NF-kappaB Signaling Pathway Probed with Natural Products

描述（由申请人提供）：转录因子NF-κ B在正常生理过程和疾病中调节基因表达中起关键作用。它是正常胚胎发生以及对感染的免疫反应所必需的。NF-κ B的失调也与从炎症到癌症的大量人类疾病有关。尽管在过去的十年中，在理解导致NF-κ B活化的早期信号传导机制方面取得了很大进展，但NF-κ B在与细胞核中的DNA结合后如何传递其信号直到最近几年才开始引起人们的关注。海洋天然产物pateamine A是一种有效的免疫抑制剂，可抑制T细胞受体刺激的白细胞介素2基因的转录。初步研究表明，帕特胺A选择性抑制NF-κ B信号传导，并在较小程度上抑制AP-1。对NF-κ B信号通路中已知步骤的系统检查显示，帕特胺A不影响早期信号步骤，直到NF-κ B与DNA结合。相反，发现帕特胺A阻断NF-κ B的反式激活活性，使得帕特胺A成为研究NF-κ B的核信号传导机制的有价值的探针。为了鉴定帕丁胺A的分子靶点，合成了生物素-帕丁胺A缀合物，其使得能够检测、分离和鉴定两种帕丁胺A结合蛋白。这些酪蛋白胺A结合蛋白以前没有被证明参与NF-κ B的调节。本申请的主要目的是确认这些蛋白质与帕太明A在体外和体内的分子相互作用，并验证这些蛋白质作为帕太明A抑制NF-κ B的介质的生理相关性。这些蛋白质在调节核信号通过NF-κ B的生理功能将进行研究。由于这些蛋白质参与了几种细胞过程，因此将研究帕丁胺A是否影响推定的帕丁胺A结合蛋白的其他已知细胞活性。最后，将进行基因芯片分析，以通过比较用帕特胺A处理的小鼠胚胎成纤维细胞的基因表达谱和来自NF-κ B和IkappaB激酶敲除动物的基因表达谱来确定帕特胺A对IkappaB-NF-κ B信号传导途径的特异性。此外，晶体和溶液的结构之间的复合物的帕特胺A和它的结合蛋白质将获得，以促进未来的设计和合成的新的和简化的帕特胺A类似物作为候选人的抗癌和免疫抑制药物的发展。

DESCRIPTION (provided by applicant): The transcription factor NF-kappaB plays key roles in regulating gene expression in both normal physiological processes and in diseases. It is required for normal embryogenesis as well as immune responses to infections. Disregulation of NF-kappaB has also been implicated in a large number of human diseases from inflammation to cancer. Although much progress has been made in the past decade on understanding the early signaling mechanisms leading to NF-kappaB activation, how NF-kappaB transmits its signal after its binding to DNA in the nucleus has only begun to attraction attention in recent years. A marine natural product named pateamine A was found to be a potent immunosuppressive agent, inhibiting the T cell receptor-stimulated transcription of interleukin-2 gene. Preliminary studies revealed that pateamine A selectively inhibits NF-kappaB signaling and to a lesser extent, that of AP-1. A systematic examination of the known steps in the NF-kappaB signaling pathway revealed that pateamine A does not affect early signaling steps up to the DNA binding by NF-kappaB. Instead, pateamine A was found to block the transactivation activity of NF-kappaB, making pateamine A a valuable probe to study the nuclear signaling mechanism of NF-kappaB. To identify the molecular targets of pateamine A, a biotin-pateamine A conjugate was synthesized, which enabled the detection, isolation and identification of two pateamine A-binding proteins. These pateamine A-binding proteins have not been previously shown to be involved in NF-kappaB regulation. The main objective of this application is to confirm the molecular interaction between these proteins and pateamine A both in vitro and in vivo and to verify the physiological relevance of these proteins as mediators of the inhibition of NF-kappaB by pateamine A. The physiological functions of these proteins in regulating nuclear signaling by NF-kappaB will be investigated. As these proteins have been implicated in several cellular processes, whether pateamine A affects the other known cellular activities of the putative pateamine A-binding proteins will be investigated. Lastly, gene chip analyses will be performed to determine the specificity of pateamine A for the IkappaB-NF-kappaB signaling pathway by comparing the gene expression profiles of mouse embryo fibroblasts treated with pateamine A and those derived from NF-kappaB and IkappaB kinase knockout animals. In addition, crystal and solution structures of the complexes between pateamine A and its binding proteins will be obtained to facilitate the future design and synthesis of novel and simplified pateamine A analogs as candidates for development of anticancer and immunosuppressive drugs.