DDX3的调控因素及生物学功用,病理学论文

　　摘要：　DEAD-box RNA解旋酶3(DEAD-box RNA helicase 3,DDX3),亦称DEAD-box RNA解旋酶3 X连锁(DEAD-box RNA helicase 3 X-linked,DDX3X),是一种高度保守并依赖ATP的RNA解旋酶,广泛分布于真核生物中,主要定位在细胞核和细胞质发挥生理作用,与RNA剪接和衰变、mRNA输出、转录、翻译相关,进一步参与细胞周期的进展、先天免疫反应、细胞凋亡、癌症发生与抑制、病毒的复制周期等多种重要的细胞生理过程.长期以来,DDX3的基因表达,调控及其功能以及它与疾病的关系一直被关注,尤其是与病毒和癌症的关系更为热点研究.因此,本文主要探讨、总结DDX3的病理生理学作用.

　　关键词：　DEAD-box RNA解旋酶3; DEAD-box RNA解旋酶3 X连锁; RNA; 肿瘤; 病毒;

　　Abstract：　DEAD-box RNA helicase 3(DDX3), also known as DEAD-box RNA helicase 3 X-linked(DDX3 X), is a highly conserved ATP-dependent RNA helicase. It widely distributes in eukaryotes, and mainly locates in the nucleus and cytoplasm to play physiological roles. It is related to RNA splicing and decay, mRNA output, transcription and translation, thus regulating cell cycle progression, innate immune response, apoptosis, cancer development and inhibition, virus replication cycle and many other important cellular physiological processes. For a long time, the gene expression, regulation and function of DDX3 and its relationship with diseases have been paid close attention to, especially the relationship with viruses and cancer has been the focus of many studies. Therefore, this article mainly reviews the pathophysiological effects of DDX3.

　　Keyword：　DEAD-box RNA helicase 3; DEAD-box RNA helicase 3 X-linked; RNA; tumor; virus;

　　DEAD-box蛋白质家族隶属于解旋酶超家族Ⅱ,是最大的解旋酶家族,因其结构特征基序Asp(D)-Glu(E)-Ala(A)-Asp(D)而得名[1,2].DEAD-box蛋白具有独特的12个保守基序,是其结合、水解ATP以及与RNA相互作用的结构基础[3].DEAD-box RNA解旋酶3(DEAD-box RNA helicase 3,DDX3)是DEAD-box蛋白家族的重要成员,具有DEAD-box RNA解旋酶3 Y连锁(DEAD-box RNA helicase 3 Y-linked,DDX3Y)和DEAD-box RNA解旋酶3 X连锁(DEAD-box RNA helicase 3 X-linked,DDX3X)两个同源物, 往往也将DDX3引为DDX3X.DDX3Y基因位于Y染色体上,仅表达雄性生殖系统[4,5];而DDX3X基因位于X染色体上,广泛表达于全身各组织,并参与细胞周期进展、先天免疫反应、细胞凋亡、癌症发生与抑制、病毒的复制周期等多种重要的细胞生理过程,因而受到了人们的广泛研究[6,7,8,9].本研究旨在系统总结DDX3的病理生理学作用,着重其与疾病的关系及生物学作用,以期探明可能的研究方向.

DDX3的调控因素及生物学功用

　　1、 DDX3简介

　　DDX3作为一种结构高度保守并依赖ATP的RNA解旋酶,广泛存在于各类真核生物中.1997年研究人员首次发现了人源DDX3蛋白,并通过原位杂交技术将其基因精确定位于Xp11.3-p11.23位点[10].人源DDX3编码基因长约16KB,共包含16个内含子及17个外显子,编码5.3KB转录本,对应662个氨基酸的多肽,富含丝氨酸(11.3%)和甘氨酸(11.4 %)[4,11].与所有的DEAD-box RNA解旋酶相似,DDX3的解旋酶核心由两个类似RecA样结构域构成,其中包含13个特征性解旋酶序列基序,并包括同义基序Ⅱ,该基序以单个字母代码读取为D-E-A-D[12,13]. DDX3解旋酶核心的两侧分别是一个序列复杂度低且具有RS样结构域的C末端和一个带有核输出信号(nuclear export signal,NES)且序列复杂度低的N末端[12,14].研究人员通过蛋白质结晶技术和X射线衍射分析,准确得到了DDX3解旋酶结构域的晶体结构,该晶体属于单斜空间群P21,其具体晶胞参数:a=43.85 ?, b=60.72 ?,c=88.39 ?,α=γ=90°,β=101.02°,并发现DDX3结晶中存在共结晶配体,进一步研究发现该配体能提高DDX3的热稳定性[15,16].DDX3作为一种穿梭蛋白定位于细胞核和细胞质,并在其中发挥相应的病理生理学功能,既往研究发现DDX3未修饰单体形式(相对分子量为73.2)过大,无法有效通过核孔被动扩散进入细胞质,需要N末端富含亮氨酸的NES与核出口受体细胞染色体维持蛋白1 (chromosomal region maintenance 1, CRM1,也称Exportin-1)结合,从而定位至核膜孔的细胞质侧[17,18].最近发现,DDX3的核输出不仅由CRM1介导,还依赖于NES和以活化GTP结合形式运行的单体鸟嘌呤核苷酸结合蛋白Ran[17].

