氧化还原对铁结合的大肠杆菌拓扑异构酶Ⅰ活性的调控
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摘要
大肠杆菌拓扑异构酶Ⅰ(E.coli TopA)属于I型拓扑异构酶,在DNA复制、转录、重组和基因表达调控等过程中发挥关键作用。E.coli TopA不仅能结合锌,还可以结合铁。细胞内过量铁可与锌竞争,通过与锌指结构域结合减弱其DNA结合能力和改变蛋白质空间构象,从而抑制TopA拓扑异构酶活性。然而,铁结合形式TopA的氧化还原特性以及氧化还原条件对其活性的影响仍不清楚。本研究通过紫外分光光谱和体外DNA拓扑异构酶活性分析,发现体外纯化得到的铁结合形式的TopA呈氧化状态,能够被二硫苏糖醇和连二亚硫酸钠还原,原本氧化状态下无活性的TopA在还原条件下,可恢复其拓扑异构酶活性。当还原剂被去除后,铁结合的TopA在空气中能够重新被氧化,且其活性重新受到抑制。这说明,氧化还原条件对铁结合的TopA功能具有可逆调节作用。通过金属-蛋白体外结合实验进一步发现,无金属结合的TopA蛋白(apo-TopA)在无氧条件下,与Fe~(2+)和Fe~(3+)均能结合,但与Fe~(2+)结合能力较弱,并且TopA结合的Fe~(3+)被还原成Fe~(2+)后,结合力显著下降,能够被铁螯合指示剂菲咯嗪快速捕获。此外,蛋白质内源性荧光光谱分析实验表明,铁结合的TopA在氧化还原的不同状态时,其在330 nm左右的荧光值有显著差异。这提示,氧化还原条件可能通过影响铁离子与TopA的结合状态,引起蛋白质空间构象改变,从而对TopA的拓扑异构酶活性进行调节。此研究表明,铁结合TopA的拓扑异构酶活性会受到细胞内氧化还原信号的可逆调控,也提示I型拓扑异构酶可能是细胞铁超载通过氧化损伤引起细胞功能障碍(或铁死亡)的靶点之一。
Escherichia coli topoisomerase Ⅰ( E. coli TopA) belongs to type I topoisomerase and plays critical roles in replication,transcription,recombination and regulation of gene expression. E. coli TopA could not only bind zinc,but also bind iron. Excessive intracellular iron would compete with zinc inbinding to TopA via targeting the zinc-finger motifs,which could weaken the DNA binding affinity and change the protein conformation of TopA, thus leading to inhibition of its topoisomerase activity.However,the redox characteristics of iron bound TopA and how redox conditions affect TopA activity still remain unknown. Here,we found that iron bound TopA was oxidized when purified in vitro,and could be reduced by dithiothreitol or sodium dithionite with the concomitant recovery of topoisomerase activity.While the reduced iron bound TopA was reoxidized upon removal of reductant,it came back to be inactive again,suggesting that the function of iron bound TopA can be reversibly regulated by redox conditions. Based on the in vitro metal binding experiments,we further found that TopA could bind both ferrous iron and ferric iron anaerobically,while the binding affinity of ferrous iron bound to TopA was weaker than that of ferric iron. When ferric iron bound TopA was treated with reductant and the irons were then readily captured by iron chelating indicator( ferrozine). Moreover,intrinsic fluorescence measurement showed that the protein conformation of iron bound TopA would be changed upon redox switch. These results indicate that the topoisomerase activity of iron bound TopA can be reversibly regulated by intracellular redox signals,which may alter the iron binding affinity and change the protein conformation,thus modulate enzyme activity. This work also suggests that topoisomerase I may be one of the oxidative damage targets during the cell dysfunction or ferroptosis induced by overload of intracellular iron.
