Regulation Of Nuclear Factor-kappa B Activity

IkappaB激酶（IKK）催化亚基IKK β在丝氨酸介导的核因子（NF）-kappaB信号传导中起关键作用，IKK β活性的缺陷似乎抑制NF-kappaB介导的炎症和致癌反应以及NF-kappaB的促存活功能。 Manumycin是一种天然的环氧醌类化合物，对法尼基转移酶表现出有效的选择性抑制活性，并已在各种实验模型中使用其抑制作用。我们最近的研究结果表明，manumycin引起了一个快速和有效的抑制TNF α刺激IKK活性在一些细胞系和大鼠肝细胞的原代培养。 此外，向异种移植有鼠B16 F10肿瘤的小鼠施用manumycin引起了有效的IKK抑制作用（Bernier等人，2006年）。 出乎意料的是，其他种类的法尼基转移酶抑制剂在这些实验条件下没有抑制作用。为了鉴定manumycin作用的分子机制，我们用IKK α和IKK β构建体转染培养的人HepG 2肝癌细胞，并证明在manumycin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的环氧醌核心的亲核攻击引起的可能性。 表达IKK β的细胞在Cys-179的激活环中突变，表现出类似的二聚体形成，而IKK β羧基末端结构域中Cys-662/716的双取代赋予了对manumycin同型二聚化的保护。 二硫苏糖醇预处理基本上阻断了manumycin介导的免疫沉淀物中共价IKK β二聚体的形成。 使用质谱技术，我们确定谷胱甘肽（GSH）作为一个分子容易通过manumycin的环氧醌功能的修改，这表明，细胞内巯基的消耗可能有助于，至少部分，manumycin的抗氧化性能。这些结果表明，manumycin通过与其作为法尼基化抑制剂的作用不同的途径在IKK/NF κ B信号转导中发挥重要的调节功能。 尽管取得了这些进展，但manumycin如何影响IKK/NF-κ B通路的功能，不仅在癌细胞中，而且在参与先天免疫反应的细胞中，仍有待了解。我们目前正在验证manumycin干扰外周血淋巴细胞中NF-κ B信号传导的假设。 正在进行生物化学和分子生物学研究，以表征由manumycin灭活IKK多聚体复合物以及与癌细胞相比其用于诱导淋巴细胞离体凋亡的途径。 我们的数据应该提供新的见解NF-κ B信号的关键方面，并可能是特别的临床意义。

The IkappaB kinase (IKK) catalytic subunit, IKKbeta, plays a key role in cytokine-mediated nuclear factor (NF)-kappaB signaling, and defects in IKKbeta activity appears to inhibit NF-kappaB-mediated inflammatory and oncogenic responses, and the pro-survival function of NF-kappaB. Manumycin is a natural epoxyquinoid compound that exhibits potent and selective inhibitory activity against farnesyltransferase, and it has been used for its antineoplastic action in a variety of experimental models. Our recent findings indicate that manumycin elicited a rapid and potent inhibition of TNF alpha-stimulated IKK activity in a number of cell lines and a primary culture of rat hepatocytes. Moreover, administration of manumycin to mice xenografted with murine B16F10 tumors caused potent IKK-suppressive effects (Bernier et al., 2006). Unexpectedly, other classes of farnesyltransferase inhibitors had no inhibitory effect under these experimental conditions. To identify the molecular mechanisms of manumycin action, we transfected cultured human HepG2 hepatoma cells 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 number of Cys-> Ala point mutants of IKKbeta were generated to investigate the possibility that IKKbeta covalent dimerization resulted from nucleophilic attack on the epoxyquinoid core of manumycin. Cells expressing IKKbeta mutated in the activation loop at Cys-179 exhibited similar dimer formation, whereas double substitution of Cys-662/716 in the carboxy-terminal domain of IKKbeta conferred protection against homotypic dimerization by manumycin. Dithiothreitol pre-treatment substantially blocked manumycin-mediated formation of covalent IKKbeta dimers in immunoprecipitates. Using mass spectrometry technique, we identified glutathione (GSH) as a molecule susceptible to modification via the epoxyquinoid function of manumycin, which suggests that the depletion of intracellular thiols could contribute, at least in part, to manumycin's antineoplastic properties. These results indicated that manumycin plays important regulatory function in IKK/NFkappaB signaling through pathways distinct from its role as farnesylation inhibitor. Despite these advances, it remains to be understood how manumycin affects the function of the IKK/NF-kappaB pathway not only in cancer cells, but also in cells involved in the innate immunity response. We are currently testing the hypothesis that manumycin interferes with NF-kappaB signaling in peripheral blood lymphocytes. Biochemical and molecular biological studies are being carried out to characterize the inactivation of the IKK multimeric complex by manumycin and the pathways that it uses to induce ex vivo apoptosis in lymphocytes as compared to cancer cells. Our data should provide novel insights into key aspects of NF-kappaB signaling, and may be of particular clinical relevance.