Mechanistic Studies of Iron Regulation in Yeast

酵母铁调节机制研究

• 批准号: 8517147
• 负责人: Caryn E Outten
• 金额: $ 23.95万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8517147
• 关键词: Address; Affect; Affinity; Aminopeptidase P; Binding; Biochemical; Biological Assay; Biological Models; Cell Nucleus; Cells; Centers for Disease Control and Prevention (U.S.); Chemistry; Collaborations; Complex; Cytosol; DNA; DNA Binding; DNA-Protein Interaction; Dimerization; Disease; Electron Nuclear Double Resonance; Eukaryota; Eukaryotic Cell; Exportins; Genetic Screening; Goals; Health; Hereditary Disease; Human; In Vitro; Iron; Iron Overload; Kinetics; Measures; Mediating; Methods; Modeling; Molecular; Molecular Genetics; Movement; Mutagenesis; Mutation; Nutrition Disorders; Pathway interactions; Process; Proteins; Recruitment Activity; Regulation; Regulon; Role; Saccharomyces cerevisiae; Signal Pathway; Spectrum Analysis; System; Testing; Work; Yeasts; design; dimer; ferryl iron; glutaredoxin; improved; in vivo; iron deficiency; iron metabolism; mutant; nucleocytoplasmic transport; paralogous gene; promoter; protein protein interaction; transcription factor

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.

描述（由申请人提供）：本提案的目标是揭示模型真核生物酿酒酵母中传感和调节细胞内铁的分子机制。为了维持最佳的细胞内铁水平，从酵母到人类的真核细胞中铁的运输和储存受到严格调节。然而，我们对细胞和分子水平上的铁调节机制的理解存在显着差距。我们将通过梳理酵母中铁调节的分子细节并定义铁信号通路中每个成分的作用来解决这些空白。在酵母中，单硫醇谷氧还蛋白 Grx3 和 Grx4、BolA 样蛋白 Fra2 和氨肽酶 P 样蛋白 Fra1 在铁响应信号通路中共同发挥作用，控制铁响应转录因子 Aft1 的核质穿梭。在铁充足的条件下，该途径诱导 Aft1（可能还有其旁系同源物 Aft2）二聚化，有利于它们定位到细胞质中。 我们已经证明 Fra2 与 Grx3 或 Grx4 形成 [2Fe-2S]2+ 桥接异二聚体，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[2Fe-2S]簇转移到Aft2，促进Aft2二聚化。 Aft1/2 二聚化反过来又被认为可以抑制铁调节子的激活。尽管我们在定义该信号通路中多个组件之间的分子相互作用方面取得了重大进展，但铁传感和调节机制的一些关键方面仍未得到解决，并将在本提案中得到解决。我们将揭示 Fe-S 从 Fra2-Grx3/4 转移到 Aft1 和 Aft2 的机制细节，并通过使用诱变、生化分析和生物物理光谱来检查 Fra1 对该过程的影响，以检查簇转移到 Aft1 和 Aft2 的动力学和效率，并识别 Grx3/Grx4/Fra1/Fra2/Aft1/Aft2 中对两者都至关重要的残基 供体-靶标识别和 Fe-S 转移（目标 1）。我们将通过确定 Fra2、Grx3/4 或 Fra1 的突变如何影响铁信号通路内的蛋白质-蛋白质相互作用、Fe 与 Aft1/2 的结合以及体内 Aft1/2 亚细胞定位和二聚化，来测试 Fra-Grx 复合物是否将 Fe-S 簇转移到 Aft1/2 并诱导体内二聚化（目标 2）。最后，我们将通过测试 Fra-Grx 介导的 Aft1/2 二聚化是否破坏 Aft1/2 向细胞核的运动、Aft1/2 与其 DNA 靶标的结合或使用体内和体外蛋白质-蛋白质和转录共激活剂的募集，来阐明 Fra-Grx 介导的 Aft1/2 二聚化抑制铁调节子激活的机制。 蛋白质-DNA 相互作用测定（目标 3）。由于该途径中的几个关键蛋白质在人类中是保守的并且对于生存至关重要，因此利用酵母系统来定义它们的功能和物理相互作用将提供对它们在人类铁代谢中的作用的基本了解。