2022,
Vol. 31
先天免疫系统是宿主抵御病原入侵的第一道防线，病原入侵会触发宿主细胞的即时反应，也称为先天免疫反应。先天免疫系统包括多种信号级联反应，这些信号级联反应是由细胞内外的模式识别受体(Pattern recognition receptor, PRRs)检测识别病原相关分子模式(Pathogen associated molecular patterns, PAMP)引发的，最终诱导下游I型干扰素(Interferon-I, IFN-I)和促炎性细胞因子的产生。其中Toll样受体(Toll-like receptor, TLR)、核苷酸结合寡聚化结构域NOD样受体(NOD-like receptor, NLR)和RIG-I样受体(RIG-I-like receptor, RLR)[1]是被广泛研究的3种模式识别受体家族。Toll样受体大多存在于细胞膜或细胞器膜上，是一类具有跨膜属性的模式识别受体，可识别多种细菌和病毒的入侵[2-3]；RIG-I样受体可以识别病毒感染时产生的核酸成分并对其做出反应[4]；NOD样受体作为调节IFN-I和NF-κB(Nuclear factor kappa B)活化的抗病毒介质，同样能被Toll样受体激活并参与抗病毒免疫调节。模式识别受体识别入侵的病原后，关键的衔接蛋白通过级联反应将免疫信号层层传递，最终将信号转导到细胞核中，激活免疫基因的转录和翻译。过度的免疫反应会影响细胞稳定，随着机体对外来病原的清除，蛋白级联反应会巧妙的进行自我限制以减轻过度免疫反应对机体的损害[5]。
蛋白翻译后修饰(Post-translational modification, PTM)通过靶向细胞内蛋白，在先天免疫反应中的病原识别和免疫调节中起到重要作用[6]。PTM主要包括泛素化修饰、磷酸化修饰以及乙酰化修饰等；泛素化作为蛋白翻译后修饰类型之一，在抗病毒信号通路中被广泛研究。泛素是一种由76个氨基酸组成的小蛋白，可以通过两种方式靶向目标蛋白，即共价键结合(锚定泛素)或非共价结合(非锚定泛素)，被泛素靶向的蛋白由泛素蛋白自身的泛素化位点决定其在信号通路中的命运[6-7]。泛素化修饰通过3种泛素酶的协同作用来启动泛素化过程：第一阶段，E1泛素激活酶在其活性位点半胱氨酸(Cys)与泛素蛋白C末端甘氨酸(Gly)之间形成硫酯键；第二阶段，泛素通过硫基化反应转移到E2泛素结合酶的Cys位点；最后阶段，泛素通过E2泛素结合酶与E3泛素连接酶的相互作用转移到靶向的蛋白上[8-10]。泛素可以通过在其自身的7个赖氨酸位点(K6 / K11 / K27 / K29 / K33 / K48 / K63)上进行泛素蛋白连接形成线性多聚泛素链[11]。在蛋白连接不同赖氨酸位点的泛素化中，K48连接的多聚泛素链通常使靶蛋白通过蛋白酶体途径被降解。而K63连接的线性多聚泛素链参与控制蛋白激酶的激活以及众多细胞信号通路的有序转导[12]。本文重点关注RLR信号通路中RIG-I、MAVS、TBK1、TRAF3和TRAF6等重要分子的泛素修饰的相关研究进展(图 1)。
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黑色箭头代表促进泛素化或激活其他蛋白；红色平头代表抑制泛素化或阻止蛋白发挥功能。 Black arrows represent promotion of ubiquitination or activation of other proteins; red flat heads represent inhibition of ubiquitination or preventing protein function. 图 1 RLR介导抗病毒免疫反应的泛素化机制示意图 Fig. 1 Schematic diagram of ubiquitination mechanism of RLR-mediated antiviral immune response |
