MECHANISMS OF HEME SIGNALING IN EUKARYOTIC CELLS

真核细胞中血红素信号传导机制

• 批准号: 6181211
• 负责人: LI ZHANG
• 金额: $ 23.64万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181211
• 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

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：这是李章五年来支持的一项提案， 研究酵母中血红素信号传导。 这部作品的重点是 因子HAP 1，控制氧感和血红素的主调节器 酿酒酵母中的信号传导。 HAP 1的靶基因包括 细胞色素和厌氧阻遏物ROX 1。 简单域结构 HAP 1的特性是很好的。 DNA结合和二聚化 结构域，属于锌指蛋白的6-cys簇家族， 含有两种重复元件的血红素反应域 结合位点和转录激活结构域。 本提案的关键 DNA的结合和活化受血红素的调节。 DATA HAP 1关联 与细胞提取物中的大复合物（HMC）的结合也受血红素调节。 因此，Zhang的血红素调节模型提出，在低血红素HAP 1中， 通过与细胞中的大复合物结合而被抑制，可能在 单体状态 该HMCc具有DNA结合活性。 高血红素水平铅 血红素与HAP 1的结合以及复合物的解离。 被压抑的 因子现在可以作为二聚体结合DNA并激活转录。 未解决 这个模型中的问题包括： HAP 1与HMC以及血红素如何调节这种相互作用？哪个蜂窝 因素，例如，HMC的组成部分，对于调节是必要的 这些因素又是如何发挥作用的呢？ 目前的提案包括4个 具体目标。 目标1确定HAP 1的最小域， 血红素调节和执行物理表征+血红素，包括一个 NMR结构测定。 目标2调查高情绪的作用 通过诱变HAP 1元素的分子量复合物， 参与HMC的形成。 第三个目标是通过以下方式研究该机制： HAP 1二聚化元件影响血红素调节和转录 activation. 这一目标包括诱变和测试， 分子内相互作用以及旨在确定 HMC中HAP 1的亚基构型。 最终的目的是利用酵母 遗传学、双杂交筛选和生物化学方法来鉴定 参与血红素调节的细胞因子。