发生 黑素体（和其他溶酶体相关的细胞器）依赖于几种蛋白质的功能， 在各种形式的Hermanky-Pudlak综合征（HPS）中突变的基因。两种最常见和最严重的形式 HPS 1和HPS 4是目前已知的最常见的两种HPS蛋白，但对这些HPS蛋白在细胞器生物发生中的功能知之甚少。我们将 测试HPS 1和HPS 4与小GT3 Rab 32一起定义转运途径的假设， Tyrp 2和其他黑素体蛋白所使用的黑素体。目的2：一项全基因组关联研究， 双孔通道2（TPC 2）作为色素沉着的遗传决定因素，但TPC 2如何调节色素沉着是一个谜。 我们的数据表明，TPC 2是黑素体膜的一个组成部分，调节黑素体功能。我们将测试 假设通过黑素体pH的变化，TPC 2控制酪氨酸酶的活性， 黑色素合成的数量和类型。此外，我们还将研究TPC 2的功能和多态性的机制， 与色素变化有关。目的3：在黑素体生物发生和黑色素合成发生后， 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， 模型已经提出。我们将测试哪一个模型是正确的，或者黑色素转移是否发生超过 一个机制。此外，在这个过程中所涉及的分子机制将进行调查。

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.