模式识别受体下游信号转导的调节对抗病毒免疫反应至关重要。作为RLR信号级联反应中的3个蛋白受体，RIG-I(Retinoic Acid-inducible Gene-I)、MDA5(Melanoma differentiation-associated protein 5)和LGP2(Laboratory of genetics and physiology 2)都包含1个具有ATP水解和RNA结合活性的DExD/H-box解旋酶结构域[4, 13]，和一个能够识别RNA底物的C端结构域(C-terminal domain，CTD)[14-15]。然而，只有RIG-I和MDA5具有N-末端caspase激活和募集结构域(Caspase activation and recruitment domain，CARD)来激活下游免疫信号转导[16]。RIG-I和MDA5均可以识别双链RNA(dsRNA)，研究发现dsRNA的长短对于RIG-I样受体的识别十分重要。RIG-I可以结合相对较短的dsRNA(< 1 kb)，MDA5可以特异性结合长双链dsRNA；将dsRNA的长度缩短后，由MDA5受体转变成RIG-I受体来识别dsRNA[17-19]。RIG-I和MDA5在识别病毒RNA时构象会发生变化，SUMO E3泛素连接酶TRIM38(Tripartite motif-containing protein 38)可以动态修饰RIG-I和MDA5的类泛素化修饰(Sumoylation)，以确保其处于最佳激活状态[20]。磷酸酶PP1(Protein phosphatase 1)的亚基PP1α和PP1γ将RIG-I和MDA5去磷酸化并使其活化[21]，K63连接的多聚泛素链被转移到RIG-I和MDA5上[22-24]，RIG-I和MDA5在线粒体内膜上招募并激活了线粒体抗病毒信号蛋白MAVS(Mitochondrial antiviral-signaling)[25-26]。在K63多聚泛素链存在的情况下，线粒体上的MAVS将转化为功能性聚集体[27]。MAVS聚集了E3泛素连接酶TRAF(TNF receptor-associated factor)家族中的TRAF2、TRAF3、TRAF6等，这些E3连接酶参与MAVS复合体形成并且促进MAVS发生K63泛素化，进而促进TBK1(TANK binding kinase 1)和IKKα/β(Inhibitor kappa B kinase α/β)复合物的磷酸化[27-30]。磷酸化后的TBK1和IKKα/β分别激活IRF(IFN regulatory factor)3/7和NF-κB，随后它们从线粒体释放并易位进入细胞核，诱导下游抗病毒基因I型干扰素(IFN-I)产生，进而促进干扰素激活基因(IFN-stimulated genes, ISGs)的转录，使免疫细胞和周围细胞处于抗病毒状态。
1.1 RIG-I的泛素化修饰TRIM25(Tripartite motif-containing protein 25)是RIG-I先天免疫信号转导的关键分子[23, 31]。研究表明TRIM25参与了RIG-I的泛素化和激活[32-33]。RIG-I激活依赖于TRIM25的调节，TRIM25的SPRY结构域与RIG-I互作，促进了RIG-I CARD结构域中Lys172位点上K63连接的多聚泛素化[22]。在研究TRIM25的功能中发现了几种影响TRIM25的E3酶活性的调节剂。NDR2(Nuclear Dbf2-related kinase 2)作为RIG-I和TRIM25的衔接蛋白促进了RIG-I/TRIM25复合物的形成，增强了TRIM25介导RIG-I连接的K63多聚泛素化[34]。RNA pull-down发现长链非编码RNA Lnczc3h7a与TRIM25结合，促进RIG-I连接K63的多聚泛素化[35]。NLRP12(NLR family pyrin domain containing 12)与TRIM25相互作用，阻止TRIM25介导RIG-I连接的K63泛素化和激活[36]。LGP2作为3个RLR模式识别受体之一，其作用机制尚未明确。研究发现LGP2与E3泛素连接酶TRIM25相互作用，抑制了TRIM25介导RIG-I的K63泛素化[37]。近来发现另一种名为Riplet，也称Ring finger protein 135(RNF135)的E3泛素连接酶，该酶与TRIM家族具有高度同源性。Riplet的C末端区域结合RIG-I并激活其K63连接的泛素化，促进RIG-I介导IFN-β启动子激活[38]。Riplet基因敲除的小鼠对水疱性口炎病毒更加敏感，证实了Riplet在抗病毒反应中的重要性[39]。RIG-I的Lys788位点对Riplet介导的K63泛素化至关重要，研究并未否定TRIM25对于RIG-I的重要性并认为Riplet可能是TRIM25激活RIG-I信号的先决条件[40]。最新的研究发现敲除TRIM25后并未影响机体在甲型流感病毒、乙型流感病毒、仙台病毒等感染中的IFN-I信号激活，而敲除Riplet削弱了RIG-I激活的IFN-I信号[41]。