| 277 | 2 | 236 |
| 下载次数 | 被引频次 | 阅读次数 |
为探究转化生长因子β激活激酶1基因(TAK1)在硬骨鱼先天免疫中的重要作用，本研究基于实验室已建的牙鲆(Paralichthys olivaceus)转录组数据库，对牙鲆TAK1基因(PoTAK1)进行了克隆及验证。研究显示，其开放阅读框(ORF)长为1 728 bp,编码575个氨基酸，蛋白分子量(Mw)约为64.33 kDa,理论等电点(pI)为6.66,编码的蛋白在N端具有一个S＿TKc结构域，在C端具有一个卷曲螺旋区域(Coiled coil region)。氨基酸多序列比对和系统发育分析表明，TAK1基因在脊椎动物中高度保守。组织表达分析显示，PoTAK1在肝脏和鳃中的表达量较高；体内攻毒实验表明，被迟缓爱德华氏菌(Edwardsiella tarda)感染后，PoTAK1在脾脏和头肾中的表达量均显著上调；体外免疫刺激实验表明，Poly I:C、TNF-α和迟缓爱德华氏菌刺激后，在各时间点牙鲆鳃细胞系中PoTAK1的表达量均显著上调。亚细胞定位显示，PoTAK1主要在细胞质中表达。双萤光素酶报告实验证明，PoTAK1能够激活MAPK通路下游AP-1转录因子的活性。体外免疫调节检测实验表明，过表达PoTAK1能够上调AP-1下游IL-1β、IL-6、IL-8和TNF-α等促炎细胞因子的转录表达。研究结果表明，PoTAK1参与了牙鲆的先天免疫反应，并对其具有一定的调节作用。本研究有助于更好地理解PoTAK1在牙鲆抵抗外界病原体入侵过程中的作用机制。
Abstract：To investigate the important role of transforming growth factor β-activated kinase 1 gene(TAK1) in innate immunity of teleost, the Japanese flounder TAK1 gene(PoTAK1) was cloned and verified based on the established transcriptome database of Japanese flounder in this study. The results showed that the length of open reading frame(ORF) of PoTAK1 was 1 728 bp, which encoded 575 amino acids. The protein molecular weight(Mw) of PoTAK1 was approximately 64.33 kDa, and the theoretical isoelectric point(pI) of PoTAK1 was 6.66. The encoded protein of PoTAK1 possesses a S＿TKc domain at the N-terminal and a coiled-coil region at the C-terminal. Amino acid multiple sequence alignment and phylogenetic analysis showed that TAK1 gene was highly conserved in vertebrates. Tissue expression analysis exhibited that the expression level of PoTAK1 was relatively high in liver and gill. The challenge experiment in vivo showed that the expression of PoTAK1 in spleen and head kidney was significantly up-regulated after Edwardsiella tarda infection. The immunostimulation experiment in vitro showed the expression of PoTAK1 was significantly up-regulated in Japanese flounder gill cell line at each time point after Poly I:C, TNF-α and E. tarda stimulation. Subcellular localization showed that PoTAK1 was mainly expressed in cytoplasm. Dual luciferase assay demonstrated that PoTAK1 can activate the activity of AP-1 transcription factor downstream of MAPK pathway. Immunomodulation assay in vitro demonstrated that the overexpression of PoTAK1 was able to up-regulate the transcriptional expression of pro-inflammatory cytokines downstream of AP-1, such as IL-1β, IL-6, IL-8, and TNF-α. The above findings indicated that PoTAK1 was involved in the innate immune response of Japanese flounder and possessed a certain regulatory role on it. In conclusion, our findings should aid to better understand the mechanism of PoTAK1 in the process of Japanese flounder against to the invasion of external pathogens.
[1] Sakurai H.Targeting of TAK1 in inflammatory disorders and cancer[J].Trends in Pharmacological Sciences,2012,33(10):522-530.
[2] Wang S,Li H Y,Lǚ K,et al.Identification and characterization of transforming growth factor β-activated kinase 1 from Litopenaeus vannamei involved in anti-bacterial host defense[J].Fish & Shellfish Immunology,2016,52:278-288.
[3] Ajibade A A,Wang H Y,Wang R F.Cell type-specific function of TAK1 in innate immune signaling[J].Trends in Immunology,2013,34(7):307-316.
[4] Yamaguchi K,Shirakabe K,Shibuya H,et al.Identification of a member of the MAPKKK family as a potential mediator of TGF-β signal transduction[J].Science,1995,270(5244):2008-2011.
[5] Dai L,Thu C A,Liu X Y,et al.TAK1,more than just innate immunity[J].IUBMB Life,2012,64(10):825-834.
[6] Ouyang C,Nie L,Gu M,et al.Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) activation requires phosphorylation of serine 412 by protein kinase A catalytic subunit α (PKACα) and X-linked protein kinase (PRKX)[J].Journal of Biological Chemistry,2014,289(35):24226-24237.
[7] Chen I T,Hsu P H,Hsu W C,et al.Polyubiquitination of transforming growth factor β-activated kinase 1 (TAK1) at lysine 562 residue regulates TLR4-mediated JNK and p38 MAPK activation[J].Scientific Reports,2015,5:12300.
