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

剖析自噬调节剂对正常人细胞黑色素生成的影响

• 批准号: 7645335
• 负责人: Anand K Ganesan
• 金额: $ 6.99万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7645335
• 关键词: Age related macular degeneration; Anabolism; Auditory; Autophagocytosis; Autophagosome; Biochemical; Biological; Biological Assay; Biological Process; Cells; Chloasma; Coupled; Disease; Electron Microscopy; Eukaryotic Cell; Event; Eye; Fibroblasts; Gene Targeting; Genes; Human; In Vitro; Individual; Labyrinth; Libraries; Lysosomes; Measures; Melanins; Melanogenesis; Melanoma Cell; Melanosomes; Methods; Modeling; Monitor; Neurologic; Neurons; Normal Cell; Normal tissue morphology; Organelles; Parkinson Disease; Pathogenesis; Pathway interactions; Physiological; Pigments; Process; Production; Proteins; Regulation; Regulator Genes; Role; Screening procedure; Skin; Small Interfering RNA; Structure; System; Validation; Vitiligo; Waardenburg syndrome; abstracting; base; cell behavior; defined contribution; functional genomics; genome-wide; in vivo; insight; keratinocyte; loss of function; melanocyte; nervous system disorder; novel; public health relevance; skin disorder; sound; therapeutic gene; therapeutic target; trafficking; ultraviolet irradiation

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.

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照射的有害影响，保护神经细胞免受有毒伤害，并且是内耳中声音传导所必需的。黑素生成的异常调节是皮肤疾病（黄褐斑和白癜风）、神经系统疾病（帕金森病）、听觉疾病（Waardenburg综合征）和眼科疾病（年龄相关性黄斑变性）的基础。广泛的研究已经确定了超过150个基因，调节人类细胞中的黑色素产生，但尚未产生一个完整的理解的发病机制的黑色素产生障碍。最近，我们将高通量基于细胞的单孔/单基因筛选平台与化学合成的小干扰RNA的全基因组阵列合成文库相结合，以鉴定人类细胞中黑素生成的94种新型调节剂。使用这种功能基因组学方法鉴定的基因靶标的二次验证显示，该方法具有低假阳性率和脱靶率。有趣的是，在这项分析中，自噬途径的几个组分被鉴定为黑色素产生的新调节剂。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。在这个提议中，我们定义了自噬调控基因控制人类皮肤色素产生的机制。初步研究将验证这些假定的自噬调节剂是否影响MNT-1细胞和正常黑素细胞中的黑色素产生和自噬。将使用定义的方法来确定自噬调节剂的耗尽对向黑素体递送特定货物的影响。将利用生物化学方法来鉴定与新型自噬调节剂相互作用的黑素体组分。最后，将利用人类皮肤等效模型来确定个体自噬调节剂对人类皮肤中黑色素产生的贡献。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。 公共卫生关系： 黑色素可保护皮肤、眼睛和神经系统免受有毒物质的侵害，在皮肤疾病（黄褐斑和白癜风）、神经系统疾病（帕金森氏病）、听觉疾病（Waardenburg综合征）和眼科疾病（年龄相关性黄斑变性）中受到异常调节。基于全基因组siRNA的功能基因组学最近确定了几种已知和推定的自噬调节剂影响人类细胞中的黑素生成。在这个提议中，我们确定了自噬的调节剂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中囊泡运输的调节以及这些过程如何影响黑素生成。