臺灣博碩士論文加值系統

RLRs (RIG-I-like receptors) 為一群位於細胞質中的模式識別受器，負責辨識暴露於細胞質內病毒的病原相關片段，訊息路徑最終活化下游兩個轉錄因子 IFN-regulatory factor 3 (IRF3) 以及 nucler factor kappa B (NF-κB)，產生第一型干擾素與促發炎細胞激素，以幫助細胞對抗病毒。先前實驗室前人研究指出，原致癌基因 PIM1 絲胺酸/蘇胺酸激酶 (serine/threonine kinase) 藉由其激酶活性負向調控RLR 訊息傳遞路徑，並且作用點可能在 RIG-I。在本實驗中我們發現 PIM1 會與 RIG-I 之多個區域進行相互作用。藉由活體外激酶試驗 (in vitro kinase assay)，我們看到 PIM1 會對全長 RIG-I 以及 RIG-I 蛋白的 N 端區域進行磷酸化，而 RIG-I 的 C 端或中央 RNA 螺旋酶區域 (RNA helicase domain) 則不會被磷酸化。此外，在活體內 (in vivo) 系統下我們也得到類似的實驗結果，顯示 RIG-I 蛋白確實是 PIM1 激酶的受質。然而，經由液相層析串聯質譜儀 (LC/MS) 分析後，我們發現 RIG-I 蛋白上有許多 PIM1 磷酸化之位點，且不侷限於 RIG-I 的 N 端區域。初步我們將 RIG-I Ser183、Thr662 及 Thr671 三點進行點突變，並由功能分析試驗顯示， RIG-I 蛋白 Ser183 位置磷酸化會抑制 RIG-I 活化 IFN-β 啟動子之活性; Thr662 胺基酸本身即具重要性; Thr671 之磷酸化則不影響 RIG-I 活性。另外，我們針對 PIM1 對不同片段 RIG-I 活性影響進行測試，結果發現 PIM1藉由其激酶活性抑制 RIG-I 之活性只針對全長之 RIG-I 而為之，顯示 RIG-I 的結構對 PIM1 激酶之調控十分重要。以往，對 PIM1 功能之了解多著重於細胞增生與細胞凋亡方面，本篇研究指出 PIM1 可能藉由其激酶活性對 RIG-I 蛋白進行磷酸化，而負向調控 RLR 訊息傳遞。

RIG-I-like receptors (RLRs) are one of the germline-encoded pattern recognition receptors (PRRs), utilized to identify viral pathogens by engaging pathogen-associated molecular patterns (PAMPs). Once recognizing the PAMPs, downstream transcriptional factors of IFN-regulatory factor 3 (IRF3) and nucler factor kappa B (NF-κB) are activated, and type I interferon (IFN) as well as proinflammatory cytokines are produced to limit viral replication.
The data from our previously study have shown that a proto-oncogene, PIM1 ( a serine/threonine kinase), may target RIG-I to down-regulate the RLR-mediated antiviral responses. In this study, we further demonstrate that PIM1 can interact with various regions of the RIG-I protein. From in vitro and in vivo assays, we showed that PIM1 phosphorylated RIG-I at its N-terminal domain, but not its C terminal domain and RNA helicase domain. These data collectively indicate that RIG-I is one of PIM1’s substrates. However, the analysis of liquid chromatography-mass spectrometry (LC/MS) indicated that PIM1 phosphorylated multiple sites of RIG-I, residing in the N-terminal domain, the helicase domain and the C-terminal domain. Site-directed mutagenesis of RIG-I at the Ser183, Thr662 and Thr671 residues was performed to reveal the significance of the phosphorylation of these sites. Our results showed that, phosphorylation of RIG-I at Ser183 dramatically suppressed RIG-I activity; the Thr662 per se was important to the RIG-I activity as mutation to either Ala or Glu significantly reduced RIG-I activity; and phosphorylation of Thr671 had no effects on RIG-I activity at all. We also examined the effect of the kinase activity of PIM1 on the signal transduction of the full-length and the truncated RIG-I. It was found that the full-length RIG-I activity was inhibited by PIM1 via the kinase activity, whereas the truncated RIG-I activities were inhibited via other mechanisms.
These data collectively indicate that PIM1 kinase may phosphorylate RIG-I to down-regulate the RIG-I-mediated antiviral responses, and the correct RIG-I conformation may be important to PIM1 phosphorylation.

致謝 i
中文摘要 ii
英文摘要 iii
目錄 v
圖表目錄 vi
英文縮寫對照表 vii
第一章 緒論 1
第一節 先天免疫系統 (innate immune system) 與第一型干擾素系統 (Type I interferon system) 1
第二節 RLR 訊息傳遞路徑與調控 6
第三節 原致癌基因 (proto-oncogene) PIM1 激酶 9
第四節 前人研究與實驗目的 15
第二章 實驗材料與方法 17
第一節 實驗材料 17
第二節 實驗方法 23
第三章 實驗結果 30
第一節 在活體內 (in vivo) PIM1 蛋白與 RIG-I 蛋白有相互作用
(interaction) 30
第二節 在活體外 (in vitro) 及活體內 (in vivo) PIM1 蛋白會對
RIG-I 蛋白進行磷酸化 (phosphorylation) 31
第三節 RIG-I Ser183 可能是 RIG-I 蛋白上 PIM1 蛋白磷酸化的位置之一 34
第四節 PIM1 可能藉由不同機制抑制 RIG-I, N-RIG 或 2CARD 所誘導之
RLR 訊息路徑 35
第四章 討論 38
圖表 42
附圖 57
參考文獻 61
圖一 建構不同片段 RIG-I (truncated RIG-I) 之示意圖與蛋白表現情形及各片段 RIG-I 活化 IFN-β 啟動子活性之情況。 42
圖二 PIM1 與 RIG-I 及不同片段間有相互作用 (interaction)。 43
圖三 帶有 GST tag 蛋白純化情形。 45
圖四 在活體外 (in vitro) 情況下，PIM1 會對全長 RIG-I 與 N-RIG 進行磷酸化。 47
圖五 在活體內 (in vivo) 情況下，PIM1 會對全長 RIG-I 及 N-RIG 進行磷酸化。 50
圖六 各片段 RIG-I與 PIM1 之間相互作用與磷酸化關係之示意圖 。 52
圖七 以 LC/MS 進行 RIG-I 蛋白上 PIM1 可能磷酸化位置之定序分析。 53
圖八 PIM1 藉由其激酶活性負向調控全長 RIG-I 誘導活化之 IFN-β 啟動子活性。 55
附圖一 由 RIG-I 或 MDA5以及 RIG-I 與 MDA5 同時辨識之病毒。 57
附圖二 RLR 訊息傳遞示意圖。 58
附圖三 PIM1 藉由其激酶活性負向調控 SeV 感染所活化之訊息路徑。 59

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