　　2、 DDX3的调控因素

　　许多因素会造成DDX3基因突变致使其功能的改变,研究人员通过外显子组测序在髓母细胞瘤、头颈部鳞状细胞癌和血液系统恶性肿瘤中均发现了DDX3突变,DDX3的突变被认为是导致肿瘤发生的重要因素[19,20,21].DDX3的转录同样受到多种因素的调节,在人乳头瘤病毒相关的肺癌中,p53 对DDX3的转录发挥调控作用,p53的状态决定了DDX3的表达水平[22,23];在乳腺癌伴缺氧条件下,低氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)通过与正常乳腺上皮MCF10A细胞中DDX3启动子区的低氧诱导因子应答元件相结合,从而诱导激活DDX3的转录,因此核DDX3也被认为是乳腺癌不良预后的预测因子之一[24,25].丙型肝炎病毒(hepatitis C virus,HCV)的结构核心蛋白被认为是第一个可以与人类DDX3相互作用的病毒蛋白,相关功能实验表明,HCV核心蛋白也许能够增强DDX3转录共激活子的功能[11].在翻译水平,激活转录因子E74 样因子3(E74-like factor 3,elF3)和Cap依赖性翻译或抑制E74 样因子4(E74-like factor 4,elF4)均调控DDX3的翻译[26,27].另外DDX3的表达还受1型艾滋病病毒(human immunodeficiency virus type 1, HIV-1)Tat(HIV-1编码的反式激活因子)刺激[28];在肝癌中,应用卡马拉素(rottlerin)可上调DDX3在肿瘤细胞中的表达[29].此外,生活习惯也密切影响着DDX3的表达,包括吸烟、饮酒及其他习惯,且男性表达水平高于女性[30].

　　3、 DDX3与疾病的关系

　　DDX3对下游众多分子具有调控作用,参与了先天免疫反应、肿瘤的发生与抑制、病毒的周期复制等多种生物学过程,并在其中发挥了重要的病理生理学作用[7,9,31].长期以来,DDX3与疾病的关系被不断探索,其与肿瘤的研究成果丰富,与病毒之间也有明确的联系.

　　3.1 、DDX3与肿瘤的关系

　　DDX3可充当酪蛋白激酶1ε(casein kinase 1ε, CK1ε)的调节亚基,直接结合CK1ε并刺激激酶活性,进一步促散乱蛋白 (disheveled protein,Dvl)磷酸化,从而使β-连环蛋白(β-catenin)移位进入细胞核并触发Wnt/β-catenin信号传导,Wnt/β-catenin信号下游有众多靶基因被调控,主要包括转录因子Snail1、基质金属蛋白酶7(matrix metalloproteinase 7,MMP7)、鼠类肉瘤病毒癌基因(kirsten rat sarcoma viral oncogene, KRAS)、成纤维细胞特异蛋白1(fibroblast-fpecific protein 1,FSP1)和纤连蛋白等,这些靶点基因大多参与上皮间质转化(epithelial-mesenchymal transformation,EMT)、侵袭、炎症、迁移、凋亡和纤维化等过程[32,33,34,35,36,37,38].Wnt/β-catenin和EMT是DDX3在肿瘤发生发展过程中发挥致癌或抑癌双重作用的关键.

