Molecular basis of selectivity for coactivator recruitment by ARNT PAS domains

描述（由申请人提供）：异常细胞生长产生缺氧微环境，抑制增殖；肿瘤通过缺氧反应途径的组成性激活来规避这种稳态调节，该途径上调无氧代谢和血管生成的基因，以促进细胞生长，否则将受到低氧的限制。缺氧反应途径受氧敏感的HIF-a（缺氧诱导因子-a）亚基和ARNT（芳烃核转运子）的转录调控，它们形成异二聚体bHLH-PAS转录激活子，结合DNA中的靶序列并招募共激活子启动转录。本提案的目的是通过检查ARNT PAS结构域共激活子募集的结构和功能基础来研究ARNT在转录激活中的作用。我们假设ARNT PAS结构域通过在这个小的模块化结构域上利用相反的接口同时介导与HIF-a和共激活子的相互作用。在Aim 1中，我们将确定ARNT的最小基序：共激活因子相互作用，并定义ARNT共激活因子特异性的分子基础。在目标2中，我们将确定ARNT的结构基础：共激活因子相互作用，并描述体内复杂破坏的功能后果。在Aim 3中，我们将筛选人工配体的ARNT PAS结构域，以识别破坏ARNT：共激活剂相互作用的小分子拮抗剂。这项工作将研究ARNT如何参与缺氧反应的转录调节，并确定阻断ARNT功能的化合物，这些化合物在预防肿瘤生长方面具有潜在的治疗价值。

DESCRIPTION (provided by applicant): Aberrant cellular growth creates oxygen-poor microenvironments that serve to inhibit proliferation; tumors circumvent this homeostatic regulation through constitutive activation of the hypoxia response pathway, which up-regulates genes for anaerobic metabolism and angiogenesis to facilitate cellular growth that would otherwise be limited by low oxygen. The hypoxia response pathway is transcriptionally regulated by an oxygen-sensitive HIF-a (hypoxia-inducible factor-a) subunit and ARNT (aryl hydrocarbon nuclear translocator), which form a heterodimeric bHLH-PAS transcriptional activator that binds target sequences in DNA and recruits coactivators to initiate transcription. The objective of this proposal is to investigate the role of ARNT in transcriptional activation by examining the structural and functional basis of coactivator recruitment by ARNT PAS domains. We hypothesize that ARNT PAS domains simultaneously mediate interaction with HIF-a and coactivators by utilizing opposing interfaces on this small, modular domain. In Aim 1, we will identify minimal motifs for ARNT:coactivator interactions and define the molecular basis of coactivator specificity for ARNT. In Aim 2, we will determine the structural basis for the ARNT:coactivator interaction and characterize functional consequences of complex disruption in vivo. In Aim 3, we will screen ARNT PAS domains for artificial ligands to identify small molecule antagonists that disrupt the ARNT:coactivator interaction. This work will examine how ARNT contributes to transcriptional regulation in response to hypoxia and identify compounds that block ARNT function with potential therapeutic value in preventing tumor growth.