Iron and copper transporter trafficking in healthy and diseased melanocytes

描述（由申请人提供）：铜和铁是许多细胞酶和转运体的辅助因子，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转运体的严格控制，这些转运体将细胞外离子输入细胞质，而铜则由atp依赖泵（ATP7A和ATP7B）将铜从细胞质中输出到分泌/内吞途径或细胞外。ATP7A缺乏导致Menkes病，这是一种严重的发育和全身性疾病，是细胞质铜积累和不能负荷分泌铜蛋白。铜从ATP7A装载到靶铜蛋白的机制，以及调节铜和铁转运体亚细胞分布的机制都还不清楚。酵母中的ATP7A同源物需要激活铜依赖的铁进口；哺乳动物体内的细胞铜和铁的输入之间是否存在这种联系尚不清楚。本研究利用黑素细胞的独特特性，其中铜是黑色素生物合成酶酪氨酸酶的辅助因子，来剖析调节铜和铁转运体定位和铜装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队列定位于依赖于其靶铜原酶、酪氨酸酶和block -1的黑素小体，block -1是水疱运输障碍Hermansky Pudlak综合征（HPS）中的一种细胞质复合物缺陷。此外，HPS黑素细胞中铜依赖性ATP7A易位缺陷和转铁蛋白受体水平升高表明，block -1可能调节铜和铁的进口。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应用于以下具体目标：1。为了验证ATP7A通过与其底物酪氨酸酶结合而定位于黑素体的假设。2. 为了验证铜的进口是由hps相关的block -1复合物调节的假设。3. 为了验证黑素细胞中的铁输入受block -1和铜输入调节的假设。Menkes病是由于许多细胞类型中铁和铜通量的遗传缺陷引起的，Hermansky Pudlak综合征是一种通常致命的遗传疾病，仅发生于某些细胞类型，包括色素细胞。这一提议验证了Hermansky Pudlak综合征是由受影响细胞类型中的铁和铜失调引起的假说。

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.