Mechanistic Studies of Iron Regulation in Yeast

酵母铁调节机制研究

• 批准号: 8656714
• 负责人: Caryn E Outten
• 金额: $ 24.82万
• 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/8656714
• 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）：该提案的这个目标是揭示在酿酒酵母模型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，铁运输和储存在从酵母到人类的艾尔真核细胞中受到严格调节。但是，我们对细胞和分子水平的铁调节机制的理解存在很大的差距。我们将通过嘲笑酵母中铁调节的分子细节并定义每个组件在铁信号传导途径中的作用来解决这些差距。在酵母中，单硫醇谷歌蛋白GRX3和GRX4，BOLA-LIKE蛋白FRA2和氨基肽酶Plike蛋白FRA1在控制铁响应转录因子Aft1的铁响应性信号途径中共同发挥作用，以控制核细胞的核细胞质量。在铁的条件下，该途径诱导了Aft1的二聚化（可能是其旁系同源物AFT2），有利于它们对胞质溶胶的定位。 我们已经证明了fra2形成[2FE-2S] 2+桥接的异二聚体，具有GRX3或GRX4，并表征了这些复合物的Fe-S配位化学。此外，我们有强有力的证据表明，[2FE-2S] FRA2-GRX3将A [2FE-2S]群集转移到Aft2，从而促进Aft2二聚化。反过来，提议AFT1/2二聚化抑制铁调节的激活。尽管我们在定义该信号通路中几个组件之间的分子相互作用方面取得了重大进展，但铁感应和调节机制的某些关键方面仍未解决，并且将在此提案中解决。我们将通过使用诱变，生物化学分析和生物物理光谱法检查Fe-S从FRA2-GRX3/4到AFT1和AFT2转移的机理细节，并确定FRA1对这一过程的影响捐助者 - 目标识别和FE-S转移（AIM 1）。我们将通过确定FRA2，GRX3/4或FRA1中的突变如何影响铁信号途径中的蛋白质 - 蛋白质相互作用，Fe与Aft1/2亚纤维细胞定位（VIV）（VIV），是否会影响FRA2，GRX3/4或FRA1中的FRA2，GRX3/4或FRA1中的突变如何影响蛋白质 - 蛋白质相互作用，从而诱导蛋白质 - 蛋白质相互作用，从而诱导蛋白质 - 蛋白质相互作用（VIV 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蛋白-DNA相互作用测定（AIM 3）。由于该途径中的几种关键蛋白质在人类中是保守的，对于生存能力至关重要，因此利用酵母系统来定义其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理解。