STRUCTURAL STUDIES ON THE BASIC HELIX-LOOP-HELIX DOMAINS OF ARNT, HIF-1 AND AHR

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。缺氧诱导因子-1(HIF-1)是一种转录调控因子，已被证明与许多与缺氧相关的疾病有关，包括心肌、脑和视网膜缺血，以及肺动脉高压和癌症。HIF-1是一种由HIF-1a和HIF-1b(也称为芳烃受体转运体，ARNT)组成的异二聚体。这两种蛋白都属于碱性-螺旋-环-螺旋Per-Arnt-Sim(BHLHPAS)转录调节蛋白家族。虽然HIF-1a是功能性HIF-1异源二聚体的缺氧反应成分，但Arnt是结构性表达的，并且由于与包括芳香烃受体(AHR)在内的其他一些bHLHPAS蛋白二聚体而在功能上混杂。ArnT/AHR二聚体参与了真核生物对二恶英和芳香烃的多药耐药反应的转录激活。人们认为，与HIF-1a一样，AHR短暂地存在于细胞质中，直到它被激活、稳定并移位到细胞核，在那里它与Arnt形成功能性二聚体并激活特定基因的转录。在这些蛋白质中，反应的特异性是通过不同的协同效应结合域结构或稳定途径来实现的，然而，DNA结合位点也有显著的特异性，这是通过高度同源的负责DNA结合和二聚化的bHLH域来实现的。这项研究的目的是探索bHLH域的结构变化导致ArnT、HIF-1和AHR的不同DNA结合位点的特异性。这些功能二聚体的DNA结合形式的结构将揭示由于序列和结构而导致的蛋白质-DNA接触的差异，并将使我们能够开始描述耐药与缺氧反应的功能特异性的结构要求。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A major adaptive response to being deprived of oxygen and nutrients is induced by hypoxia-inducible factor 1 (HIF-1), a transcriptional regulator, which has been demonstrated to be involved in a host of hypoxia related diseases including myocardial, cerebral, and retinal ischemias, as well as pulmonary hypertension and cancer. HIF-1 is a heterodimer comprised of HIF-1a and HIF-1b (also known as the aryl hydrocarbon receptor translocator, ARNT). Both proteins belong to the basic-helix-loop-helix Per-Arnt-Sim (bHLH PAS) family of transcriptional regulator proteins. While HIF-1a is the hypoxically responsive component of the functional HIF-1 heterodimer, ARNT is expressed constitutively and is functionally promiscuous as a result of dimerizing with a number of other bHLH PAS proteins including the Aryl Hydrocarbon Receptor (AHR). The ARNT/AHR dimer is involved in the transcriptional activation of multidrug resistance response to dioxins and aryl hydrocarbons in eukaryotes. It is believed that, like HIF-1a, AHR exists transiently in the cytoplasm of the cell until it is activated, stabilized and translocated to the nucleus, where it forms a functional dimer with ARNT and activates transcription of specific genes. Specificity of response in these proteins is achieved through variety of coeffector binding domain structures or stability pathways, however, there is also a significant specificity for DNA binding sites achieved through the highly homologous bHLH domains responsible for DNA binding and dimerization. This purpose of the studies outlined in this proposal are to explore the variations in structure in the bHLH domains that lead to specificity for different DNA binding sites for ARNT, HIF-1 and AHR. The structures of the DNA bound forms of these functional dimers will reveal differences in protein-DNA contacts resulting from sequence and structure, and will allow us to begin to delineate the structural requirements for functional specificity in drug resistance versus the hypoxic response.