E2泛素结合酶Ube2D3(Ubiquitin-conjugating enzyme E2D3)和Ube2N协同E3泛素连接酶Riplet激活RIG-I，Ube2D3-Riplet促进RIG-I连接的K63泛素化，而Ube2N-Riplet促进未锚定多泛素链的生成，激活RIG-I信号[42]。另外两种E3泛素连接酶MEX3C(Mex-3 RNA-Binding Family Member C)和TRIM4被确定参与RIG-I连接K63多聚泛素链的形成，并促进下游信号传导[24, 43]。USP14(Ubiquitin-specific proteases 14)也被认为是抗病毒反应的负调节剂之一，它能与RIG-I互作并消除其K63连接的多聚泛素化[44]。
RIG-I连接的K48泛素化减弱了RLR信号传导。RNF125通过K48连接的多聚泛素链降解RIG-I和MDA5蛋白并抑制IFN-I激活[45]。非典型激酶Riok3(RIO Kinase 3)招募E3泛素连接酶TRIM40，促进K27和K48连接的多聚泛素化降解RIG-I和MDA5[46]。病毒也能通过对靶蛋白的降解进行免疫逃避，如猪急性腹泻综合征冠状病毒的N蛋白与RIG-I相互作用并促进其K48连接的泛素化，诱导RIG-I的蛋白酶体途径降解[47]。USP4的过表达显著增强了RIG-I触发的IFN-β信号传导，并通过去除RIG-I连接的K48泛素化稳定RLR信号转导，抑制了VSV病毒的复制[48]。蛋白激酶抑制剂PRKRIR(Protein-kinase, IFN-inducible double-stranded RNA dependent inhibitor, and repressor of P58 repressor)通过阻断RIG-I连接的K48泛素化，阻止了RIG-I通过蛋白酶体途径降解，增强了RIG-I的稳定性[49]。研究发现，未锚定泛素链也积极参与IFN-I信号传导。锚定的K48多聚泛素链被认为参与蛋白酶体途径降解，与此不同的是，未锚定的K48泛素化修饰可能具有正向调节作用。TRIM6合成未锚定的K48连接的多聚泛素链激活IKKε后，促进STAT1(Signal transducer and activator of transcription 1)磷酸化[50]。TRIM6与DHX16(DEAH box polypeptide 16)和RIG-I内源性互作，其合成的未锚定K48连接的泛素链能促进DHX16与RIG-I的结合并介导IFN-I的产生和ISGs高表达[51]。
1.2 MAVS的泛素化修饰目前研究显示，MAVS的泛素化修饰多发生在病毒感染期间，在静息状态时一般很少受到泛素化修饰。线粒体上的MAVS作为RLR信号通路中的一个关键衔接蛋白，被多种病毒作为目标以各种方式进行攻击[52]。MAVS蛋白连接的K63泛素化对于抗病毒信号传导十分关键。例如，TRIM31通过促进MAVS连接的K63泛素化增强了MAVS多聚体的形成[53]。病毒感染后增强了USP18与MAVS的互作，并促进了MAVS连接的K63泛素化，而敲除USP18的小鼠更容易受到病毒感染[54]。仙台病毒感染多种免疫细胞后促进去泛素化酶YOD1(Ubiquitin thioesterase OTU1)mRNA高表达，YOD1仅在病毒感染后与内源MAVS互作，以消除MAVS连接的K63泛素化并影响多聚体形成，这是为数不多的有关去泛素化酶靶向MAVS负调控IFN信号的研究[55]。SARS-CoV病毒基因组编码的辅助蛋白ORF-9b可以通过泛素化修饰调节机制降解MAVS，从而抵抗抗病毒免疫反应[56]。TRIM25除了参与激活RIG-I并增强其与MAVS的结合外，还促进MAVS的Lys7和Lys10位点上的泛素化并诱导其降解。该研究证明MAVS多聚体的消失并不代表抑制信号传导，反而能够快速释放MAVS聚合体并激发了下游IRF3的磷酸化[57]。多个E3泛素连接酶参与MAVS的蛋白酶体降解，例如，RNF5促进MAVS的Lys362和Lys461位点发生K48泛素化[58]；而在MAVS的Lys371和Lys420位点，PCBP2(PolyIC binding protein 2)招募包含HECT域的E3泛素连接酶AIP4(Atrophin-1-interacting protein 4)促进MAVS连接的K48泛素化并使其降解[59]；RACK1(Receptor for activated C kinase 1)促进了MAVS连接的K48泛素化，进而降低了MAVS介导的抗病毒信号转导，并且减弱了MAVS连接的K63泛素化从而降低其活性[60]；Ndfip1(Nedd4 family interacting protein 1)与MAVS结合，并募集E3泛素连接酶Smurf1(SMAD specific E3 ubiquitin protein ligase 1)和Smurf2从而促进MAVS发生泛素化降解[61-62]。