NOVEL RECEPTORS OF VITAMIN A IN THE CYTOPLASM

描述（由申请人提供）：我们对营养维生素A缺失引起的免疫缺陷机制的研究发现了类维生素A和锌在信号转导中的前所未有的作用。当被第二个类似信使的活性氧激活时，丝氨酸/苏氨酸激酶需要结合的维生素A作为辅助因子。我们的目标已经从蛋白激酶C上受体位点的定义进展到对生物学意义的探究，以及类视黄酮结合域本身的功能。这些与锌协同结构相吻合，被称为锌指，在许多信号分子中普遍存在。尽管它们的外观是刚性结构，但我们相信它们的真正目的是充当可逆铰链。这个“关键”假设是该提议的核心，它是基于活性氧激活PKC需要释放Zn2+离子的发现。出乎意料的是，在统一的逻辑下，经典的第二信使二酰基甘油和肿瘤促进剂佛波酯也会引起锌的动员。体外和体内证据表明锌的释放、配位中心的溶解和PKC的展开之间存在因果关系，这是催化能力的前奏。结合结构生物学、生物化学、遗传学和影像学方法，我们建议研究锌指作为铰链样元件参与PKC的展开和激活的作用。四种相互关联的方法将被应用于定义：通过核磁共振（Aim 1）导致锌指的结构变化，通过质谱（Aim 2）导致锌指分解的共价生化变化，通过诱变锌指与PKC其他区域的关系（Aim 3），通过FRET成像在体内锌指铰链的构象变化。这些研究也将揭示活性氧第二信使功能的机制。视黄醇和锌离子在信号转导中的作用以及如何看待锌指的基本范式转变的前景是很高的。此外，我们对维生素A和锌的功能的研究与营养、免疫系统和癌症直接相关。

DESCRIPTION (provided by applicant): Our inquiries into the mechanism of the immunodeficiency caused by nutritional vitamin A depletion have uncovered an unprecedented role for retinoids and zinc in signal transduction. When activated by the second messenger-like reactive oxygen species, serine/threonine kinases required bound vitamin A as co-factor. Our objectives have progressed from the definition of the receptor sites on Protein Kinase C to inquiries on the biological significance, and to the function of the retinoid binding domains themselves. These coincide with zinc-coordinated structures, named zinc-fingers, that are prevalent in a multitude of signalling molecules. Despite their appearance as rigid structures we believe their true purpose is to serve as reversible hinges. This "linchpin" hypothesis, central to the proposal, is based on the findings that activation of PKC by reactive oxygen entails the release of Zn2+ ions. Unexpectedly, but with unifying logic, the classic 2nd messenger, diacylglycerol, and the tumor promoter, phorbol ester, also cause zinc mobilization. Evidence raised in vitro and in vivo suggest a causal relationship between zinc release, dissolution of the coordination center, and unfolding of PKC as a prelude for catalytic competence. Using a combination of structural biological, biochemical, genetic and imaging approaches we propose to investigate the role of zinc-fingers as hinge-like elements involved in the unfolding and activation of PKC. Four interrelated approaches will be applied to define: Structural changes of zinc-fingers by NMR (Aim 1), Covalent biochemical changes that lead to zinc-finger disassembly by Mass Spectrometry (Aim 2), Relationship of zinc-fingers to other domains of PKC by mutagenesis (Aim 3), Conformation change of zinc-finger hinge in vivo by FRET imaging. These studies will also uncover the mechanism underlying the 2nd messenger function of reactive oxygen. The prospects are high for a fundamental paradigm shift on the role of retinol and zinc ions in signal transduction and how zinc-fingers are to be viewed. Furthermore, our work on the function of vitamin A and zinc is of immediate relevance to nutrition, the immune system and cancer.