Dissecting the Impact of Autophagy Regulators on Melanogenesis in Normal Human Ce

描述(由申请人提供)： 摘要黑色素保护皮肤和眼睛免受紫外线辐射的有害影响，保护神经细胞免受有毒侮辱，并且是内耳传导声音所必需的。黑素生成的异常调节是皮肤疾病(黄褐斑和白癜风)、神经疾病(帕金森氏病)、听觉障碍(瓦登堡综合征)和眼科疾病(老年性黄斑变性)的基础。广泛的研究已经确定了150多个调节人类细胞黑色素产生的基因，但还没有完全了解黑色素产生障碍的发病机制。最近，我们将一个基于细胞的高通量一孔/一基因筛选平台与一个全基因组阵列的化学合成小干扰RNA文库相结合，在人类细胞中鉴定了94个新的黑素合成调控因子。对使用这种功能基因组学方法确定的基因靶点进行二次验证表明，该方法具有低的假阳性和脱靶率。有趣的是，在这项分析中，自噬途径的几个成分被确定为黑色素产生的新调节因子。对这些假定的自噬调节因子的详细验证表明，这些基因在体外和体内都影响黑素生成。在这个提案中，我们定义了自噬调节基因控制人类皮肤中色素产生的机制。初步研究将验证这些假定的自噬调节因子是否影响MNT-1细胞和正常黑素细胞中黑色素的产生和自噬。将利用一种明确的方法来确定自噬调节器枯竭对向黑素小体运送特定货物的影响。生物化学方法将被用来鉴定与新型自噬调节剂相互作用的黑素小体成分。最后，将利用人类皮肤等效模型来确定个体自噬调节因子对人类皮肤黑色素产生的贡献。通过这些研究，我们希望确定黑素细胞如何利用自噬机制将特定的蛋白质货物运送到体内的黑素小体。 公共卫生相关性： 项目简介(相关)黑色素保护皮肤、眼睛和神经系统免受有毒侮辱，并在皮肤病(黄褐斑和白癜风)、神经系统疾病(帕金森氏病)、听力障碍(瓦登堡综合征)和眼科疾病(老年性黄斑变性)中受到异常调节。基于siRNAi的全基因组功能基因组学最近确定了几个已知的和可能的自噬调节因子影响人类细胞中的黑素生成。在这项提案中，我们确定了自噬的调节者也如何影响黑色素的产生。通过这些研究，我们将深入了解黑素细胞囊泡运输的调节，以及这些过程是如何影响黑素生成的。

DESCRIPTION (provided by applicant): Abstract Melanin protects the skin and eyes from the harmful effects of UV irradiation, protects neural cells from toxic insults, and is required for sound conduction in the inner ear. Aberrant regulation of melanogenesis underlies skin disorders (melasma and vitiligo), neurologic disorders (Parkinson's disease), auditory disorders (Waardenburg's syndrome), and opthalmologic disorders (age related macular degeneration). Extensive studies have identified over 150 genes that regulate melanin production in human cells, but have not yet yielded a complete understanding of the pathogenesis of disorders of melanin production. Recently, we combined a high-throughput cell-based one-well/one-gene screening platform with a genome-wide arrayed synthetic library of chemically synthesized small interfering RNAs to identify 94 novel regulators of melanogenesis in human cells. Secondary validation of gene targets identified using this functional genomics approach revealed that the approach had a low-false positive and off-target rate. Intriguingly, several components of the autophagy pathway were identified as novel regulators of melanin production in this analysis. Detailed validation of these putative autophagy regulators revealed that these genes impacted melanogenesis both in vitro and in vivo. In this proposal, we define the mechanism by which autophagy regulatory genes control pigment production in human skin. Initial studies will validate whether these putative autophagy regulators impact both melanin production and autophagy in MNT-1 cells and normal melanocytes. A defined approach will be utilized to determine the impact of depletion of autophagy regulators on the delivery of specific cargo to the melanosome. Biochemical approaches will be utilized to identify melanosome components that interact with novel autophagy regulators. Finally, a human skin equivalent model will be utilized to determine the contribution of individual autophagy regulators to melanin production in human skin. Through these studies, we hope to determine how the melanocyte utilizes the autophagy machinery to deliver specific protein cargo to the melanosome in vivo. PUBLIC HEALTH RELEVANCE: Project Narrative (relevance) Melanin protects the skin, eyes, and neurologic system from toxic insults and is aberrantly regulated in skin disorders (melasma and vitiligo), neurologic disorders (Parkinson's disease), auditory disorders (Waardenburg's syndrome) and opthalmologic disorders (age related macular degeneration). Genome-wide siRNAi-based functional genomics recently determined that several known and putative autophagy regulators impact melanogenesis in human cells. In this proposal, we determine how regulators of autophagy also impact melanin production. Through these studies we will gain insight into the regulation of vesicular trafficking in melanocytes and how these processes impact melanogenesis.