病毒诱导OTUD1(OTU deubiquitinase 1)高表达，OTUD1通过增强Smurf1对MAVS的K48泛素化，促进MAVS的蛋白酶体降解[63]；RNF115能够与MAVS互作，并且调节稳态中MAVS的K48泛素化，RNF115的缺失增强了RNA病毒触发的抗病毒信号传导[64]。SeV和VSV病毒感染免疫细胞MEF或BMDCs后，增强了OTUD4与MAVS的互作，消除MAVS连接的K48泛素化并抑制MAVS蛋白降解；在OTUD4失活后，这种能力被显著减弱，这也是少数有关去泛素化酶靶向MAVS正向调节IFN信号的报道[65]。
1.3 TBK1的泛素化修饰TBK1是参与先天免疫反应的重要蛋白，包含1个N末端激酶结构域，1个泛素样结构域(Ubiquitin-like domain，ULD)和2个C末端卷曲螺旋结构域(CCD1和CCD2)。A20和TAX1BP1(Tax1-binding protein 1)通过破坏TBK1-IKKi复合体的形成并且抑制K63泛素化，抑制病毒感染触发的IRF3激活，从而阻断抗病毒信号传导，但并未发现A20具体调控TBK1泛素化的机制[66]。E3泛素连接酶Nrdp1(Neuregulin receptor degradation protein-1)促进TBK1的K63泛素化，对IFN信号传递起到正向作用；A20通过抑制Nrdp1介导的TBK1激活，减少了IFN-β的产生[67-68]。MIB2(Mindbomb E3 ubiquitin protein ligase 2)被认为可能是参与MAVS介导的TBK1激活的另一种E3泛素连接酶，它与MAVS结合后促进了TBK1连接的K63泛素化，从而激活下游IRF3/7[69]。E3泛素连接酶RNF128是TBK1激活的正调节因子，RNF128的敲降或缺失会减弱IRF3激活和IFN-β的产生，RNF128与TBK1结合并通过增强TBK1连接的K63泛素化促进IFN-I信号传导[70]。UBE2S作为E2泛素结合酶招募去泛素化酶USP15去除TBK1连接的K63泛素化，抑制IFN-I信号[71]。非洲猪瘟病毒pI215L蛋白招募E3泛素连接酶RNF138抑制TBK1连接的K63泛素化，抑制TBK1的活性并进行免疫逃避[72]。
NLRP4(NLR family pyrin domain containing 4)通过靶向TBK1作为I型干扰素信号的负调节剂，NLRP4招募E3泛素连接酶DTX4(Deltex E3 ubiquitin ligase 4)，促进TBK1连接的K48泛素化和蛋白降解，防止抗病毒免疫反应的过度激活[73]。DYRK2(Dual-specificity tyrosine-Y-phosphorylation-regulated kinase 2)磷酸化TBK1的Ser527位点对于招募NLRP4和DTX4降解TBK1至关重要，并以激酶活性依赖性方式促进TBK1连接的K48泛素化[74]。此外TRIP(Tumor necrosis factor interacting protein)作为被病毒诱导的E3泛素连接酶，通过促进TBK1连接的K48泛素化和蛋白酶体降解负调控抗病毒免疫反应[75]。USP38在TBK1的Lys670位点上特异性地切割了K33连接的多泛素链，并促进DTX4和TRIP介导的TBK1连接的K48泛素化[76]。蛋白激酶MAP4K1(Mitogen-activated protein kinase kinase kinase kinase 1)参与先天抗病毒免疫反应调控，MAP4K1与TBK1互作并在E3泛素连接酶DTX4的帮助下促进TBK1连接的K48泛素化降解[77]。USP1与UAF1(USP1-associated factor 1)形成去泛素化酶蛋白复合体，与TBK1结合并去除了TBK1连接的K48泛素化，稳定了TBK1的蛋白表达[78]。
