New Pathway of Vitamin A Action.

描述(由申请人提供)：一个世纪以来，营养维生素A的枯竭已经被认为会导致一种影响多个器官的复杂的缺乏综合症。缺乏维甲酸(RA)导致转录错误是最好的研究机制。然而，发育、生长、生殖和免疫方面的缺陷是缺乏维生素A本身的后果，因为RA不能完全扭转这些症状。我们和其他人已经描述了在细胞质中运行的视黄醇通路，它独立于转录。此外，我们已经确定CRAF和PKC家族的丝氨酸/苏氨酸激酶是维生素A作用的直接靶点。维生素A结合它们的调节域，并结合维生素A使这些酶敏化氧化还原介导的激活。氧化还原激活是指通过活性氧物种(ROS)作为第二信使而进行的另一种途径。主要目的是通过建立因果关系来证明维生素A在氧化还原信号中所起的重要作用。由于ROS同时激活几种激酶，我们建议使用遗传学方法分别研究两种异构体--α和β对维生素A的依赖性。我们将通过扫描突变来绘制维生素A结合位点的图谱，CRAF已经成功(AIM#1)。利用缺失视黄醇结合位点的突变体PKCalpha和theta，将通过转基因和敲入、细胞系和小鼠在体内和体外探索其生物学功能。IL-2的产生和T细胞的增殖是依赖PKCtheta的免疫重要过程(AIM#2)。我们假设ROS在维生素A的帮助下产生共价修饰，导致锌指的解体，作为激酶展开和激活的前奏。目的#3致力于相关半胱氨酸修饰的生化分析。目的#4将利用活体成像技术，重点研究视黄醇作为辅助因子来调节锌的释放和氧化还原激活后锌指的构象变化。这一结果将建立维生素A的新作用机制，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，并为癌症的预防和治疗开辟新的途径。

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.