NOVEL RECEPTORS OF VITAMIN A IN THE CYTOPLASM

描述(申请人提供)：我们对维生素A营养缺乏导致免疫缺陷的机制的研究发现，维甲酸和锌在信号转导中具有前所未有的作用。当被第二信使样活性氧激活时，丝氨酸/苏氨酸激酶需要结合维生素A作为辅助因子。我们的目标已经从定义蛋白激酶C上的受体位置，到探讨其生物学意义，以及类维A酸类结合域本身的功能。这些结构与锌的配位结构相吻合，这种结构被称为锌指，这种结构普遍存在于多种信号分子中。尽管它们看起来像刚性结构，但我们相信它们的真正目的是充当可逆铰链。这一“关键”假设是这一提议的核心，它是基于这样的发现，即活性氧激活PKC需要释放锌离子。出乎意料的是，按照统一的逻辑，经典的第二信使二酰甘油和肿瘤促进剂佛波酯也会引起锌动员。体外和体内的证据表明，锌的释放、协调中心的溶解和PKC的展开之间存在因果关系，这是催化活性的前奏。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元件在PKC的展开和激活中的作用。四个相互关联的方法将被用来定义：核磁共振(Aim 1)中锌指的结构变化(Aim 1)，质谱学中导致锌指分解的共价生化变化(Aim 2)，突变中锌指与PKC其他结构域的关系(Aim 3)，FRET成像中锌指铰链在体内的构象变化。这些研究还将揭示活性氧第二信使功能的潜在机制。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根本范式转变的前景很高。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.