Regional Academic Health Center, Medical Research Division, University of Texas Health Science Center at San Antonio, Edinburg, Texas 78541.
Regional Academic Health Center, Medical Research Division, University of Texas Health Science Center at San Antonio, Edinburg, Texas 78541; Department of Medicine/Division of Diabetes, University of Texas Health Science Center, San Antonio, Texas 78229.
J Biol Chem. 2012 Oct 12;287(42):35756-35767. doi: 10.1074/jbc.M112.397703. Epub 2012 Aug 27.
Cullin-RING E3 ligases (CRLs) are a class of ubiquitin ligases that control the proteasomal degradation of numerous target proteins, including IκB, and the activity of these CRLs are positively regulated by conjugation of a Nedd8 polypeptide onto Cullin proteins in a process called neddylation. CRL-mediated degradation of IκB, which normally interacts with and retains NF-κB in the cytoplasm, permits nuclear translocation and transactivation of the NF-κB transcription factor. Neddylation occurs through a multistep enzymatic process involving Nedd8 activating enzymes, and recent studies have shown that the pharmacological agent, MLN4924, can potently inhibit Nedd8 activating enzymes, thereby preventing neddylation of Cullin proteins and preventing the degradation of CRL target proteins. In macrophages, regulation of NF-κB signaling functions as a primary pathway by which infectious agents such as lipopolysaccharides (LPSs) cause the up-regulation of proinflammatory cytokines. Here we have analyzed the effects of MLN4924, and compared the effects of MLN4924 with a known anti-inflammatory agent (dexamethasone), on certain proinflammatory cytokines (TNF-α and IL-6) and the NF-κB signaling pathway in LPS-stimulated macrophages. We also used siRNA to block neddylation to assess the role of this molecular process during LPS-induced cytokine responsiveness. Our results demonstrate that blocking neddylation, either pharmacologically or using siRNA, abrogates the increase in certain proinflammatory cytokines secreted from macrophages in response to LPS. In addition, we have shown that MLN4924 and dexamethasone inhibit LPS-induced cytokine up-regulation at the transcriptional level, albeit through different molecular mechanisms. Thus, neddylation represents a novel molecular process in macrophages that can be targeted to prevent and/or treat the LPS-induced up-regulation of proinflammatory cytokines and the disease processes associated with their up-regulation.
Cullin-RING E3 连接酶（CRLs）是一类泛素连接酶，可控制包括 IκB 在内的众多靶蛋白的蛋白酶体降解，这些 CRL 的活性通过 Cullin 蛋白上 Nedd8 多肽的缀合来正向调节，这一过程称为 Neddylation。CRL 介导的 IκB 降解，通常与细胞质中的 NF-κB 相互作用并保留 NF-κB，从而允许 NF-κB 转录因子的核易位和转录激活。Neddylation 通过涉及 Nedd8 激活酶的多步酶促过程发生，最近的研究表明，药理学药物 MLN4924 可以有效地抑制 Nedd8 激活酶，从而防止 Cullin 蛋白的 Neddylation 和 CRL 靶蛋白的降解。在巨噬细胞中，NF-κB 信号通路的调节作为主要途径，通过该途径，诸如脂多糖（LPSs）等感染因子引起促炎细胞因子的上调。在这里，我们分析了 MLN4924 的作用，并将 MLN4924 的作用与已知的抗炎药（地塞米松）的作用进行了比较，研究了其对 LPS 刺激的巨噬细胞中某些促炎细胞因子（TNF-α和 IL-6）和 NF-κB 信号通路的影响。我们还使用 siRNA 阻断 Neddylation 来评估该分子过程在 LPS 诱导的细胞因子反应性中的作用。我们的结果表明，无论是通过药理学方法还是使用 siRNA 阻断 Neddylation，都可以消除 LPS 刺激的巨噬细胞中某些促炎细胞因子分泌的增加。此外，我们已经表明，MLN4924 和地塞米松通过不同的分子机制抑制 LPS 诱导的细胞因子上调。因此，Neddylation 代表了巨噬细胞中的一种新的分子过程，可以靶向该过程以预防和/或治疗 LPS 诱导的促炎细胞因子上调及其上调相关的疾病过程。
