Regulation Of Nuclear Factor-kappa B Activity

IkappaB激酶(IKK)催化亚基在细胞因子介导的核因子-kappaB信号转导中起着关键作用，而核因子-kB功能的丧失似乎可以抑制炎症和肿瘤的发生。曼努霉素A是一种天然环氧奎宁类化合物，是一种高效、选择性的法尼基转移酶抑制剂，具有抗肿瘤活性。我们最近的研究结果表明，甘露霉素A能迅速、选择性和有效地抑制一些细胞系和原代培养的大鼠肝细胞中由肿瘤坏死因子α刺激的IKK活性。出乎意料的是，其他类别的法尼基转移酶抑制剂没有抑制作用。为了探讨甘露霉素A作用的分子机制，我们用构建的IKKalpha和IKKβ载体转染人肝癌细胞株HepG2，发现在甘露霉素A的存在下，IKK活性受到直接抑制，并伴随有稳定的同型IKKβ二聚体的形成。通过构建IKKβ的ALA点突变体来研究IKKβ的共价二聚化可能是由于亲核攻击MANUMYCIN A的环喹类核心而导致的。在Cys-179的激活环中突变的表达IKKβ的细胞表现出相似的二聚体形成，而Cys-662和？716的双重取代对MANUMYCIN A的同型二聚反应具有保护作用。二硫苏糖醇可以阻断MANUMYCIN A的存在，直接抑制IKK活性和形成稳定的IKKβ二聚体。在甘露霉素A处理的细胞中，IKK与适配蛋白IKKGamma/Nemo的相互作用也被破坏。重要的是，给接种了小鼠B16F10肿瘤的小鼠注射甘露霉素A可以产生有效的IKK抑制效应。二硫苏糖醇可以中和甘露霉素A，这一事实使我们利用质谱仪研究了甘露霉素A与两个硫醇分子之间加合物的形成。因此，甘露霉素A通过不同于法尼化抑制物的途径在IKK信号转导中起着重要的调节作用。 我们计划使用体内方法来研究甘露霉素A对IKKβ激活的影响，IKKβ是炎症诱导的胰岛素抵抗的主要贡献者。将收集各种组织(例如，肝脏、肌肉、胰腺和脂肪)，并对其进行蛋白质印迹、体外IKK活性和EMSA(核因子-kappaB)研究。预计我们的结果将证明IKKbeta在体内是manumycin A的直接靶点。未来的计划包括研究甘露霉素A在调节生理和病理生理学促炎状态(如饮食诱导的肥胖和衰老)中的胰岛素反应性的作用。

IkappaB kinase (IKK) catalytic subunits play a key role in cytokine-mediated nuclear factor (NF)-kappaB signaling, and a loss of NF-kB function appears to inhibit inflammation and oncogenesis. Manumycin A, a natural epoxyquinoid compound, is a potent and selective farnesyltransferase inhibitor with antitumor activity. Our recent findings indicate that treatment with manumycin A resulted in a rapid, selective and potent inhibition of TNF alpha-stimulated IKK activity in a number of cell lines and a primary culture of rat hepatocytes. Unexpectedly, other classes of farnesyltransferase inhibitors had no inhibitory effect. To identify the molecular mechanisms of manumycin A action, we transfected human HepG2 hepatoma cell line with IKKalpha and IKKbeta constructs and demonstrated direct inhibition of IKK activity with concomittant formation of stable homotypic IKKbeta dimers in the presence of manumycin A. A number of Cys-> Ala point mutants of IKKbeta were generated to investigate the possibility that IKKbeta covalent dimerization results from nucleophilic attack on the epoquinoid core of manumycin A. Cells expressing IKKbeta mutated in the activation loop at Cys-179 exhibited similar dimer formation, whereas double substitution of Cys-662 and ?716 conferred protection against homotypic dimerization by manumycin A. Direct inhibition of IKK activity and formation of stable IKKbeta dimers were observed in the presence of manumycin A that could be blocked by dithiothreitol. IKK interaction with the adaptor protein IKKgamma/NEMO was also disrupted in manumycin A-treated cells. Importantly, administration of manumycin A to mice xenografted with murine B16F10 tumors caused potent IKK-suppressive effects. The fact that dithiothreitol could neutralize manumycin A, led us to examine the formation of an adduct between manumycin A and two thiol molecules utilizing mass spectrometry. Thus, manumycin A plays important regulatory function in IKK signaling through pathways distinct from its role as farnesylation inhibitor. We are planning to use an in vivo approach to investigate the effects of manumycin A on the activation of IKKbeta, a major contributor to inflammation-induced insulin resistance. Various tissues will be collected (e.g., liver, muscle, pancreas and fat) and processed for Western blot, in vitro IKK activity and EMSA (NF-kappaB) studies. It is anticipated that our results will demonstrate that IKKbeta is a direct target of manumycin A in vivo. Future plans include the study of the effect of manumycin A in the regulation of insulin responsiveness in physiological and pathophysiological pro-inflammatory states such as diet-induced obesity and aging.