1.4 TRAF的泛素化修饰肿瘤坏死因子受体相关因子家族共有6个已知蛋白(TRAF1-6)，参与RLR，NLR和TLR 3种模式识别受体下游蛋白信号级联反应，调节NF-κB激活和IFN-I生成。TRAF蛋白家族具有RING指结构域和多个锌指结构位点，这是E3泛素连接酶的典型特征之一。TRAF2、TRAF4、TRAF5和TRAF6在自身赖氨酸位点上进行K63泛素化连接，激活自身传递信号的功能[79-82]。TRAF3和TRAF6是RLR途径中参与MAVS激活的蛋白，TRAF3通过激活TBK1/IRF3来促进IFN-I表达，TRAF6则通过激活MEKK1(MAPK/ERK kinase kinase 1)进而激活NF-κB，促进IFN-I的表达[66, 83]。
病毒感染后增强了内源UBL4A(Ubiquitin-like protein 4a)与TRAF6互作，促进TRAF6连接的K63泛素化，从而正向调节抗病毒信号中TRAF6的活性[84]。TRIM24直接靶向介导TRAF3在K429/K436位点上连接的K63泛素化，并且促进MAVS与TBK1的结合以激活下游抗病毒信号[85]。DUBA(Deubiquitinating enzyme A)作为调节IFN-I产生的去泛素酶，能特异性将TRAF3连接的K63泛素化进行切割，抑制IFN-I信号激活[86]。病毒入侵后OTUB1(Otubain-1)和OTUB2靶向TRAF3和TRAF6，并同时对TRAF3和TRAF6进行去泛素化，抑制IFN-I信号激活[87]。HSCARG(NmrA-like family domain-containing protein 1，NMRAL1)靶向TRAF3并招募OTUB1并对其进行去泛素化，从而避免过度的抗病毒先天免疫反应[88]。MCPIP1(MCP-induced protein 1)以DUB的方式抑制RLR信号通路中TRAF家族蛋白的K63泛素化，进而抑制了IFN-β的产生[89]。AIP4广泛且多重地抑制NLR，RLR和TLR介导的免疫信号传导。E3泛素连接酶cIAP1(cellular inhibitor of apoptosis proteins 1)在病毒感染期间促进TRAF3/6的激活，而AIP4促进cIAP1发生溶酶体降解，作为cIAP1抑制剂来减弱IFN-I和NF-κB的活化[29, 90]。在斑马鱼中，精氨酸甲基转移酶prmt2(protein arginine methyltransferase 2)通过与TRAF6的C末端结合，阻止其自身的K63泛素化，进而影响抗病毒信号转导[91]。
TRAF连接的K48泛素化同样是调控抗病毒信号的重要因素，例如，OTUD1通过去泛素化上调细胞内Smurf1的蛋白水平，增强了Smurf1与MAVS、TRAF3和TRAF6的结合，从而促进MAVS/TRAF3/TRAF6复合体中蛋白的K48泛素化并引发泛素-蛋白酶体降解[63]；泛素连接酶Parkin通过促进TRAF3发生K48泛素化，降低蛋白的稳定性来调节RLR信号转导[92]。
2 展望在众多的蛋白翻译后修饰中，泛素化修饰在调节抗病毒先天免疫反应中起到关键性作用，大多数参与信号级联反应的蛋白能够被泛素化修饰，它们的激活过程也受到严格控制，并通过严谨的负反馈调节机制来防止过度免疫，这种动态调整为保护机体起到了积极作用。RIG-I、MAVS、TBK1和TRAF3/6连接的K63泛素化积极参与信号传导过程，一些E3泛素连接酶和去泛素化酶家族蛋白可以促进K63的泛素化从而正向调节抗病毒信号通路。同时也发现病毒能直接通过操控宿主泛素修饰系统抑制先天免疫，或间接利用宿主机制干扰细胞的抗病毒信号传导逃避先天免疫。如部分被病毒诱导后的蛋白促进靶蛋白的K48泛素化和泛素-蛋白酶体途径降解。这要求更加精确判断靶点蛋白，确定E3s和DUBs的作用靶点。此外，未锚定泛素链的研究目前很少，但是其机制更加值得深入研究，如未锚定K48泛素链与蛋白酶体降解途径无关，而且具有正向的调控作用，这也将是泛素化的研究重点之一。越来越多的研究正在关注蛋白的泛素修饰机制，更加全面深入的发现具有特异性的E3泛素连接酶和去泛素化家族蛋白并探究其生物学机制，将为疾病治疗和免疫防控提供更多的解决方案。在鱼类先天免疫领域，泛素化修饰研究尚处于起步阶段，鱼类的E3泛素连接酶家族以及去泛素化蛋白家族相关研究也相对较少。本文通过对哺乳动物先天免疫泛素化修饰机制的深入了解及探讨，以期有助于推动泛素化修饰在鱼类先天免疫反应中的作用及机制研究。
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