鑑定 C 型肝炎病毒低度及高度磷酸化態非結構性蛋白質 5A 之交互作用蛋白質

Identification of the interacting proteins of the hypo- and hyper- phosphorylated HCV NS5A

潘亭均(Ting-Chun Pan)

指導教授 ： 余明俊

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803592

摘要

C 型肝炎病毒(Hepatitis C virus, HCV)之非結構性蛋白 5A (non-structure protein 5A, NS5A)是一個對病毒繁殖重要的磷酸化蛋白，它有兩個磷酸化態: 低度及高度磷酸化 NS5A，被認為會招募不同的交互作用宿主蛋白來執行不同的 功能。我們先前的研究指出第 235 號絲氨酸(S235)的磷酸化是最主要造成的 NS5A 高度磷酸化的事件，S235 磷酸化是由第一型酪蛋白激酶(casein kinase I isoform alpha)負責，其需要第 232 絲氨酸(S232)先被磷酸化。然而，有兩個 議題仍待解決:1)哪些蛋白質會被低度及高度磷酸化的 NS5A 招募來協助它們 的功能，2)哪一個激酶負責 S232 磷酸化，進而造成 NS5A 的高度磷酸化態。在 本篇研究中，我們建立的一個結合了兩步驟之磁珠上蛋白酶消化及同位素雙甲基 標定的親和性純化質譜儀方法，並用了抗低度及抗高度磷酸化態 NS5A 之專一性 抗體來鑑定低度及高度磷酸化態 NS5A 的交互作用蛋白，我們鑑定到第二型聚合 酶相關因子一複合體(polymerase II-associated factor I complex, Paf1 complex)的 成員為潛在的高度磷酸化 NS5A 的交互作用蛋白:我們也結合了貝氏定理之激酶 排序，鑑定出 DNA 依賴型蛋白質激酶之催化單元(DNA-dependent protein kinase catalytic subunit, PRKDC)為低度磷酸化 NS5A 的交互作用蛋白，與此一致地， 抑制激酶活性或抑制激酶基因轉譯皆降低 S232 磷酸化，上述結果暗示了一個 PRKDC 磷酸化 S232 的新角色。

關鍵字

C型肝炎病毒 ； 蛋白質交互作用 ； 蛋白質質譜 ； 非結構性蛋白5A ； 磷酸化

並列摘要

The hepatitis C virus (HCV) non-structural 5A protein (NS5A) is a phosphoprotein critical to virus propagation. It has two phosphorylation states: hypo- and hyper- phosphorylated NS5A which are believed to recruit different host interacting proteins to execute distinct functions. Our previous studies showed that serine 235 (S235) phosphorylation is a primary event accounting for hyper-phosphorylated NS5A. S235 is phosphorylated by casein kinase I isoform alpha that requires S232 to be phosphorylated/primed first. However, two issues remain to be solved: 1) what proteins hypo- and hyper-phosphorylated NS5A recruit to facilitate their functions in HCV- infected cells, 2) which kinase is responsible for S232 phosphorylation, thus leading to NS5A hyper-phosphorylation. Here, we establish an affinity purification-mass spectrometry (AP-MS) approach combining on-bead two step digestion and stable isotope dimethyl labeling and use hypo- and hyper-phosphorylated NS5A-specific antibodies to identify the interactors of hypo- and hyper-phosphorylated NS5A. We identified the polymerase II-associated factor I complex (Paf1 complex) proteins as potential interactors of hyper-phosphorylated NS5A. Moreover, by combining the AP- MS result and a kinase ranking based on Bayes’ theorem, we identified DNA-dependent protein kinase catalytic subunit (PRKDC) as an interactor of hypo-phosphorylated NS5A. In line with this, inhibition and knockdown of PRKDC reduce S232 phosphorylation. These results suggested a novel role of the Paf1 complex in HCV infection and a new function of PRKDC in phosphorylate NS5A at S232.

並列關鍵字

HCV ； protein-protein interaction ； protein mass spectrometry ； NS5A ； phosphorylation

參考文獻

Reference

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