【作者】 刘如娟；

【导师】 龚为民；

【作者基本信息】 中国科学技术大学 ， 生化与分子生物学， 2008， 博士

【摘要】 腺苷酸激酶广泛存在于生物体内,参与细胞内能量和核苷酸代谢。它们催化可逆的磷酸转移反应,将三磷酸腺苷酸或鸟苷酸上的γ磷酸转移到单磷酸腺苷酸上,该反应需要镁离子的辅助。腺苷酸激酶属于单磷酸核苷酸激酶家族,该家族还包括鸟苷酸激酶,胸腺嘧啶激酶,和鸟嘧啶/胞嘧啶激酶。所有这些酶有共同典型的α/β折叠,其中α-螺旋包围着以β-片组成的核心,氨基端有共同的磷酸供体结合的P-loop。所有的腺苷酸激酶由三个结构域组成:CORE结构域,LID结构域,和一个单磷酸腺苷酸结合的NMP_bind结构域。根据LID结构域的长短可以将腺苷酸激酶分为长亚型和短亚型。短亚型腺苷酸激酶的LID结构域由柔性的环组成,而长亚型腺苷酸激酶的LID结构域由四片反平行的折叠片组成。AK4是目前发现的AK家族中唯一一个没有AK催化活性的特殊成员,AK4的生理功能以及失活的原因至今仍一无所知。因此本论文对AK4的结构和功能以及催化机理进行了全面的研究。AK4中Gln159不能像其它AK中的Arg一样与转移中的磷酸基团相互作用形成氢键而稳定反应中间态,因此像其它AK一样的磷酸转移反应不能在AK4中发生,这就是为什么母体AK4无论在体外酶活实验,还是在体内突变互补实验中都没有表现出任何AK的活性。虽然,AK4没有表现出AK的催化活性,然而从AK4与GP5的晶体结构,以及AK4（Q159R）突变体能够有AK催化活性等实验结果都可以看出,AK4本身还具有底物结合能力。AK4（Q159R）突变体能够将GTP上的磷酸基团转移到AMP上,因此AK4可以结合GTP,AMP,GDP和ADP等核苷酸。因此,由于参与催化磷酸转移的重要氨基酸Arg被Gln取代,母体AK4不具有AK催化活性,但仍然具有核苷酸结合能力,我们可以推测,AK4在体内可以作为核苷酸的载体而发挥作用。通过检测AK4的mRNA和蛋白水平,我们发现AK4的表达水平在细胞压力（如Hypoxia）存在下都会大幅升高。因此,AK4有可能是细胞压力反应蛋白,在细胞存在压力情况下表达上升。对本底AK4表达水平高的HEK293细胞进行AK4 siRNA实验表明,AK4的siRNA对HEK293细胞的生长,形态,存活有很大影响,且随AK4表达抑制程度增高影响越大;而且AK4蛋白表达被抑制会导致HEK293细胞很难形成克隆。因此可以看出,AK4对HEK293细胞的生长及发育过程是十分重要的。在本底AK4表达水平低的细胞SH-SY5Y中过表达AK4蛋白实验表明,显微镜下观察,过表达AK4的SH-SY5Y细胞的形态和密度与对照组并无明显差异;MTT测细胞活性实验也进一步证明,过表达AK4对SH-SY5Y存活率没有明显影响。但是,在细胞压力（H₂O₂处理）情况下,AK4过表达的细胞要比对照组细胞存活率高,换言之,AK4过表达可以保护SH-SY5Y细胞对抗H2O2引起的细胞死亡。上述实验结果可以看出,AK4可能是细胞压力反应的蛋白,AK4的表达对细胞生长及发育很重要,且AK4的表达对细胞起保护作用。本文进一步研究AK4在体内发挥作用的可能的机制,我们试图寻找AK4相互作用的蛋白。通过对AK4-FLAG的免疫共沉淀及质谱分析得到线粒体内膜蛋白ANT可能参与AK4的相互作用,然后进行反向的免疫共沉淀实验进一步确定了AK4与ANT的相互作用。在细胞面对氧化压力的情况下,ANT与AK4之间相互作用会增强,因此我们推测,ANT与AK4的相互作用有可能参与到AK4在氧化压力下保护细胞的过程中。为进一步了解AK4与其它蛋白的相互作用,我们通过体外表达纯化人源的AK4,以及与AK4序列高度相似的AK3的His标签融合蛋白,在体外进行了His的pull-down实验。体外的His-tagged pull-down进一步确定了AK4与ANT的相互作用,而且,还发现了新的与AK4相互作用的蛋白ATP合成酶F1复合物的alpha和beta亚基。另外据报导ANT在线粒体内膜上与ATP合成酶形成大的复合体（ATP synthasome）,因此,对AK4相互作用蛋白的研究可以提示,AK4有可能参与到与细胞线粒体内ATP合成及转运相关途径中。在AK4中,171位的Leu突变为Pro导致HingeⅣ区域结构发生变化,HingeⅣ的结构也伴随着HingeⅢ的结构发生变化,这种Hinge区域的结构变化最终导致AK4整体结构发生大规模构象变化。这种“twisted-and-closed”构象的AK4（L171P）的结构为蛋白质结构域之间的运动以及蛋白构象变化提供了直接的模型,揭示了Hinge区域如何直接调节蛋白的结构变化。而且在AK家族中,提供了一个新的AK结构构象,而且这种“关闭”结构并不是由底物或其类似物的结合引起的。对AK4这个不具有酶学活性的特殊AK进行了可能的生理功能研究,发现AK4更可能是细胞内的调节蛋白。对AK4结构的研究可以看出,AK4的结构具有非常大的柔性,结构域之间的相对运动幅度较大,对AK4的结构的研究结果为AK4作为一个调节蛋白发挥功能的假设提供了又一证据。综上所述,本文对AK4进行了较全面的结构和功能的研究。通过突变及结构分析,AK4虽不具有AK催化活性,但仍然具有核苷酸结合能力,推测AK4在体内可以作为核苷酸的载体而发挥作用。通过对细胞生物学的研究得出AK4受细胞压力反应调节,AK4的表达对细胞生长及发育至关重要。对AK4相互作用蛋白的研究可以提示,AK4有可能参与到与细胞线粒体内ATP合成及转运相关途径中。对AK4（L171P）的结构研究为蛋白质结构域之间的运动以及蛋白构象变化提供了直接的模型,揭示了Hinge区域如何直接调节蛋白的构象变化。更多还原

