圖註：斑馬魚C1q-like的敲降造成胚胎的發育缺陷和caspase 3/9活性的改變
生物谷報導：近十幾年來，由於水產動物重大病害頻頻發生以及病急亂用藥、濫用藥引起的水產品食品安全等隱患，國際上已形成了進行魚類等水產動物免疫學研究的交叉學科，且尤為重視先天性免疫機制研究。近年來，中科院水生所淡水生態與生物技術國家重點實驗室在國家基礎研究計劃973項目的支持下，已在漁業生物技術研究方向滋生出新的進行魚類先天性免疫調控機制研究的生長點，建立了研究魚類抗病毒基因的細胞模型，在魚類病毒病的免疫遺傳機理研究上取得突破，已克隆出一批與魚類先天性免疫相關基因或抗病相關基因，引起國際同行的關注。最近，由桂建芳研究員主持的魚類發育遺傳學學科組和張奇亞研究員主持的水生病毒學學科組通過交叉合作，已開始深入到魚類先天性免疫相關基因的功能研究方面，取得了兩項重要進展。
第一項進展主要是由博士研究生梅潔等完成，美國加州大學洛杉磯校區林碩教授提供了斑馬魚突變體，為合作者之一。該研究是在實驗室前期從鯽魚中鑑定出一個與補體C1q類似的C1q-like基因並發現它可能與免疫和細胞凋亡有關的基礎上，進一步利用體外培養細胞和斑馬魚來進行功能研究。體外研究表明，C1q-like能抑制誘導劑誘導的caspase-3和9的活性，進而抑制凋亡。Morpholino介導的基因敲除分析揭示斑馬魚C1q-like有抗凋亡作用，發現C1q-like敲降後，調亡因子表達上調、細胞周期紊亂、血細胞發生相關基因表達下調，血色素合成減少、腦血管發育異常。研究還進一步發現，所有抑制C1q-like造成的功能缺陷，都能通過抑制p53的表達來挽救。紫外線照射和細菌感染均能誘導C1q-like的過量表達，而且抑制C1q-like的表達胚胎更易於受到細菌的侵襲而死亡。因此，C1q-like 應是斑馬魚胚胎中一個新的細胞存活調控因子，在胚胎發育特別是腦發育過程抑制p53依賴的和caspase 3/9介導的細胞凋亡中起了重要的抗凋亡和保護作用。該研究成果已在美國發育生物學會會刊愛思唯爾期刊《發育生物學》（Developmental Biology）在線發表。
第二項進展主要是由朱蓉博士和張義兵博士等共同完成。該研究主要是圍繞魚類中是否存在dsRNA依賴的蛋白激酶PKR以及魚類PKR是否具有抗病毒功能而展開的。通過建立由滅活病毒誘導的牙鮃囊胚培養細胞SMART cDNA文庫，篩選克隆得到牙鮃PKR基因。該基因具有哺乳類PKR保守的結構域，包括N端的兩個dsRNA結合區，以及C端激酶區。dsRNA結合區能結合dsRNA，而激酶區能抑制翻譯。在正常細胞中，PKR僅具有低水平的本底表達。在大菱鮃彈狀病毒感染過程中，PKR表達上調，同時伴隨著eIF2α磷酸化增強。在細胞中過量表達野生型PKR也能導致eIF2α磷酸化水平增強，且抑制病毒增殖；而當過量表達喪失激酶活性的PKR突變體時，則無此效應。進而，RNA幹擾實驗證實PKR能通過磷酸化eIF2α發揮抑制蛋白翻譯的功能。上述研究結果首次證實魚類確實存在與哺乳類一致、且具有相似功能的PKR介導的抗病毒途徑，首次揭示了魚類PKR基因的功能結構域及其抗病毒功能。該研究結果已由美國微生物學會主辦的權威雜誌之一《病毒學雜誌》（Journal of Virology）在線發表。
關於這兩項研究成果的意義，這兩篇論文的通訊作者桂建芳研究員介紹說，魚類先天性免疫相關基因的鑑定特別是其功能和作用機制研究是國際上的研究熱點之一，這一方面是因為魚類作為相對原始的脊椎動物，對
其先天性免疫基因功能及其作用機制的剖析將有助於了解脊椎動物免疫系統的發生和進化，另一方面魚類作為人類的主要蛋白來源之一，魚類的健康養殖和安全水產品的可持續發展更需要我們闡明魚類先天性免疫機制，制定出更為健康高效的養殖對策和措施。（生物谷www.bioon.com）
生物谷推薦原始出處：
Developmental Biology， doi:10.1016/j.ydbio.2008.04.022，Jie Mei，Jian-Fang Gui
C1q-like inhibits p53-mediated apoptosis and controls normal hematopoiesis during zebrafish embryogenesis
Jie Meia, Qi-Ya Zhanga, Zhi Lia, Shuo Linb and Jian-Fang Guia, ,
aState Key Laboratory of Freshwater Ecology and Biotechnology, Center for Developmental Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Wuhan 430072, China
bDepartment of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095, USA
Except for the complement C1q, the immunological functions of other C1q family members have remained unclear. Here we describe zebrafish C1q-like, whose transcription and translation display a uniform distribution in early embryos, and are restricted to mid-hind brain and eye in later embryos. In vitro studies showed that C1q-like could inhibit the apoptosis induced by ActD and CHX in EPC cells, through repressing caspase 3/9 activities. Moreover, its physiological roles were studied by morpholino-mediated knockdown in zebrafish embryogenesis. In comparison with control embryos, the C1q-like knockdown embryos display obvious defects in the head and craniofacial development mediated through p53-induced apoptosis, which