Molecular basis of selectivity for coactivator recruitment by ARNT PAS domains

描述(由申请人提供)：异常的细胞生长创造了缺氧的微环境，从而抑制了增殖；肿瘤通过结构性激活缺氧反应途径来绕过这种动态平衡调节，缺氧反应途径上调无氧代谢和血管生成的基因，以促进细胞生长，否则细胞生长将受到低氧的限制。低氧反应途径由氧敏感的HIF-a(低氧诱导因子-a)亚单位和ArnT(芳烃核转运体)转录调控，形成一个异源二聚体bHLH-PAS转录激活子，与DNA中的靶序列结合并招募辅助激活子启动转录。这个建议的目的是通过研究Arnt PAS结构域招募辅助激活因子的结构和功能基础，来研究Arnt在转录激活中的作用。我们假设Arnt Pas结构域通过利用这个小的模块化结构域上的相反接口同时调节与HIF-a和共激活因子的相互作用。在目标1中，我们将确定ARNT：辅助激活子相互作用的最小基序，并定义ARNT辅助激活子特异性的分子基础。在目标2中，我们将确定ARNT：共激活子相互作用的结构基础，并表征体内复杂干扰的功能后果。在目标3中，我们将筛选Arnt PAS结构域的人工配体，以确定破坏Arnt：辅活化子相互作用的小分子拮抗剂。这项工作将研究Arnt如何在低氧反应中参与转录调节，并确定阻断Arnt功能的化合物在防止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.