NF-kappaB Signaling Pathway Probed with Natural Products

描述(申请人提供)：转录因子NF-kappaB在调节正常生理过程和疾病中的基因表达方面发挥关键作用。它是正常胚胎发育和对感染的免疫反应所必需的。核因子-kappaB的失调也与从炎症到癌症的大量人类疾病有关。尽管在过去的十年里，人们在了解导致核转录因子-kappaB活化的早期信号机制方面取得了很大的进展，但核转录因子-kappaB在与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注意。一种名为帕特明A的海洋天然产物被发现是一种有效的免疫抑制剂，可以抑制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，帕替胺A选择性地抑制核因子-kappaB信号转导，在较小程度上抑制AP-1信号转导。对已知的核因子-kappaB信号通路步骤的系统研究表明，帕特胺A不影响核因子-kappaB与DNA结合的早期信号步骤。相反，帕特胺A被发现阻断了核因子-kappaB的反式激活活性，使其成为研究核因子-kappaB核信号机制的有价值的探针。为了确定帕金森胺A的分子靶标，合成了生物素-甲基苯丙胺A偶联物，从而能够检测、分离和鉴定两种帕金森胺A结合蛋白。这些帕特胺A结合蛋白以前没有被证明参与了核因子-kappaB的调节。本研究的主要目的是证实这些蛋白质在体内外与帕金森胺A之间的分子相互作用，并验证这些蛋白质作为介体对核因子-kappaB抑制的生理相关性。这些蛋白质在核因子-kappaB调节核信号中的生理功能将被研究。由于这些蛋白与几个细胞过程有关，因此我们将研究帕特胺A是否会影响其他已知的帕特胺A结合蛋白的细胞活性。最后，基因芯片分析将通过比较帕特胺A处理的小鼠胚胎成纤维细胞与来自NF-kappaB和IkappaB激酶基因敲除动物的基因表达谱，来确定帕特胺A在IkappaB-NF-kappaB信号通路中的特异性。此外，还将获得帕特胺A与其结合蛋白之间的络合物的晶体和溶液结构，以便于将来设计和合成新型和简化的帕特胺A类似物，作为开发抗癌和免疫抑制药物的候选药物。

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.