New Pathway of Vitamin A Action.

维生素 A 作用的新途径。

• 批准号: 6967756
• 负责人: ULRICH G HAMMERLING
• 金额: $ 34.76万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6967756
• 关键词: RNA interference; binding sites; biological signal transduction; cell line; cofactor; fluorescence microscopy; free radical oxygen; gel mobility shift assay; genetically modified animals; green fluorescent proteins; immunoprecipitation; isozymes; laboratory mouse; mass spectrometry; matrix assisted laser desorption ionization; oxidation reduction reaction; phosphorylation; protein kinase C; protein structure; retinoids; site directed mutagenesis; vitamin A deficiency

DESCRIPTION (provided by applicant): Nutritional vitamin A depletion has been known for a century to cause a complex deficiency syndrome that affects multiple organs. Lack of retinoic acid (RA) leading to faulty transcription is the best studied mechanism. However, deficiencies in development, growth, reproduction and immunity were consequences of absence of vitamin A itself, since RA could not fully reverse these symptoms. We and others have described retinoid pathways that operate in the cytoplasm, independently of transcription. Further, we have identified serine/threonine kinases of the cRaf and PKC families as direct targets of vitamin A action. Vitamin A binds their regulatory domains and bound vitamin A sensitizes these kinases for redox-mediated activation. Redox activation refers to the alternative pathway operating through reactive oxygen species {ROS) as second messenger. The main objective is to prove the essential role that vitamin A plays in redox signaling, by establishing cause /effect relationships. Because ROS activates several kinases at once we propose to study the vitamin A dependence of two isoforms, alpha and theta, separately using a genetic approach. We will map the vitamin A binding site by scanning mutagenesis, already successful with cRaf (AIM #1). Using mutant PKCalpha and theta where retinolbinding sites are deleted, the biological function will be probed in vivo and in vitro with transgenic, as well as knock-in, cell lines and mice. IL-2 production and T cell proliferation are PKCtheta-dependent immunologically significant processes (AIM # 2). We hypothesize that ROS with help of vitamin A produces covalent modifications, leading to disassembly of the zinc-finger as prelude to kinase unfolding and activation. AIM # 3 is devoted to biochemical analyses of pertinent cysteine-modifications. Aim # 4 will focus on retinol as co-factor in mediating the release of zinc and conformation change in the zinc-finger consequent to redox activation, using intra-vital imaging. The results will establish a new mechanism of action of vitamin A, help understand vitamin A deficiency syndromes in reproduction, development and immunity, and open new approaches to prevention and treatment of cancer.

描述（由申请人提供）：世纪以来，已知营养性维生素A缺乏会导致影响多个器官的复杂缺乏综合征。缺乏视黄酸（RA）导致错误的转录是最好的研究机制。然而，发育、生长、生殖和免疫缺陷是缺乏维生素A本身的后果，因为RA不能完全逆转这些症状。我们和其他人已经描述了在细胞质中独立于转录而起作用的类维生素A通路。此外，我们已经确定cRaf和PKC家族的丝氨酸/苏氨酸激酶作为维生素A作用的直接靶点。维生素A结合它们的调节结构域，结合的维生素A使这些激酶对氧化还原介导的活化敏感。氧化还原活化是指通过活性氧（ROS）作为第二信使进行操作的替代途径。主要目的是通过建立因果关系来证明维生素A在氧化还原信号传导中的重要作用。由于活性氧激活几种激酶一次，我们建议研究维生素A依赖的两种亚型，α和θ，分别使用遗传方法。我们将通过扫描诱变绘制维生素A结合位点，这在cRaf（AIM #1）中已经成功。使用其中视黄醇结合位点缺失的突变体PKCalpha和theta，将用转基因以及敲入细胞系和小鼠在体内和体外探测生物学功能。IL-2产生和T细胞增殖是PKC θ依赖性的免疫学重要过程（AIM#2）。我们假设ROS在维生素A的帮助下产生共价修饰，导致锌指的分解，作为激酶解折叠和激活的前奏。AIM#3致力于相关半胱氨酸修饰的生物化学分析。目的4将集中在视黄醇作为辅助因子介导锌的释放和构象变化的锌指随之而来的氧化还原活化，使用活体成像。研究结果将建立维生素A作用的新机制，有助于了解维生素A缺乏综合征在生殖、发育和免疫方面的作用，并为预防和治疗癌症开辟新途径。