An Oxygen-Sensing Network Involving Heme and Chaperones

描述（由申请人提供）：本提案的广泛、长期目标是阐明真核生物中控制氧传感、血红素信号传导和伴侣作用的分子机制。氧对于维持许多生物体中的关键细胞功能至关重要，而血红素对于氧的感知和利用至关重要。在人类中，氧感应和调节或血红素合成的缺陷会导致人类严重的疾病，包括癌症、卟啉症以及呼吸和血液疾病。因此，了解氧气是如何被感知的，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至关重要。该建议使用酵母作为研究真核生物中的氧传感、血红素信号和伴侣作用的模型系统。在酵母中，血红素通过控制血红素激活蛋白Hapl的转录活性来介导许多基因的氧调节。分子伴侣Hsp 90和Hsp 70与Hapl结合并促进Hapl活性的血红素调节。本研究中的实验将严格验证以下假设：（1）Hapl-多分子伴侣复合物直接与DNA结合并促进转录激活和抑制;（2）氧水平通过血红素合成来感知;（3）血红素和Hapl在整体氧感知和整体转录调控中起关键作用。 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相关的分子机制，以及（3）确定血红素和Hapl在氧传感和调节中的整体作用。生物化学、遗传学和先进的微阵列技术和计算算法将用于实现这些目标。所获得的知识将有助于理解高等真核生物中氧传感、血红素信号和分子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.