蛋白激酶A和水杨酸类药物调控蛋白激酶TAK1激活的新型分子机制

发布时间：2018-07-06 20:24

本文选题：TAK1 + 激酶　；参考：《浙江大学》2015年博士论文

【摘要】：转化生长因子-β激活激酶1(TAK1)是NF-κB信号通路(如Toll样受体和白介素1受体信号)激活过程中的关键激酶。尽管现有证据表明TAK1的激活需要位于其活化环的第184位和第187位苏氨酸的磷酸化,但人们对TAK1完全激活的分子机制仍缺乏深入了解。本研究发现TAK1的C末端的卷曲螺旋结构域所介导的TAK1蛋白二聚化和自身磷酸化调控了第187位苏氨酸的磷酸化。更重要的是,在多种炎症信号如TNF-α、LPS和IL-1β刺激下,TAK1的完全激活还需要其第412位丝氨酸的磷酸化。体外的激酶实验和在体内利用shRNA干扰内源基因表达的方法发现cAMP依赖的蛋白激酶催化亚基α(PKACα)和X连锁蛋白激酶(PRKX)催化了该位点的磷酸化,并参与调节TLR/IL-1R信号的激活和下游炎性细胞因子的产生。利用Morpholino干扰体内基因表达和回补突变体实验证实了斑马鱼的TAK1蛋白上相对应的第391位丝氨酸位点也在NF-κB激活过程中发挥重要作用。本研究揭示出了PKACα和PRKX通过磷酸化调节TAK1激酶活力来调控天然免疫信号的新的分子机制。水杨酸及其衍生物作为非甾体类抗炎药物已经成为人类消炎、镇痛和预防心血管疾病乃至癌症的常用药物,这部分归功于这类药物能够抑制NF-κB的激活。虽然十多年前就有报道认为水杨酸能够通过竞争ATP抑制IKKβ激酶的活性,但此机制仍无法解释许多现象。本研究发现在TNF-a或IL-1β刺激下,水杨酸钠和阿司匹林可以抑制IKK激酶上游的TAK1激酶的激活。进一步的细胞生化试验表明水杨酸类药物在体外没有直接抑制TAK1和IKKβ激酶的活性,而是通过抑制体内泛素化过程抑制了TAKl激酶及其下游信号的激活。体外的泛素化试验也表明水杨酸类药物可以在体外直接抑制K63连接的和线性化的多聚泛素链合成。在LPS诱导的小鼠急性肺损伤疾病模型中,高剂量水杨酸类药物抑制了肺部组织细胞的NF-κB信号激活和泛素化过程介导的TNFR信号复合体的组装,最终达到缓解肺部炎症反应的效应。本研究首次发现水杨酸类药物通过抑制泛素化系统调控TAK1激酶和下游信号激活,为阿司匹林等药物的药理作用提供了新的理论基础,并提供了以泛素化系统为靶点设计抗炎药物的新思路。
[Abstract]:Transforming growth factor- 尾 -activated kinase 1 (TAK1) is a key kinase in the activation of NF- 魏 B signaling pathways, such as Toll-like receptor and interleukin-1 receptor signal. Although the available evidence suggests that the activation of TAK1 requires phosphorylation of threonine at the 184th and 187th sites of its activation ring, the molecular mechanism of full activation of TAK1 is still poorly understood. In this study, we found that tak 1 protein dimerization and self phosphorylation regulated the phosphorylation of threonine at site 187 mediated by the crimp helix domain at the C-terminal of TAK1. More importantly, the complete activation of TAK1 under the stimulation of various inflammatory signals such as TNF- 伪, LPS and IL-1 尾 also requires the phosphorylation of its 412 site serine. Kinase assay in vitro and shRNA interference of endogenous gene expression in vivo revealed that camp dependent protein kinase catalyzed the phosphorylation of protein kinase 伪 (PKAC 伪) and X-linked protein kinase (PRKX). And involved in regulating the activation of TLR / IL-1 R signal and the production of downstream inflammatory cytokines. Morpholino interference gene expression and complementary mutants experiments confirmed that the 391-site serine site on the TAK1 protein of zebrafish also plays an important role in the activation of NF- 魏 B. This study revealed a new molecular mechanism of PKAC 伪 and PRKX regulating innate immune signal by phosphorylation of TAK1 kinase. Salicylic acid and its derivatives as non-steroidal anti-inflammatory drugs have become common drugs for anti-inflammatory analgesia and prevention of cardiovascular diseases and even cancer partly due to their ability to inhibit the activation of NF- 魏 B. Although it has been reported for more than a decade that salicylic acid can inhibit the activity of IKK 尾 kinase by competing ATP, this mechanism still cannot explain many phenomena. In this study, it was found that sodium salicylate and aspirin inhibited the activation of TAK1 kinase upstream of IKK kinase under the stimulation of TNF-a or IL-1 尾. Further cell biochemical tests showed that salicylic acid drugs did not directly inhibit the activities of TAK1 and IKK 尾 kinase in vitro, but inhibited the activation of Takl kinase and its downstream signal by inhibiting the process of ubiquitin in vivo. Ubiquitin assay in vitro also showed that salicylic acid could directly inhibit the synthesis of K63 linked and linearized polyubiquitin chains in vitro. In the model of acute lung injury induced by LPS, high dose salicylic acid drugs inhibited the activation of NF- 魏 B signal and the assembly of TNFR signal complex mediated by Ubiquitin process in lung tissue cells. This study for the first time found that salicylic acid drugs regulate the activation of TAK1 kinase and downstream signal by inhibiting the ubiquitin system, which provides a new theoretical basis for the pharmacological action of aspirin and other drugs. A new way to design anti-inflammatory drugs using ubiquitin system as target is provided.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R96

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