[8] Delaney J R,Mlodzik M.TGF-β activated kinase-1:New insights into the diverse roles of TAK1 in development and immunity[J].Cell Cycle,2006,5(24):2852-2855.
[9] Shim J H,Xiao C C,Paschal A E,et al.TAK1,but not TAB1 or TAB2,plays an essential role in multiple signaling pathways in vivo[J].Genes & Development,2005,19(22):2668-2681.
[10] Zhao F,Li Y W,Pan H J,et al.Grass carp (Ctenopharyngodon idella) TRAF6 and TAK1:Molecular cloning and expression analysis after Ichthyophthirius multifiliis infection[J].Fish & Shellfish Immunology,2013,34(6):1514-1523.
[11] Li Y W,Li X,Wang Z,et al.Orange-spotted grouper Epinephelus coioides Tak1:Molecular identification,expression analysis and functional study[J].Journal of Fish Biology,2015,86(2):417-430.
[12] Jang J H,Kim H,Cho J H.Molecular cloning and functional characterization of TRAF6 and TAK1 in rainbow trout,Oncorhynchus mykiss[J].Fish & Shellfish Immunology,2019,84:927-936.
[13] Park S B,Aoki T,Jung T S.Pathogenesis of and strategies for preventing Edwardsiella tarda infection in fish[J].Veterinary Research,2012,43(1):67.
[14] Isshik T,Nishizawa T,Kobayashi T,et al.An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed Japanese flounder Paralichthys olivaceus in Japan[J].Diseases of Aquatic Organisms,2001,47(2):87-99.
[15] Moustafa E M,Naota M,Morita T,et al.Pathological study on the scuticociliatosis affecting farmed Japanese flounder (Paralichthys olivaceus) in Japan[J].Journal of Veterinary Medical Science,2010,72(10):1359-1362.
[16] Xu T T,Zhang X H.Edwardsiella tarda:An intriguing problem in aquaculture[J].Aquaculture,2014,431:129-135.
[17] Kim J H,Sohn S,Kim S K,et al.Effects on hematological parameters,antioxidant and immune responses,AChE,and stress indicators of olive flounders,Paralichthys olivaceus,raised in bio-floc and seawater challenged by Edwardsiella tarda[J].Fish & Shellfish Immunology,2020,97:194-203.
[18] 周丽，宫庆礼，俞开康.牙鲆的疾病[J].青岛海洋大学学报，1997(2):45-52.Zhou L,Gong Q L,Yu K K.Diseases of Japanese flounder,Paralichthys olivaceus[J].Journal of Ocean University of Qingdao,1997(2):45-52.
[19] Ellis A E.Innate host defense mechanisms of fish against viruses and bacteria[J].Developmental & Comparative Immunology,2001,25(8-9):827-839.
[20] Magnadottir B.Innate immunity of fish (overview)[J].Fish & Shellfish Immunology,2006,20(2):137-151.
[21] Plouffe D A,Hanington P C,Walsh J G,et al.Comparison of select innate immune mechanisms of fish and mammals[J].Xenotransplantation,2005,12(4):266-277.
[22] Park S I.Disease control in korean aquaculture[J].Fish Pathology,2009,44(1):19-23.
[23] 曹丹丹，刘金相，王志刚，等.牙鲆NOD2基因的表达分析及在抗迟缓爱德华氏菌感染过程中的功能[J].渔业科学进展，2018,39(3):53-64.Cao D D,Liu J X,Wang Z G,et al.Expression analysis and functional characterization of NOD2 in the resistance of Japanese flounder (Paralichthys olivaceus) to Edwardsiella tarda infection[J].Progress in Fishery Sciences,2018,39(3):53-64.
[24] 王欣桐，王宣刚，孙敏敏，等.牙鲆PIK3R3-like基因的表达分析及重组表达[J].中国海洋大学学报(自然科学版),2021,51(11):49-58.Wang X T,Wang X G,Sun M M,et al.Identification,expression analysis and inducible expression of PIK3R3-like in Japanese flounder (Paralichthys olivaceus) [J].Periodical of Ocean University of China,2021,51(11):49-58.
[25] Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method[J].Methods,2001,25(4):402-408.
[26] Kajino-Sakamoto R,Inagaki M,Lippert E,et al.Enterocyte-derived TAK1 signaling prevents epithelium apoptosis and the development of ileitis and colitis[J].Journal of Immunology,2008,181(2):1143-1152.
[27] Omori E,Matsumoto K,Sanjo H,et al.TAK1 is a master regulator of epidermal homeostasis involving skin inflammation and apoptosis[J].Journal of Biological Chemistry,2006,281(28):19610-19617.
[28] Ma F Y,Tesch G H,Ozols E,et al.TGF-β1-activated kinase-1 regulates inflammation and fibrosis in the obstructed kidney[J].American Journal of Physiology-Renal Physiology,2011,300(6):1410-1421.
[29] Paquette N,Conlon J,Sweet C,et al.Serine/threonine acetylation of TGFβ-activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(31):12710-12715.