　　在KRAS突变的结直肠癌细胞中,质粒构建与转染结果表明DDX3可通过CK1ε/Dvl2轴激活β-catenin/T细胞因子(T cell factor,TCF),进而促进细胞侵袭和异种移植肺肿瘤的结节形成[39];而在KRAS野生型结直肠癌细胞中,发现DDX3可通过DDX3/KRAS/ROS/HIF-1α/DDX3级联反馈环增强细胞侵袭性[40].以上研究明确DDX3的致癌作用,解释了高表达DDX3结直肠癌患者临床总体生存率较差的原因[41].大型队列分析发现低表达DDX3的结直肠癌患者预后也较差,且癌细胞向远处转移更加频繁.进一步实验发现低表达DDX3可激活Snail/E-钙粘着蛋白(E-cadherin)信号通路,从而促进了结直肠癌转移[42].因此,DDX3也被认为具有抑癌作用.

　　在非小细胞肺癌中,应用质粒构建等方法,使E6癌蛋白和p53突变共同诱导DDX3失活,MDM2/Slug/E-cadherin途径就被激活,表现为肿瘤恶性程度增加和预后不良,反映了DDX3的抑癌作用[22];而在小细胞肺癌中,高表达DDX3与癌细胞的发生发展紧密相关[43].尽管DDX3在肺癌中发挥双重作用,但目前被视为肺癌的治疗靶点,本研究认为不应一概而论,临床应用应遵循个体化精准医疗.

　　在肝癌细胞中检测到过表达DDX3,经锚定非依赖性生长分析,其被鉴定为肝癌发生中的转化基因[44];Chang等[45]发现,在乙型肝炎病毒(hepatitis B virus,HBV)阳性的原发性肝癌(hepatocellular carcinoma,HCC)患者中,DDX3表达水平较低,而在HCV阳性的患者中则不表达,进一步通过集落形成实验和质粒构建发现,DDX3主要通过与特异性蛋白1(specificity protein 1,SP1)结合而增强p21(waf1/cip1)启动子活性的转录来发挥其对肿瘤生长的抑癌作用[46].由于DDX3同样与病毒有着直接关联,故在研究肝癌时不应忽略病毒的效应.

　　在口腔鳞状细胞癌中,非吸烟患者的组织免疫组化染色表明,低表达的DDX3严重影响总体生存率[30];对吸烟患者而言,高表达DDX3与淋巴转移相关并可预测不良生存率[47].这种现象可能是因为吸烟作为一种诱因造成DDX3突变,使其由抑癌转变为致癌.

　　荧光素酶报告表明,被HIF-1α上调后的DDX3通过诱导EMT进程的方式增强乳腺癌侵袭[22];髓母细胞瘤细胞活力增加则是由于DDX3突变与突变的β-catenin结合增加了TCF启动子的反式激活[48].目前,DDX3对于癌症的发生发展起着促进或抑制双重作用,明确两者间的关系一直备受关注,理清此关系能为癌症的治疗提供更多的可行途径.

　　3.2 、DDX3与病毒感染的关系

　　在免疫反应中,DDX3发挥着重要的抗病毒作用,主要调节视黄酸诱导型基因I(retinoic acid-inducible gene I,RIG-I)样受体介导的抗病毒应答,激活的RIG-I结合线粒体抗病毒信号(mitochondrial antiviral signaling protein,MAVS)衔接蛋白,最终致使I-κB激酶-ε(I-kappa-B kinase-ε,IKKε)和TANK结合激酶1(tank binding kinase-1,TBK1)复合物的募集, IKKε/TBK1复合物能使干扰素调节因子3(interferon regulatory factors 3,IRF3)、干扰素调节因子7(interferon regulatory factors 7,IRF7)磷酸化和核易位,从而激活Ⅰ型干扰素(type-I interferon,IFN-I)转录并产生IFN-β,同时DDX3亦为IKKε/TBK1复合物的重要组成部分[17,49,50,51].DDX3同HCV、HIV-1、HBV、甲型流感病毒、日本脑炎病毒、登革热病毒和西尼罗河病毒等多种病毒密切相关.全基因组遗传筛选结果表明DDX3是HCV感染的宿主因子,对HCV复制极其重要[52].对于HIV-1,DDX3作为其辅助因子,能携同CRM1介导HIV-1 Rev RNA的核输出并促进mRNA的翻译[28].在HBV中,DDX3能结合并抑制HBV的DNA聚合酶/逆转录酶,从而减少HBV的复制[53].近期通过免疫沉淀实验发现,作为一种抗病毒蛋白,DDX3可与甲型流感病毒NS1和NP蛋白相互作用,发挥抗病毒作用,DDX3与日本脑炎病毒5′和3′非翻译区结合调控病毒感染[54,55].近期还发现了DDX3的N端能相互作用于登革热病毒衣壳,且可能与该病毒感染后期存在联系[56].总之,研究人员明确了DDX3是一种有效的抗病毒蛋白,并将其作为许多病毒的治疗靶点,靶向DDX3研究有效的抗病毒药物能为HIV等流行病提供解决方案.

