NOVEL RECEPTORS OF VITAMIN A IN THE CYTOPLASM

描述（由申请人提供）：我们对营养性维生素A缺乏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。我们的目标已经从蛋白激酶C上受体位点的定义进展到对生物学意义的询问，以及维甲酸结合域本身的功能。这些与锌配位结构一致，称为锌指，在许多信号分子中普遍存在。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作为可逆铰链。这一“关键”假设是该提议的核心，其基础是活性氧激活PKC需要释放Zn 2+离子。出乎意料的是，但与统一的逻辑，经典的第二信使，甘油二酯，和肿瘤促进剂，佛波酯，也导致锌动员。在体外和体内提出的证据表明，锌的释放，溶解的协调中心，并展开的PKC作为催化能力的前奏之间的因果关系。使用结构生物学，生物化学，遗传学和成像方法的组合，我们建议调查锌指的作用，铰链样元素参与PKC的展开和激活。本研究将采用四种相互关联的方法来定义锌指的结构变化：核磁共振法研究锌指的结构变化（目的1），质谱法研究锌指的共价生物化学变化导致锌指的解体（目的2），突变法研究锌指与PKC其他结构域的关系（目的3），FRET成像法研究锌指铰链在体内的构象变化。这些研究还将揭示活性氧的第二信使功能的机制。关于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很高。此外，我们关于维生素A和锌的功能的工作与营养、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.