Aldehyde dehydrogenase: A novel regulator of melanin biogenesis

描述（由申请人提供）：虽然遗传和生物化学研究已经鉴定出调节色素产生的基因，但黑素细胞将紫外线照射与黑素产生偶联的机制尚未确定。进行全基因组siRNA筛选以鉴定调节黑素细胞和黑素瘤细胞中黑素生成的人基因。验证研究确定，这种新的，无偏见的方法具有较低的假阳性和脱靶率，并确定了调节黑色素分泌和黑色素生物合成的新基因。在该分析中鉴定的两个经验证的基因靶标，醛脱氢酶1和醛脱氢酶9，是参与脂质过氧化反应代谢副产物的细胞基因。初步数据表明，Aldhl和AldhQ影响正常黑素细胞和黑素瘤细胞中酪氨酸酶和MITF的表达。该提案的总体目标是了解Aldhl和Aldh9调节黑色素生物合成的机制。具体目标1将确定Aldhl和AldhQ调节酪氨酸酶和MITF蛋白水平的机制。具体目标2将基于siRNA的功能丧失方法与化学方法结合以定量黑色素以确定Aldh活性是否影响褐黑素产生。具体目标3将功能丧失方法与标准平板测定结合，以确定UVA和UVB诱导的色素产生是否需要Aldh活性。后续实验将确定Aldhl和Aldh9是否通过维持低细胞内活性氧和脂质过氧化产物水平来维持黑色素产生的许可环境。具体目标4将使用新的成像模式和shRNA功能丧失技术来确定Aldhl和Aldh9是否影响人类皮肤等效模型中的色素产生。通过这些研究，我们将确定Aldhl和Aldh9调节黑色素产生的机制，并更全面地了解UV诱导的色素产生的分子介质。相关性（见说明）：紫外线诱导的黑色素生成保护皮肤免受紫外线的致癌作用。虽然已经确定了调节色素产生的核心基因，但对黑色素细胞如何响应紫外线产生更多黑色素知之甚少。在这个提议中，我们表征了UV诱导的晒黑的假定调节因子（Aldhl和Aldh9），并确定这些基因是否调节人类皮肤模型中的晒黑。

DESCRIPTION (provided by applicant): While genetic and biochemical investigations have led to the identification of genes that regulate pigment production, the mechanisms by which melanocytes couple UV irradiation to melanin production has not been identified. Genome wide siRNA screening was performed to identify human genes that regulate melanogenesis in melanocytes and melanoma cells. Validation studies determined that this novel, unbiased approach had a low false positive and off-target rate, and identified novel genes that regulate melanin secretion and melanin biosynthesis. Two validated gene targets identified in this analysis, aldehyde dehydrogenase 1 and aldehyde dehydrogenase 9, are cellular genes involved in metabolizing byproducts of lipid peroxidation reactions. Preliminary data indicates that Aldhl and AldhQ impact the expression of tyrosinase and MITF in normal melanocytes and melanoma cells. The overall goal of this proposal is to understand the mechanism by which Aldhl and Aldh9 regulate melanin biosynthesis. Specific aim 1 will determine the mechanism by which Aldhl and AldhQ regulate tyrosinase and MITF protein levels. Specific aim 2 couples siRNA based loss of function approaches with chemical approaches to quantitate melanin to determine if Aldh activity impacts pheomelanin production. Specific aim 3 will couple loss of function approaches with standard plate assays to determine whether Aldh activity is required for UVA and UVB induced pigment production. Followup experiments will determine if Aldhl and Aldh9 maintain a permissive environment for melanin production by maintaining low intracellular levels of reactive oxygen species and lipid peroxidation products. Specific aim 4 will use novel imaging modalities and shRNA loss of funciton technology to determine whether Aldhl and Aldh9 impact pigment production in a human skin equivalent model. Through these studies, we will determine the mechanism by which Aldhl and Aldh9 regulate melanin production and gain a more complete understanding of the molecular mediators of UV induced pigment production. RELEVANCE (See instructions): UV induced melanin production protects the skin from the carcinogenic effects of UV light. While core genes that regulate pigment production have been identified, little is understood about how melanocytes produce more melanin in response to UV. In this proposal, we characterize putative regulators (Aldhl and Aldh9) of UV induced tanning and determine whether these genes regulate tanning in human skin models.