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.
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