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蛋白的辅因子，但也是有毒的氧化还原反应性过渡金属。因此，它们的细胞水平受到将细胞外离子输入细胞质的特定转运蛋白的严格控制，并且对于铜，受到将铜从细胞质输出到分泌/内吞途径或细胞外的ATP依赖性泵（ATP 7A和ATP 7 B）的严格控制。ATP 7A缺乏导致门克斯病，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全身性疾病。铜从ATP 7A装载到靶铜蛋白的机制和调节铜和铁转运蛋白亚细胞分布的机制都不清楚。酵母中的ATP 7A同源物需要激活铜依赖性铁输入;哺乳动物中细胞铜和铁输入之间是否存在这种联系尚不清楚。该建议利用黑素细胞的独特性质，其中铜是黑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和铜加载到分泌蛋白上的分子机制。初步数据表明，黑素细胞中的ATP 7A队列定位于黑素体，这取决于其靶向铜酶、酪氨酸酶和BLOC-1，BLOC-1是一种在囊泡转运障碍、Hermansky Pudlak综合征（HPS）中有缺陷的细胞质复合物。此外，铜依赖性ATP 7A易位缺陷和HPS黑素细胞中转铁蛋白受体水平升高表明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ATP 7A通过与其底物酪氨酸酶结合而定位于黑素体的假设。2.检验铜的进口受HPS相关的BLOC-1复合物调控的假设。3.为了验证黑素细胞中的铁输入受BLOC-1和铜输入调节的假设。总结：Menkes病是由于许多细胞类型中的铁和铜通量的遗传缺陷，而Hermansky Pudlak综合征是一种仅限于某些细胞类型（包括色素细胞）的致命遗传疾病。该提案测试了Hermansky Pudlak综合征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.