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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。缺氧诱导因子1 （HIF-1）是一种转录调节因子，对缺氧和营养剥夺的主要适应性反应，已被证明参与许多缺氧相关疾病，包括心肌、脑和视网膜缺血，以及肺动脉高压和癌症。HIF-1是由HIF-1a和HIF-1b（也称为芳烃受体转运体，ARNT）组成的异源二聚体。这两种蛋白都属于碱性-螺旋-环-螺旋Per-Arnt-Sim （bHLH PAS）转录调节蛋白家族。虽然HIF-1a是功能性HIF-1异源二聚体的低氧响应成分，但由于与包括芳烃受体（AHR）在内的许多其他bHLH PAS蛋白二聚，ARNT是组成性表达的，并且在功能上是混杂的。ARNT/AHR二聚体参与真核生物对二恶英和芳烃多药耐药反应的转录激活。人们认为，AHR与HIF-1a一样，在细胞的细胞质中短暂存在，直到被激活、稳定并易位到细胞核中，在细胞核中与ARNT形成功能性二聚体并激活特定基因的转录。这些蛋白的特异性反应是通过各种效应结合域结构或稳定性途径实现的，然而，通过负责DNA结合和二聚化的高度同源bHLH结构域实现的DNA结合位点也具有显著的特异性。本提案中概述的研究目的是探索bHLH结构域的结构变化，这些结构域导致ARNT， HIF-1和AHR对不同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序列和结构引起的蛋白质-DNA接触的差异，并将使我们能够开始描述耐药性与缺氧反应中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.