Mechanistic Studies of Iron Regulation in Yeast

描述(申请人提供)：这项建议的目标是揭示在真核生物酿酒酵母模型中感应和调节细胞内铁的分子机制。为了维持细胞内最佳的铁水平，铁的运输和储存在从酵母到人类的真核细胞中受到严格调控。然而，我们在细胞和分子水平上对铁的调节机制的理解存在着显著的差距。我们将通过梳理酵母中铁调节的分子细节并确定铁信号通路中每个成分的作用来解决这些差距。在酵母中，单硫醇谷氧还蛋白Grx3和Grx4、类BoLA蛋白Fra2和类氨基肽酶P蛋白Fra1共同作用于铁响应信号通路，该信号通路控制铁响应转录因子Aft1的核质穿梭。在铁充足的条件下，这一途径诱导Aft1(可能还有它的副产物Aft2)的二聚化，有利于它们定位到胞浆。 我们证明了FRA2与Grx3或Grx4形成[2Fe-2S]2桥联杂二聚体，并表征了这些配合物的Fe-S配位化学。此外，我们有强有力的证据表明，[2Fe-2S]Fra2-Grx3将一个[2Fe-2S]簇转移到Aft2，促进了Aft2的二聚化。在1/2二聚化之后，又提出了抑制铁调节子激活的建议。尽管我们在确定这一信号通路中几个组分之间的分子相互作用方面取得了重大进展，但铁感应和调控机制的一些关键方面仍然没有解决，将在本提案中解决。我们将揭示Fe-S从Fra2-Grx3/4转移到Aft1和Aft2的机制细节，并通过突变、生化分析和生物物理光谱确定Fra1对这一过程的影响，以检测簇转移到Aft1和Aft2的动力学和效率，并确定Grx3/Grx4/Fra1/Fra2/Aft1/Aft2中对供体-靶标识别和Fe-S转移(目标1)至关重要的残基。我们将通过确定Fra2、Grx3/4或Fra1的突变如何影响铁信号通路中的蛋白质-蛋白质相互作用、铁与Aft1/2的结合以及Aft1/2在体内的亚细胞定位和二聚化来测试Fra-GRX复合体是否将Fe-S簇转移到Aft1/2并在体内诱导二聚化(AIM 2)。最后，我们将通过检测Fra-GRX介导的Aft1/2二聚化是否扰乱Aft1/2向核的运动、Aft1/2与其DNA靶标的结合或利用体内和体外蛋白质-蛋白质和蛋白质-DNA相互作用分析(AIM 3)来阐明Fra-GRX介导的Aft1/2二聚化抑制铁调节子激活的机制(Aim 3)。由于这一途径中的几个关键蛋白在人类中是保守的，对生存是必不可少的，利用酵母系统来定义它们的功能和物理相互作用将提供一个对它们在人类铁代谢中的作用的基本了解。 公共卫生相关性：缺铁和铁超载都是严重的人类健康问题：缺铁是世界上最常见和最普遍的营养紊乱，而铁过载紊乱是美国最常见的遗传性疾病之一。这项提案旨在以面包师酵母为模型系统，定义保持最佳细胞内铁水平的传感和控制机制。

DESCRIPTION (provided by applicant): This goal of this proposal is to uncover the molecular mechanisms for sensing and regulating intracellular iron in the model eukaryote S. cerevisiae. To maintain optimal intracellular iron levels, iron transport and storage is tightly regulated in al eukaryotic cells ranging from yeast to humans. However, there are significant gaps in our understanding of iron regulation mechanisms at the cellular and molecular level. We will address these gaps by teasing out the molecular details of iron regulation in yeast and defining the roles of each component in the iron signaling pathway. In yeast, the monothiol glutaredoxins Grx3 and Grx4, the BolA- like protein Fra2, and the aminopeptidase P-like protein Fra1 function together in an iron-responsive signaling pathway that controls nucleocytoplasmic shuttling of the iron-responsive transcription factor Aft1. Under iron replete conditions, this pathway induces dimerization of Aft1 (and presumably its paralog Aft2), favoring their localization to the cytosol. We have demonstrated that Fra2 forms [2Fe-2S]2+-bridged heterodimers with Grx3 or Grx4 and characterized the Fe-S coordination chemistry of these complexes. In addition, we have strong evidence that [2Fe-2S] Fra2-Grx3 transfers a [2Fe-2S] cluster to Aft2, facilitating Aft2 dimerization. Aft1/2 dimerization, in turn, is proposed to inhibit activation of the iron regulon. Despite our significant progress in defining the molecular interactions between several components in this signaling pathway, some key aspects of the iron sensing and regulation mechanism remain unresolved and will be addressed in this proposal. We will uncover the mechanistic details of Fe-S transfer from Fra2-Grx3/4 to Aft1 and Aft2 and determine the impact of Fra1 on this process by using mutagenesis, biochemical analysis, and biophysical spectroscopy to examine the kinetics and efficiency of cluster transfer to Aft1 and Aft2 and identify residues in Grx3/Grx4/Fra1/Fra2/Aft1/Aft2 that are critical for both donor-target recognition and Fe-S transfer (Aim 1). We will test whether the Fra-Grx complex transfers an Fe-S cluster to Aft1/2 and induces dimerization in vivo by determining how mutations in Fra2, Grx3/4, or Fra1 affect protein-protein interactions within the iron signaling pathway, Fe binding to Aft1/2, and Aft1/2 subcellular localization and dimerization in vivo (Aim 2). Finally, we will elucidate the mechanism by which Fra-Grx mediated Aft1/2 dimerization inhibits activation of the iron regulon by testing if Fra-Grx-mediated dimerization of Aft1/2 disrupts movement of Aft1/2 to the nucleus, binding of Aft1/2 to its DNA targets, or recruitment of transcriptional co-activators using both in vivo and in vitro protein-protein and protein-DNA interaction assays (Aim 3). Since several key proteins in this pathway are conserved in humans and essential for viability, exploiting the yeast system to define their functional and physical interactions will provide a fundamental understanding of their roles in human iron metabolism. PUBLIC HEALTH RELEVANCE: Both iron deficiency and iron overload are significant human health issues: iron deficiency is the most common and widespread nutritional disorder in the world, while iron overload disorders are some of the most common genetic disorders in the U.S. This proposal is designed to define the sensing and control mechanisms for maintaining optimal intracellular iron levels using bakers' yeast as a model system.