Role of FA-BRCA pathway in stem cell resistance to acetaldehyde

项目摘要 乙醛既是一种外源性致癌物，又是一种内源性代谢产物。而乙醛在 酒精饮料的消费是众所周知的肝毒素和贡献者的发育异常 与胎儿酒精综合征有关，乙醛的内源性产生只是最近才与遗传疾病有关， 范可尼贫血。乙醛形成DNA加合物，包括链间DNA交联，由范可尼修复 贫血/BRCA途径。酒精代谢还诱导线粒体自噬，或线粒体自噬，作为细胞保护因子。 机制最近描述了范可尼贫血/BRCA蛋白在线粒体自噬中的一种全新功能，强调了 FA/BRCA对乙酰丙酮酸诱导的细胞损伤的潜在作用。范可尼贫血和胎儿酒精 综合征以先天性异常为特征，范可尼贫血和酒精易导致重叠集合 上皮肿瘤 细胞对乙醛的敏感性知之甚少，并且可能受内源性乙醛产生的影响。 乙醛、代谢、共价DNA和蛋白质加合物的产生以及修复途径。天真和准备 胚胎干细胞代表发育中的胚胎中植入前和植入后的多能群体。我们证明 幼稚的胚胎干细胞对1 mM乙醛具有惊人的耐受性，而引发的胚胎干细胞对乙醛的耐受性非常好。 敏感（事实上，我们的合作者，卡罗尔·韦尔博士，常规使用乙醛来选择任何可能出现的引发细胞， 在幼稚胚胎干细胞培养期间）。在初步的数据中，我们表明这两种类型的胚胎干细胞都能产生 乙醛，并表现出不同程度的乙醛与DNA加合物的基础水平。令人惊讶的是，天真的抵抗 胚胎干细胞对乙醛的耐受性并不因乙醛去代谢乙醛脱氢酶的抑制而降低。 在这个建议中，我们将探讨胚胎干细胞中的差异乙醛抗性的机制。 具体来说，在目标1中，我们将利用敏感的DNA内收体分析来研究范可尼贫血/BRCA的作用。 乙醛抗性中的DNA修复途径或线粒体DNA修复。在目标2中，我们将研究 通过FA/BRCA途径对乙醛的线粒体吞噬反应。乙醛抑制sirtuin 3， 脱乙酰酶，最有可能通过耗尽其NAD+底物，导致超乙酰化的线粒体蛋白质组。乙酰化 抑制线粒体自噬。我们将确定FA/BRCA途径是否减少线粒体蛋白乙酰化，从而 通过线粒体自噬实现质量控制。拟议中的研究将产生重要的新信息， 在饮酒和乙醛遗传综合征的背景下，细胞对乙醛的反应 超敏反应

Project Summary Acetaldehyde is both an exogenous carcinogen and endogenous metabolite. While acetaldehyde formed after consumption of alcoholic beverages is well-known as a hepatotoxin and contributor to the developmental abnormalities associated with fetal alcohol syndrome, endogenous production of acetaldehyde was only recently linked to the genetic disease, Fanconi anemia. Acetaldehyde forms DNA adducts, including interstrand DNA crosslinks, repaired by the Fanconi anemia/BRCA pathway. Alcohol metabolism also induces mitochondrial autophagy, or mitophagy, as a cytoprotective mechanism. An entirely new function for Fanconi anemia/BRCA proteins in mitophagy was recently described, highlighting a potential role for the FA/BRCA in responding to acetaldehyde-induced cellular injury. Both Fanconi anemia and fetal alcohol syndrome are characterized by congenital anomalies, and Fanconi anemia and alcohol predispose to an overlapping collection of epithelial neoplasms. Cellular sensitivity to acetaldehyde is poorly understood, and is likely affected by endogenous production of acetaldehyde, metabolism, generation of covalent DNA and protein adducts, and repair pathways. Naïve and primed embryonic stem cells represent pre- and post-implantation pluripotent populations in the developing embryo. We demonstrate that naïve embryonic stem cells are strikingly tolerant of 1 mM acetaldehyde, while primed embryonic stem cells are exquisitely sensitive (in fact, our collaborator, Dr. Carol Ware, uses acetaldehyde routinely to select against any primed cells that may arise during culture of naïve embryonic stem cells). In preliminary data, we show that both types of embryonic stem cell generate acetaldehyde and exhibit basal levels of acetaldehyde adducts with DNA to different degrees. Surprisingly, resistance of naïve embryonic stem cells to acetaldehyde is not diminished by inhibition of acetaldehyde-metabolizing aldehyde dehydrogenases. In this proposal, we will investigate the mechanisms of differential acetaldehyde resistance in embryonic stem cells. Specifically, in Aim 1 we will utilize sensitive DNA adductomic profiling to examine the role of the Fanconi anemia/BRCA DNA repair pathway or mitochondrial DNA repair in acetaldehyde resistance. In Aim 2, we will investigate the regulation of the mitophagic response to acetaldehyde by the FA/BRCA pathway. Acetaldehyde inhibits sirtuin 3, the mitochondrial deacetylase, most likely by depleting its NAD+ substrate, leading to a hyperacetylated mitochondrial proteome. Acetylation inhibits mitophagy. We will determine whether the FA/BRCA pathway reduces mitochondrial protein acetylation, thus enabling quality control via mitophagy. The proposed research will generate important new information for understanding cellular responses to acetaldehyde, both in the context of alcohol consumption and genetic syndromes of acetaldehyde hypersensitivity.