Regulation Of Nuclear Factor-kappa B Activity

IkappaB激酶（IKK）催化亚基在丝氨酸介导的核因子（NF）-kappaB信号传导中起关键作用，并且NF-kB功能的丧失似乎抑制炎症和肿瘤发生。Manumycin A是一种天然环氧醌类化合物，是一种有效的选择性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，治疗与manumycin A导致一个快速，选择性和有效的抑制TNF α刺激IKK活性在一些细胞系和大鼠肝细胞的原代培养。出乎意料的是，其他种类的法尼基转移酶抑制剂没有抑制作用。为了确定manumycin A作用的分子机制，我们用IKK α和IKK β构建体转染人HepG 2肝癌细胞系，并证明在manumycin A存在下直接抑制IKK活性并伴随形成稳定的同型IKK β二聚体。产生了许多IKK β的Cys-> Ala点突变体，以研究IKK β共价二聚化是由对manumycin A的epoquinoid核心的亲核攻击引起的可能性。细胞表达IKK β突变的激活环在Cys-179表现出类似的二聚体形成，而双取代Cys-662和？716赋予针对manumycin A的同型二聚化的保护作用。IKK与接头蛋白IKK γ/NEMO的相互作用也在manumycin A处理的细胞中被破坏。因此，manumycin A通过不同于其作为法尼基化抑制剂的作用的途径在IKK信号传导中发挥重要的调节功能。 我们目前正在使用体内方法来研究manumycin A影响IKK β激活的假设，IKK β是炎症诱导的胰岛素抵抗的主要因素。在皮下植入的鼠B16 F10黑色素瘤中注射不同浓度的manumycin A。在几只老鼠的背上切除肿瘤块，称重，目前正在进行蛋白质印迹、体外IKK活性和EMSA（NF-κ B）研究。预期我们的结果将证明IKK β是manumycin A在体内的直接靶点。未来的计划包括manumycin A在生理和病理生理促炎状态（如饮食诱导的肥胖和衰老）中调节胰岛素反应性的作用研究。

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. IKK interaction with the adaptor protein IKKgamma/NEMO was also disrupted in manumycin A-treated cells. Thus, manumycin A plays important regulatory function in IKK signaling through pathways distinct from its role as farnesylation inhibitor. We are currently using an in vivo approach to investigate the hypothesis that manumycin A affects the activation of IKKbeta, a major contributor to inflammation-induced insulin resistance. Injection of various concentrations of manumycin A has been carried out in murine B16F10 melanoma implanted s.c. in the back of several mice. The tumor masses were excised, weighted and are currently being 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.