Iron and copper transporter trafficking in healthy and diseased melanocytes

说明(申请人提供)：铜和铁是许多细胞酶和转运蛋白的辅因子，但也是有毒的氧化还原反应过渡金属。因此，它们的细胞水平受到特定的转运蛋白的严格控制，这些转运蛋白将细胞外离子输入细胞质，而铜的水平则受到依赖于ATP的泵(ATP7A和ATP7B)的严格控制，后者将铜从胞浆输出到分泌/内吞途径或细胞外。ATP7A缺乏导致门克斯病，这是一种严重的细胞质铜积累和无法加载分泌型铜蛋白的发育和系统性疾病。无论是铜从ATP7A装载到靶蛋白CuProteins的机制，还是那些调控铜和铁转运蛋白的亚细胞分布的机制都不清楚。酵母中的ATP7A同源基因是激活依赖铜的铁进口所必需的；哺乳动物中是否存在细胞铜和铁进口之间的这种联系尚不清楚。这项建议利用黑素细胞的独特性质，其中铜是黑色素生物合成酶酪氨酸酶的辅因子，以剖析调节铜和铁转运蛋白定位以及铜负载到分泌蛋白上的分子机制。初步数据表明，黑素细胞中的ATP7A序列定位于黑素体，取决于其目标铜酶酪氨酸酶和BLOC-1，BLOC-1是水泡运输障碍Hermansky Pudlak综合征(HPS)中存在缺陷的细胞质复合体。此外，HPS黑素细胞中铜依赖的ATP7A易位缺陷和转铁蛋白受体水平的升高表明，BLOC-1可能调节铜和铁的输入。我们在HPS和黑素细胞生物学方面的独特专业知识以及我们的合作者在铁和铜代谢方面的专业知识将被应用于以下具体目标：1.验证ATP7A通过与其底物酪氨酸酶结合而定位于黑素体的假设。2.验证铜进口受HPS相关的BLOC-1复合体调控的假设。3.验证黑素细胞铁输入受BLOC-1和铜输入调控的假说。摘要：门克斯病是由于许多细胞类型的铁和铜通量的遗传缺陷所致，而赫曼斯基·普德拉克综合征是一种通常只有某些细胞类型的致命遗传疾病，包括色素细胞。这一建议验证了赫曼斯基·普德拉克综合症是由受影响细胞类型中的铁和铜失调引起的假设。

DESCRIPTION (provided by applicant): Copper and iron are cofactors for many cellular enzymes and transporters, but are also toxic redox-reactive transition metals. Their cellular levels are thus tightly controlled by specific transporters that import extracellular ion to the cytoplasm, and for copper by ATP-dependent pumps (ATP7A and ATP7B) that export copper from the cytosol to the secretory/endocytic pathway or outside the cell. ATP7A deficiency causes Menkes Disease, a severe developmental and systemic disorder of cytoplasmic copper accumulation and failure to load secretory cuproproteins. Neither the mechanisms by which copper is loaded from ATP7A into target cuproproteins nor those regulating the subcellular distribution of copper and iron transporters are understood. The ATP7A orthologue in yeast is needed to activate copper-dependent iron import; whether such a link between cellular copper and iron import exists in mammals is not known. This proposal exploits unique properties of melanocytes, in which copper is a cofactor for the melanin biosynthetic enzyme, tyrosinase, to dissect molecular mechanisms regulating copper and iron transporter localization and copper loading onto secretory proteins. Preliminary data suggest that an ATP7A cohort in melanocytes localizes to melanosomes dependent on its target cuproenzyme, tyrosinase, and on BLOC-1, a cytoplasmic complex defective in the vesicular transport disorder, Hermansky Pudlak Syndrome (HPS). Moreover, a copper-dependent ATP7A translocation defect and elevated transferrin receptor levels in HPS melanocytes suggest that BLOC-1 may regulate copper and iron import. Our unique expertise in HPS and melanocyte cell biology and our collaborator's expertise in iron and copper metabolism will be applied to the following Specific Aims: 1. To test the hypothesis that ATP7A localizes to melanosomes by associating with its substrate, tyrosinase. 2. To test the hypothesis that copper import is regulated by the HPS-associated BLOC-1 complex. 3. To test the hypothesis that iron import in melanocytes is regulated by BLOC-1 and by copper import. Summary: Menkes Disease is due to a genetic defect in iron and copper flux in many cell types, and Hermansky Pudlak Syndrome is an often lethal genetic disorder of only certain cell types, including pigment cells. This proposal tests the hypothesis that Hermansky Pudlak Syndrome results from iron and copper dysregulation in the affected cell types.