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MECHANISMS OF HEME SIGNALING IN EUKARYOTIC CELLS

真核细胞中血红素信号传导机制

基本信息

批准号：
6386230
负责人：
LI ZHANG
金额：
$ 24.03万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-05-01 至 2004-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6386230
关键词：
Saccharomyces cerevisiae biological signal transduction cytochromes dimer heme nuclear magnetic resonance spectroscopy protein structure function regulatory gene site directed mutagenesis transcription factor yeast two hybrid system yeasts

项目摘要

DESCRIPTION: This is a proposal by Li Zhang for five years of support to investigate heme signaling in yeast. The work focuses on the transcription factor HAP1, a master regulator controlling oxygen sensing and heme signaling in Saccharomyces cerevisiae. Target genes for HAP1 include cytochromes and the anaerobic repressor ROX1. The simple domain structure of HAP1 is well-characterized. There is a DNA binding and dimerization domain, belonging to the 6-cys cluster family of zinc finger proteins, a heme responsive domain with two types of repeated elements including heme binding sites, and a transcription activation domain. Key to this proposal DNA binding and activation are modulated by heme. DATA HAP1 association with a large complex (HMC) in cell extracts is also regulated by heme. Thus, Zhang's model for heme regulation proposes that in low heme HAP1 repressed by association with a large complex in the cell, possibly in a monomeric state. This HMCc has DNA binding activity. High heme levels lead to heme binding to HAP1 and dissociation of the complex. The de-repressed factor can now bind DNA as a dimer and activate transcription. Unresolved questions in this model include: what is the nature of the association of HAP1 with HMC and how does heme regulate this interaction?, which cellular factors, for example, components of HMC, are necessary for the regulation and how do these factors function? The current proposal consists of 4 specific aims. Aim 1 determines the minimal domain of HAP1 necessary for heme regulation and performs physical characterizations + heme, including a NMR structural determination. Aim 2 investigates the role of the high molecular weight complex by mutagenizing HAP1 elements proposed to be involved in HMC formation. The third aim investigates the mechanism by which HAP1 dimerization elements affect heme regulation and transcriptional activation. This aim includes both mutagenesis and tests for inter and intramolecular interactions as well as experiments designed to determine the subunit configuration of HAP1 in the HMC. The final aim employs yeast genetics, a two-hybrid screen, and biochemical approaches to identify the cellular factors involved in heme regulation.

描述：这是一份由李章提出的五年支持研究酵母中的血红素信号。这项工作的重点是转录 HAP1因子，一种控制氧气感应和血红素的主调节因子酿酒酵母中的信号转导。HAP1的靶基因包括细胞色素和厌氧抑制物ROX1。简单的域结构 HAP1的基因序列已被很好地描述。存在DNA结合和二聚化结构域，属于锌指蛋白的6-半胱氨酸簇家族，a 含两种重复元素的血红素反应结构域，包括血红素结合位点和转录激活结构域。这项提案的关键 DNA的结合和激活受血红素的调节。数据HAP1关联在细胞提取物中有一个大的复合体(HMC)，也受血红素的调节。因此，张的血红素调控模型提出，在低血红素中，HAP1 通过与细胞中的大型复合体的关联来抑制，可能在单体状态。该HMCC具有DNA结合活性。高水平的血红素会导致使血红素与HAP1结合并解离该复合体。被解压的人因子现在可以以二聚体的形式与DNA结合并激活转录。未解决此模型中的问题包括：关联的性质是什么 HAP1和HMC以及血红素是如何调节这种相互作用的？因素，例如，HMC的组成部分，是监管所必需的这些因素是如何发挥作用的呢？目前的提案包括4个明确的目标。目标1确定HAP1的最小结构域血红素调节，并执行物理特性+血红素，包括核磁共振结构测定。目标2调查HIGH的作用建议通过诱变HAP1元件形成分子量复合体参与了HMC的形成。第三个目的是通过以下方式来研究这种机制哪些HAP1二聚化元件影响血红素的调节和转录激活。这一目标既包括诱变，也包括内部和外部试验。分子内相互作用以及旨在确定 HMC中HAP1的亚基配置。最终目标是使用酵母遗传学，双杂交筛选，和生化方法来识别参与血红素调节的细胞因子。