　　4 、DDX3的生物学作用

　　4.1、参与RNA活动

　　RNA诸多代谢活动均有DDX3参与,包括RNA转录和剪接、mRNA转运和翻译等.DDX3参与RNA转录表现在对转录因子的影响,涉及的转录因子包括上文所提及的Snail1、IRF3、IRF7和SP1[34,46,49].在DDX3的C端,富含精氨酸-丝氨酸的RS结构域能与核出口因子Tip相关蛋白(tip-associated protein,TAP)相互作用,而TAP能直接结合mRNA继而介导mRNA输出,TAP还有助于mRNP(信使核糖核蛋白,包含蛋白质、snRNA和mRNA)核出口,故DDX3可参与mRNA的转运[57,58].斑点印迹分析法结果发现DDX3能与N6-甲基腺嘌呤(N6-methyladenosine,m6A)的去甲基化酶烷基化修复同源蛋白5相互作用并影响其活性,通过调节mRNA的去甲基化发挥对mRNA的修饰作用[59,60].HCV核心可同DDX3的C端(氨基酸553-622)结合,HCV核心的N末端(氨基酸1-59)介导它们的相互作用,这种相互作用的影响被认为是HCV的mRNA剪接,转录和翻译调控的操纵,最终影响HCV复制[61,62]. 质谱分析结果发现DDX3能依赖外显子连接复合体与剪接的mRNA相结合,剪接因子还有其RS结构域,故DDX3可能有助于RNA 剪接[63].DDX3也是胞质应激颗粒的组成部分,暗示了DDX3在翻译调控中的作用[64].事实上,DDX3能与eIF4E及其他几种翻译起始因子包括eIF3,eIF2a,eIF4a,eIF4G和poly(A)结合蛋白1(Poly(A)-binding protein 1,PABP1)相互作用,增强含有结构化的5’非翻译区的mRNA翻译,其他研究则发现DDX3可与eIF3和40S核糖体相互作用以支持功能性80S核糖体的组装,此外,DDX3还能通过与Tat蛋白交互增强病毒mRNA翻译[26,65,66,67,68,69];同时DDX3也能充当翻译抑制因子,双顺反子报告表明DDX3将eIF4E捕获于翻译非活动复合物中以阻滞与eIF4G的交互,从而抑制了Cap依赖性翻译[26].

　　4.2、调节细胞周期

　　DDX3同样对细胞周期发挥着重要的调节作用.既往发现DDX3定位不仅与CRM1相关,同时会随细胞周期的进程而变化,G0期定位于细胞核,而G1/S期则主要定位于细胞质,其定位的改变是同生物学功能相对应的[70].在有丝分裂的前期或中期,细胞核内DDX3积累增加,此现象可能与其在有丝分裂期间中心体调节和染色体分离有关,Pek等[71]提示DDX3在HeLa细胞的前期或前中期定位于接近染色体聚集的位置[72];Chen等[73]经免疫荧光实验发现DDX3与中心体共定位;Brennan[72]的图像还提示DDX3在有丝分裂中期至胞质分裂期间与纺锤体相关联,此外 DDX3与中心体相关的p53共定位,DDX3下调致染色体错位、分离缺陷和多极有丝分裂,并造成G2/M延迟和细胞死亡等均佐证了此观点[73].与Chen等[73]的研究一致,Brennan等[72]的研究也显示,敲除DDX3会明确减缓细胞周期G2/M期的进展,这表明可能需要增加DDX3的表达和核积累,细胞才能顺利通过G2/M期的进展.起初人们发现在DDX3敲低的细胞中,细胞周期加速并提早向S期过渡,此过程伴随着细胞周期蛋白D1(G1早期至中期的调节剂)的增加[74].进一步研究表明,过表达的DDX3会提高p21并下调细胞周期蛋白D1,导致S期阻滞,DDX3的Q基序上的苏氨酸204被细胞周期蛋白B/cdc2磷酸化,导致DDX3的调节功能丧失[75,76].然而,细胞周期蛋白B的翻译尤其取决于DDX3的水平[77].此外,DDX3亦可通过促进细胞周期蛋白E1的翻译调控细胞周期[78].最新研究也认为,DDX3的抑制作用会对所有阶段的细胞产生影响,并导致细胞周期的整体进程延迟[6].

