An Oxygen-Sensing Network Involving Heme and Chaperones

描述(申请人提供)：这项提议的广泛的、长期的目标是阐明真核生物中氧感应、血红素信号和伴侣作用的分子机制。在许多生物体内，氧气对维持关键的细胞功能至关重要，而血红素是氧气感知和利用的核心。在人类中，氧气感知和调节或血红素合成的缺陷会导致人类的严重疾病，包括癌症、门静脉症以及呼吸系统和血液系统疾病。因此，了解氧气是如何感受到的，以及血红素和分子伴侣如何促进氧气和全球基因调控，对于改善人类健康非常重要。这项建议使用酵母作为一个模型系统来研究真核生物中的氧气感应、血红素信号和伴侣作用。在酵母中，血红素通过控制血红素激活蛋白Hap1的转录活性来调节许多基因的氧调节。分子伴侣Hsp90和Hsp70与Hap1结合，促进Hap1活性的血红素调节。在这一提议中的实验将严格检验以下假设：(1)Hapl-多环蛋白复合体直接与DNA结合，促进转录失活和抑制；(2)氧水平通过血红素合成来感知；(3)Hapl和Hapl在全球氧气感知和全球转录调控中发挥关键作用。 其具体目的是(1)阐明和比较HAPL激活和抑制转录的分子机制，(2)剖析细胞内血红素水平与氧水平联系的分子机制，(3)确定HAPL和HAPL在氧感知和调节中的整体作用。将使用生化、遗传和先进的微阵列技术和计算算法来实现这些目标。所获得的知识应该有助于理解高等真核生物中氧感应、血红素信号转导和伴侣作用的分子机制。

DESCRIPTION (provided by applicant): The broad, long-term objective of this proposal is to elucidate the molecular mechanisms governing oxygen sensing, heme signaling and chaperone action in eukaryotes. Oxygen is vital for maintaining critical cellular functions in many living organisms, and heme is central to oxygen sensing and utilization. In humans, defects in oxygen sensing and regulation or in heme synthesis cause serious diseases in humans, including cancers, porphyrias, and respiratory and hematological diseases. Thus, understanding how oxygen is sensed and how heme and molecular chaperones promote oxygen and global gene regulation is important for improving human health. This proposal uses yeast as a model system for investigating oxygen sensing, heme signaling and chaperone action in eukaryotes. In yeast, heme mediates oxygen regulation of many genes by controlling the transcriptional activity of the heme activator protein Hapl. Molecular chaperones Hsp90 and Hsp70 bind to Hapl and promote heme regulation of Hapl activity. Experiments in this proposal will rigorously test the following hypotheses: (1) Hapl-multichaperone complexes bind directly to DNA and promote transcriptional aactivation and repression; (2) the oxygen level is sensed through heme synthesis; and (3) heme and Hapl play key roles in global oxygen sensing and global transcription regulation. The specific aims are (1) to clarify and compare the molecular mechanisms by which Hapl activates and represses transcription, (2) to dissect the molecular mechanism by which intracellular heme level is linked to oxygen level, and (3) to determine the global roles of heme and Hapl in oxygen sensing and regulation. Biochemical, genetic, and advanced microarray technologies and computational algorithms will be used to accomplish these aims. The gained knowledge should facilitate the understanding of the molecular mechanisms of oxygen sensing, heme signaling and chaperone action in higher eukaryotes.