【Abstract】 Aden ylate kinases（AKs） are ubiquitous enzymes which are involved in energy metabolism and homoeostasis of cellular adenine nucleotide composition.They catalyze reversible transfer ofγ-phosphate group from Mg2+ATP（or GTP） to AMP, releasing Mg2+ADP（or GDP） and ADP.AKs belong to the nucleoside monophosphate kinase（NMPK） family that includes other members such as guanylate kinases,thymidylate kinases,and UMP/CMP kinases.All of these enzymes share a common typicalα/βfold that consists of aβ-sheet CORE surrounded byα-helices,with a P-loop motif at the N-terminus that binds the phosphoryl donor. All AKs contain a central CORE domain,a LID domain,and an AMP-binding domain called NMPbind.The CORE and NMPbind domains are conserved in all AKs, whereas the LID domain is quite different.Six AK genes named AK1-AK6 have been identified in vertebrates.Adenylate kinase 4（AK4） is a unique member with no enzymatic activity in vitro in the adenylate kinase（AK） family although it shares high sequence homology with other AKs.It remains unclear what physiological function AK4 might play or why it is enzymatically inactive.Most residues within active site are conserved in human AK4 except residue 159,where an arginine conserved in other AKs is replaced with a glutamine.Structural analysis revealed that,while AK4 retains the capability of binding nucleotides,AK4 has a giutamine residue instead of a key arginine residue in the active site well conserved in other AKs.Mutation of the glutamine residue to arginine（Q159R） restored the adenylate kinase activity with GTP as substrate.In this study,we strive to establish the functional importance of AK4.When cells were exposed to hypoxia,the AK4 protein level increased significantly Moreover,we tested the AK4 levels in a commonly used transgenic mouse model of ALS,a neurodegenerative disease in which oxidative stress has been implicated,the AK4 level was significantly higher in the spinal cords of ALS mice.The results support that AK4 protein levels increase in response to different stress in cultured cells as well as in an animal model of human disease,suggesting that AK4 might provide protective benefits to cells under stress.Laterly,we showed that small hairpin RNA （shRNA）-mediated knockdown of AK4 in HEK293 cells with high levels of endogenous AK4 resulted in reduced cell proliferation and cell death.Furthermore, we found that AK4 over-expression in the neuronal cell line SH-SY5Y with low endogenous levels of AK4 protected cells from H2O2 induced cell death.These results indicate that the enzymatically inactive AK4 is a cell stress protective protein critical to cell survival and proliferation.Proteomic studies revealed that the mitochondrial ADP/ATP translocases（ANTs） interacted with AK4 and more interaction can be detected when cells were exposed to H2O2 treatment.It is likely that AK4 function by modulating its interaction with the mitochondrial inner membrane protein ANT.In vitro pull-down experiments further confirmed the protein-protein interactions between AK4 and ANT,further more,the a andβsubuinits of mitochondrial ATP synthase F1 complex are also found to be involved in the protein interactions with AK4.ANTs are important in mitochondrial ADP/ATP transport in the cell energy metabolism system,which also be reported to form into big complex（ATP sythasome） with mitochondrial ATP synthase.In other words,the proteins identified to be interacted with AK4 play key roles in process of mitochondrial ATP synthesis and translocation.Combined with the result of AK4 retains the capability of binding nucleotides,which suggests that AK4 may be also involved in the process of mitochondrial ATP synthesis and translocation through interaction with ANT and ATP synthase.It is well known that motion of LID and NMP-binding(NMP_bind) domains in adenylate kinase（AK） is important in ligand binding and catalysis.However,the nature of such domain motions is poorly characterized.One of the critical hinge regions is hingeⅣ,which connects the CORE and LID domains.In addition,the hingeⅣcontains a strictly conserved residue,L 171,in the AK family.To investigate the role of hingeⅣ,crystal structure of human adenylate kinase 4（AK4） L171P mutant was determined.This mutation dramatically changes the orientation of the LID domain,which could be described as a novel twisted-and-closed conformation in contrast to the open and closed conformations in other AKs.This mutant provides a new example of domain motions in AK family.Collectively,these results indicate that the enzymatically inactive AK4 is a cell stress protective protein critical to cell survival and proliferation and it is likely to function by modulating its interaction with the mitochondrial inner membrane protein ANT and ATP synthase.So it is possibly that AK4 involved in the process of mitochondrial ATP synthesis and translocation through interaction with ANT and ATP synthase.The crystal structures of AK4（L171P） provide a direct model of hinge regions regulate protein conformational change.更多还原