　　4.3、调节细胞凋亡

　　细胞凋亡为细胞生命重要的进程,受多方面各个因素的调节,DDX3是众多调节分子中的一员.现有研究表明,DDX3能抑制细胞凋亡,既可调控外源性凋亡信号,亦能选择性调控内源性凋亡信号.2D凝胶位移和质谱分析表明,DDX3被鉴定为TNF相关凋亡诱导配体受体2(TNF-related apoptosis-inducing ligand receptor 2,TRAIL-R2)相关蛋白,TRAIL-R2信号转导涉及DDX3的解离,此过程抵消了死亡信号.DDX3还与死亡受体、糖原合成酶激酶-3 (serine protein kinase 3,GSK3)和细胞凋亡抑制蛋白-1 (Cellular inhibitor of apoptosis protein 1,cIAP-1)形成死亡拮抗复合物,调控外源性凋亡信号抑制凋亡[79].研究表明,百草枯可能通过上调死亡受体5和抑制抗凋亡蛋白DDX3/GSK3来诱导外源性细胞凋亡途径,进而减少抗凋亡复合物的产生,为抵抗百草枯肺毒性提供了可能途径[80].DNA损伤后,内源性凋亡信号可被DDX3选择性调控.在无功能p53或表达突变的细胞中,DDX3通过抑制半胱氨酸蛋白酶-3(caspase-3)而抑制细胞凋亡;相反,在p53野生型表达的细胞中,DDX3通过促caspase-7结合p53,增加p53在胞内的积累,正向调节喜树碱诱导的凋亡信号[8].在DDX3不表达的情况下,Caspase-6和Caspase-9激活被阻断,从而可保护DDX3敲低细胞不受细胞凋亡的影响[26].综上,DDX3在细胞凋亡的调节中既能抗凋亡,亦能促凋亡.

　　5 、总结与展望

　　众多研究不断加深着对DDX3的认知,它在多种生理病理进程中发挥着极其重要的作用.研究主要通过实验探究DDX3的作用,多在质粒构建、细胞转染、蛋白印迹法、免疫组化分析和荧光素酶报告分析等技术上增减或按研究进行改进,但也会出现部分相反的结果,例如促癌与抑癌,因此,在进行研究时更应关注实验的限定条件,准确的条件限制能得到更为准确的结果,更加明确DDX3的病理生理学作用.

　　DDX3可调控肿瘤、病毒感染等疾病的发生发展,具有促进或抑制癌细胞发生发展的双重作用,目前被认为是癌症的治疗靶点.DDX3的抗病毒功能及本身的生物学特性,使HIV、HCV等全球性传染性疾病的治疗有了新的途径.因此研发DDX3抑制剂靶向相关疾病的治疗,是DDX3公认的研究方向.迄今为止,人们通过对DDX3病理生理学的研究已经研发出了RK-33、FE-15、NZ51、酮咯酸盐、阿霉素等多种抑制剂[81,82,83,84].例如应用最多的RK-33能结合DDX3的ATP结合域,从而阻断其在肺癌、尤文氏肉瘤、乳腺癌、结直肠癌和前列腺癌细胞中解旋酶的活性,同时RK-33可使高表达DDX3癌细胞的细胞周期G1停滞和细胞凋亡[85,86,87,88,89].因此,对于DDX3高效抑制剂的开发是未来长期内的突破点,同时抑制剂的研发方法也值得深入探究.最新的DDX3抑制剂被应用在抗病毒感染中,提示进一步研发相关制剂调控DDX3也是病毒类疾病的有效治疗途径[54,90].除此之外,DDX3是否参与各类新型病毒的感染与复制,是否可成为治疗靶标,值得进一步探讨.

　　DDX3对细胞周期、细胞凋亡的作用提示,靶向调控DDX3以抑制癌症细胞周期增殖或促进癌细胞凋亡,能为癌症的治疗提出新方案.目前,关于DDX3翻译后修饰的研究较少,而DDX3本身具有众多潜在的翻译后修饰位点,故通过翻译后修饰调控DDX3的活性及功能可能为相关疾病的治疗提供可靠途径.分布广泛且参与细胞各项生命活动的DDX3是值得被研究的,尽管已经有了许多研究成果,但因其具有双刃剑的特性,故需要更加精准的研究以明确其病理生理学作用,从而为与DDX3相关的疾病治疗提供更加可靠的策略.

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