本文選題：TAK1 + 激酶　； 參考：《浙江大學(xué)》2015年博士論文

【摘要】：轉(zhuǎn)化生長(zhǎng)因子-β激活激酶1(TAK1)是NF-κB信號(hào)通路(如Toll樣受體和白介素1受體信號(hào))激活過(guò)程中的關(guān)鍵激酶。盡管現(xiàn)有證據(jù)表明TAK1的激活需要位于其活化環(huán)的第184位和第187位蘇氨酸的磷酸化,但人們對(duì)TAK1完全激活的分子機(jī)制仍缺乏深入了解。本研究發(fā)現(xiàn)TAK1的C末端的卷曲螺旋結(jié)構(gòu)域所介導(dǎo)的TAK1蛋白二聚化和自身磷酸化調(diào)控了第187位蘇氨酸的磷酸化。更重要的是,在多種炎癥信號(hào)如TNF-α、LPS和IL-1β刺激下,TAK1的完全激活還需要其第412位絲氨酸的磷酸化。體外的激酶實(shí)驗(yàn)和在體內(nèi)利用shRNA干擾內(nèi)源基因表達(dá)的方法發(fā)現(xiàn)cAMP依賴(lài)的蛋白激酶催化亞基α(PKACα)和X連鎖蛋白激酶(PRKX)催化了該位點(diǎn)的磷酸化,并參與調(diào)節(jié)TLR/IL-1R信號(hào)的激活和下游炎性細(xì)胞因子的產(chǎn)生。利用Morpholino干擾體內(nèi)基因表達(dá)和回補(bǔ)突變體實(shí)驗(yàn)證實(shí)了斑馬魚(yú)的TAK1蛋白上相對(duì)應(yīng)的第391位絲氨酸位點(diǎn)也在NF-κB激活過(guò)程中發(fā)揮重要作用。本研究揭示出了PKACα和PRKX通過(guò)磷酸化調(diào)節(jié)TAK1激酶活力來(lái)調(diào)控天然免疫信號(hào)的新的分子機(jī)制。 水楊酸及其衍生物作為非甾體類(lèi)抗炎藥物已經(jīng)成為人類(lèi)消炎、鎮(zhèn)痛和預(yù)防心血管疾病乃至癌癥的常用藥物,這部分歸功于這類(lèi)藥物能夠抑制NF-κB的激活。雖然十多年前就有報(bào)道認(rèn)為水楊酸能夠通過(guò)競(jìng)爭(zhēng)ATP抑制IKKβ激酶的活性,但此機(jī)制仍無(wú)法解釋許多現(xiàn)象。本研究發(fā)現(xiàn)在TNF-a或IL-1β刺激下,水楊酸鈉和阿司匹林可以抑制IKK激酶上游的TAK1激酶的激活。進(jìn)一步的細(xì)胞生化試驗(yàn)表明水楊酸類(lèi)藥物在體外沒(méi)有直接抑制TAK1和IKKβ激酶的活性,而是通過(guò)抑制體內(nèi)泛素化過(guò)程抑制了TAKl激酶及其下游信號(hào)的激活。體外的泛素化試驗(yàn)也表明水楊酸類(lèi)藥物可以在體外直接抑制K63連接的和線(xiàn)性化的多聚泛素鏈合成。在LPS誘導(dǎo)的小鼠急性肺損傷疾病模型中,高劑量水楊酸類(lèi)藥物抑制了肺部組織細(xì)胞的NF-κB信號(hào)激活和泛素化過(guò)程介導(dǎo)的TNFR信號(hào)復(fù)合體的組裝,最終達(dá)到緩解肺部炎癥反應(yīng)的效應(yīng)。本研究首次發(fā)現(xiàn)水楊酸類(lèi)藥物通過(guò)抑制泛素化系統(tǒng)調(diào)控TAK1激酶和下游信號(hào)激活,為阿司匹林等藥物的藥理作用提供了新的理論基礎(chǔ),并提供了以泛素化系統(tǒng)為靶點(diǎn)設(shè)計(jì)抗炎藥物的新思路。
[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- 偽, 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 偽 (PKAC 偽) 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 偽 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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R96

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