[30] Xu Y R,Lei C Q.TAK1-TABs complex:A central signalosome in inflammatory responses[J].Frontiers in Immunology,2020,11:608976.
[31] Fan Y H,Yu Y,Shi Y,et al.Lysine 63-linked polyubiquitination of TAK1 at lysine 158 is required for tumor necrosis factor α- and interleukin-1β-induced IKK/NF-κB and JNK/AP-1 activation[J].Journal of Biological Chemistry,2010,285(8):5347-5360.
[32] Mao R F,Fan Y H,Mou Y G,et al.TAK1 lysine 158 is required for TGF-β-induced TRAF6-mediated Smad-independent IKK/NF-κB and JNK/AP-1 activation[J].Cellular Signalling,2011,23(1):222-227.
[33] Yu Y,Ge N L,Xie M,et al.Phosphorylation of Thr-178 and Thr-184 in the TAK1 T-loop is required for interleukin (IL)-1-mediated optimal NFκB and AP-1 activation as well as IL-6 gene expression[J].Journal of Biological Chemistry,2008,283(36):24497-24505.
[34] Sakurai H,Miyoshi H,Mizukami J,et al.Phosphorylation-dependent activation of TAK1 mitogen-activated protein kinase kinase kinase by TAB1[J].FEBS Letters,2000,474(2-3):141-145.
[35] Singhirunnusorn P,Suzuki S,Kawasaki N,et al.Critical roles of threonine 187 phosphorylation in cellular stress-induced rapid and transient activation of transforming growth factor-β-activated kinase 1 (TAK1) in a signaling complex containing TAK1-binding protein TAB1 and TAB2[J].Journal of Biological Chemistry,2005,280(8):7359-7368.
[36] Takaesu G,Kishida S,Hiyama A,et al.TAB2,a novel adaptor protein,mediates activation of TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signal transduction pathway[J].Molecular Cell,2000,5(4):649-658.
[37] Kondo M,Osada H,Uchida K,et al.Molecular cloning of human TAK1 and its mutational analysis in human lung cancer[J].International Journal of Cancer,1998,75(4):559-563.
[38] Meng F,Kang M J,Liu L,et al.Characterization of the TAK1 gene in Apis cerana cerana (AccTAK1) and its involvement in the regulation of tissue-specific development[J].BMB Reports,2011,44(3):187-192.
[39] Qi Z T,Xu Y,Wang X,et al.TLR13,TLR22,TRAF6,and TAK1 in the soiny mullet (Liza haematocheila):Molecular characterization and expression profiling analysis[J].Developmental & Comparative Immunology,2020,112:103774.
[40] Bao S Y,Sun Q X,Yao C L.The interaction of TAK1 and TAB1 enhances LPS-induced cytokine release via modulating NF-κB activation (Larimichthys crocea)[J].Fish & Shellfish Immunology,2018,74:450-458.
[41] Secombes C J,Wang T.The innate and adaptive immune system of fish[M]//Infectious Disease in Aquaculture:Prevention and Control.UK:Woodhead Publishing Limited,2012:3-68.
[42] Wang C Y,Peng J,Zhou M Y,et al.TAK1 of black carp positively regulates IRF7-mediated antiviral signaling in innate immune activation[J].Fish & Shellfish Immunology,2019,84:83-90.
[43] Collings D A.Subcellular localization of transiently expressed fluorescent fusion proteins[J].Methods in Molecular Biology,2013,1069:227-258.
[44] Wang S,Li H Y,Chen R J,et al.TAK1 confers antibacterial protection through mediating the activation of MAPK and NF-κB pathways in shrimp[J].Fish & Shellfish Immunology,2022,123:248-256.
[45] Boshtam M,Asgary S,Kouhpayeh S,et al.Aptamers against pro- and anti-inflammatory cytokines:A review[J].Inflammation,2017,40(1):340-349.
[46] Kim E Y,Moudgil K D.Immunomodulation of autoimmune arthritis by pro-inflammatory cytokines[J].Cytokine,2017,98:87-96.
[47] Gao Y,Huang Y J,Zhao Y,et al.LL202 protects against dextran sulfate sodium-induced experimental colitis in mice by inhibiting MAPK/AP-1 signaling[J].Oncotarget,2016,7(39):63981-63994.
[48] Ondrey F G,Dong G,Sunwoo J,et al.Constitutive activation of transcription factors NF-κB,AP-1,and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines[J].Molecular Carcinogenesis,1999,26(2):119-129.
[49] Smolinska M J,Page T H,Urbaniak A M,et al.Hck tyrosine kinase regulates TLR4-induced TNF and IL-6 production via AP-1[J].Journal of Immunology,2011,187(11):6043-6051.
基本信息:
中图分类号:S917.4
引用信息:
[1]李恒顺,司瑜,王宣刚,等.牙鲆TAK1基因的表达分析及免疫功能探究[J],2023,53(03):152-165.
基金信息:
国家自然科学基金项目(31802327)资助~~
2022-05-03
2022
2022-06-25
2022-09-02
2022
1