was confirmed by the in vitro transcribed C1q-like mRNA or p53 MO co-injection. TUNEL assays revealed extensive cell death, and caspase 3/9 activity measurement also revealed about two folds increase in C1q-like morphant embryos, which was inhibited by p53 MO co-injection. Real-time quantitative PCR showed the up-regulation expression of several apoptosis regulators such as p53, mdm2, p21, Bax and caspase 3, and down-regulation expression of hbae1 in the C1q-like morphant embryos. Knockdown of C1q-like in zebrafish embryos decreased hemoglobin production and impaired the organization of mesencephalic vein and other brain blood vessels. Interestingly, exposure of zebrafish embryos to UV resulted in an increase in mRNA expression of C1q-like, whereas over-expression of C1q-like was not enough resist to the damage. Furthermore, C1q-like transcription was up-regulated in response to pathogen Aeromonas hydrophila, and embryo survival significantly decreased in the C1q-like morphants after exposure to the bacteria. The data suggested that C1q-like might play an antiapoptotic and protective role in inhibiting p53-dependent and caspase 3/9-mediated apoptosis during embryogenesis, especially in the brain development, and C1q-like should be a novel regulator of cell survival during zebrafish embryogenesis.
Journal of Virology，doi:10.1128/JVI.02385-07，Rong Zhu，Jian-Fang Gui
Functional domains and the antiviral effect of the dsRNA-dependent protein kinase PKR from Paralichthys olivaceusState Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Wuhan 430072, China
* To whom correspondence should be addressed. Email: jfgui@ihb.ac.cn .
Abstract
The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2). However, little is known about the data related to low vertebrate including fish. Recently, identification of PKR-like or PKZ has addressed the question of whether there is orthologous PKR in fish. Herein, we identify the first fish PKR gene from Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows conserved structure characteristic of mammalian PKRs, with the N-terminal region for dsRNA binding and the C-terminal region for inhibition of protein translation. The catalytic activity of PoPKR is further validated to require for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2 phosphorylation and inhibits replication of SMRV (Scophthalmus maximus rhabdovirus) in FEC cells, whereas the phosphorylation and antiviral effect are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2. The interaction between PoPKR and eIF2 is demonstrated by coimmunoprecipitation assays, and transfection of PoPKR-specific siRNA further reveals that the enhanced eIF2 phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of PKR-mediated antiviral pathway in fish, and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.