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標題: | 絲氨酸229磷酸化的動態循環對於C型肝炎病毒非結構性蛋白質5A的連續磷酸化以及病毒複製極為重要 Dynamic cycles of serine 229 phosphorylation are critical to sequential phosphorylation of the HCV non-structural protein 5A and viral replication |
作者: | Chia-Ni Tsai 蔡佳倪 |
指導教授: | 余明俊(Ming-Jiun Yu) |
關鍵字: | C型肝炎病毒,磷酸化,非結構性蛋白質5A,連續磷酸化,病毒複製, hepatitis C virus,phosphorylation,non-structural protein 5 A,sequential phosphorylation,viral replication, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | C型肝炎病毒(hepatitis C virus, HCV)的非結構性蛋白5A(non-structural protein 5A,NS5A)對於HCV病毒的生命週期非常重要。NS5A能被加上磷酸根進而形成兩種磷酸化態,分別為低度磷酸化態以及高度磷酸化態,以免疫墨點法觀查到此兩種狀態分別位於56 kDa以及58 kDa的位置。許多文獻指出,透過連續磷酸化位於第一低複雜度序列 (low complexity sequence I) 區域中的絲氨酸群,可以讓NS5A被高度磷酸化,並參與在病毒的複製與病毒顆粒的組裝,但「連續磷酸化」的機制尚未明瞭。在先前的研究中,利用三個對NS5A中第232、235、以及238絲氨酸位點具有專一性的抗體,討論出絲氨酸232的磷酸化會透過第一型酪蛋白激酶(casein kinase I,CKI)進行連續磷酸化(sequential phosphorylation)調控絲氨酸235及238的磷酸化,並因此而造成高度磷酸化的出現。值得注意的是,CKI偏好特定的序列來進行磷酸化(pS/T-X-X-S/T),而位於絲氨酸232前面的絲氨酸229剛好符合此序列,由生物體外激脢實驗(in vitro kinase assay)得知絲氨酸229磷酸化會藉由CKI引發絲氨酸232的磷酸化。在本研究中,我們試圖去測試絲氨酸229究竟有無被磷酸化,那它是否為開啟連續磷酸化的第一個胺基酸呢?將絲氨酸229突變為丙氨酸(S229A)使229不能被磷酸化後會降低NS5A的連續磷酸化;然而用天冬氨酸模擬磷酸化(S229D)則能回復從絲氨酸232開始到絲氨酸238的連續磷酸化,意味著絲氨酸229磷酸化能夠開啟如此的連續性的磷酸化。利用對絲氨酸229具專一性的抗體發現絲氨酸229的磷酸化程度相當低,並在56及58 kDa都被偵測到,意外的是,在絲氨酸235天冬氨酸突變的NS5A中,絲氨酸229磷酸化被大幅度地提升了,這樣的發現意味著也許在連續磷酸化進行到絲氨酸235時,絲氨酸229也許要被去磷酸化。由以上結果推測絲氨酸229能被動態地磷酸化與去磷酸化並且對於NS5A連續磷酸化非常重要。除此之外,229丙氨酸及229天冬氨酸突變皆會改變複製複合體(replication complex)的蛋白質(NS5A、NS3)在細胞內的分布,這樣的改變可能會導致複製複合體的功能受到影響,與這樣的推測相符,229丙氨酸及229天冬氨酸突變皆大幅度地降低報告病毒(reporter virus)的活性。綜合以上的結果推測出絲氨酸229能被動態地磷酸化與去磷酸化,並且對於非結構性蛋白質5A的連續磷酸化以及病毒的生命週期極為重要。 The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein critical to the viral life cycle. NS5A is a phosphoprotein with two phosphorylation states: hypo-phosphorylation (p56) and hyper-phosphorylation (p58) involved in HCV replication and assembly. It has been shown that p58 is a result of phosphorylation on a cluster of serine residues in NS5A low-complexity sequence I (LCS I). The serine cluster was thought to undergo sequential phosphorylation, but the phosphorylation cascade remained to be elucidated. Using antibodies specific to S232, S235, and S238, we have previously shown that S232 primes NS5A phosphorylation sequentially from S235 to S238 by casein kinase I (CKI). Notably, S229 is also located in the CKI motif (pS/T-X-X-S/T), and an in vitro kinase assay suggest that S229 phosphorylation primes S232 phosphorylation. In my thesis, we aimed to examine whether S229 is phosphorylated and whether S229 phosphorylation initiates NS5A sequential phosphorylation from S232 to S238. Phosphoablatant alanine mutation at S229 (S229A) reduced whereas phosphomimetic aspartate mutation at S229 (S229D) rescued phosphorylation of S232, S235, and S238, suggesting that S229 phosphorylation triggered NS5A sequential phosphorylation from S232 to S238. Using an S229 phosphorylation-specific antibody, we found that the level of S229 phosphorylation was quite low but detected in both p56 and p58; however, S229 phosphorylation was highly elevated in S235 phosphoablatant alanine mutant NS5A (S235A), indicating that S229 phosphorylation should be de-phosphorylated when sequential phosphorylation from S232 propagate to S235. Thus, we infer that S229 undergoes dynamic cycles of phosphorylation and de-phosphorylation that are important to NS5A sequential phosphorylation. We also found that S229A or S229D mutation altered the intracellular distribution of replication complex components (NS5A and NS3), that would interfere with the functions of the replication complex. In line with this, S229A and S229D mutation disturbed reporter virus activity. In summary, the above results suggest that S229 is crucial to NS5A sequential phosphorylation and HCV life cycle. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72235 |
DOI: | 10.6342/NTU201803753 |
全文授權: | 有償授權 |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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