Escherichia coli topoisomerase Ⅰ( E. coli TopA) belongs to type I topoisomerase and plays critical roles in replication,transcription,recombination and regulation of gene expression. E. coli TopA could not only bind zinc,but also bind iron. Excessive intracellular iron would compete with zinc inbinding to TopA via targeting the zinc-finger motifs,which could weaken the DNA binding affinity and change the protein conformation of TopA, thus leading to inhibition of its topoisomerase activity.However,the redox characteristics of iron bound TopA and how redox conditions affect TopA activity still remain unknown. Here,we found that iron bound TopA was oxidized when purified in vitro,and could be reduced by dithiothreitol or sodium dithionite with the concomitant recovery of topoisomerase activity.While the reduced iron bound TopA was reoxidized upon removal of reductant,it came back to be inactive again,suggesting that the function of iron bound TopA can be reversibly regulated by redox conditions. Based on the in vitro metal binding experiments,we further found that TopA could bind both ferrous iron and ferric iron anaerobically,while the binding affinity of ferrous iron bound to TopA was weaker than that of ferric iron. When ferric iron bound TopA was treated with reductant and the irons were then readily captured by iron chelating indicator( ferrozine). Moreover,intrinsic fluorescence measurement showed that the protein conformation of iron bound TopA would be changed upon redox switch. These results indicate that the topoisomerase activity of iron bound TopA can be reversibly regulated by intracellular redox signals,which may alter the iron binding affinity and change the protein conformation,thus modulate enzyme activity. This work also suggests that topoisomerase I may be one of the oxidative damage targets during the cell dysfunction or ferroptosis induced by overload of intracellular iron.
引文
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[15]Ren B,Duan X,Ding H.Redox control of the DNA damageinducible protein Din G helicase activity via its iron-sulfur cluster[J].J Biol Chem,2009,284(8):4829-4835
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[17]Landry AP,Ding H.Redox control of human mitochondrial outer membrane protein Mito NEET[2Fe-2S]clusters by biological thiols and hydrogen peroxide[J].J Biol Chem,2014,289(7):4307-4315
[18]Liu F,Rehmani I,Esaki S,et al.Pirin is an iron-dependent redox regulator of NF-kappa B[J].Proc Natl Acad Sci U S A,2013,110(24):9722-9727
[19]Dixon SJ,Lemberg KM,Lamprecht MR,et al.Ferroptosis:an iron-dependent form of nonapoptotic cell death[J].Cell,2012,149(5):1060-1072
[20]Dixon SJ,Stockwell BR.The role of iron and reactive oxygen species in cell death[J].Nat Chem Biol,2014,10(1):9-17
[21]Yang WS,Stockwell BR.Ferroptosis:death by lipid peroxidation[J].Trends Cell Biol,2016,26(3):165-176
[22]Xie Y,Hou W,Song X,et al.Ferroptosis:process and function[J].Cell Death Differ,2016,23(3):369-379
[23]Friedmann Angeli JP,Schneider M,Proneth B,et al.Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice[J].Nat Cell Biol,2014,16(12):1180-1191
[24]Matak P,Matak A,Moustafa S,et al.Disrupted iron homeostasis causes dopaminergic neurodegeneration in mice[J].Proc Natl Acad Sci U S A,2016,113(13):3428-3435
[25]Jha JC,Banal C,Chow BS,et al.Diabetes and kidney disease:Role of oxidative stress[J].Antioxid Redox Signal,2016,25(12):657-684
[26]Lu J,Holmgren A.The thioredoxin antioxidant system[J].Free Radic Biol Med,2014,66:75-87
[27]Bresciani G,da Cruz IB,Gonzalez-Gallego J.Manganese superoxide dismutase and oxidative stress modulation[J].Adv Clin Chem,2015,68:87-130
[28]Couto N,Wood J,Barber J.The role of glutathione reductase and related enzymes on cellular redox homeostasis network[J].Free Radic Biol Med,2016,95:27-42
[29]Dooley CT,Dore TM,Hanson GT,et al.Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators[J].J Biol Chem,2004,279(21):22284-22293
[30]Schmidt R,Schippers JH.ROS-mediated redox signaling during cell differentiation in plants[J].Biochim Biophys Acta,2015,1850(8):1497-1508
[31]Reczek CR,Chandel NS.ROS-dependent signal transduction[J].Curr Opin Cell Biol,2015,33:8-13
[2]Schoeffler AJ,Berger JM.DNA topoisomerases:harnessing and constraining energy to govern chromosome topology[J].Q Rev Biophys,2008,41(1):41-101
[3]Forterre P,Gadelle D.Phylogenomics of DNA topoisomerases:their origin and putative roles in the emergence of modern organisms[J].Nucleic Acids Res,2009,37(3):679-692
[4]Tse-Dinh YC,Beran-Steed RK.Escherichia coli DNA topoisomerase I is a zinc metalloprotein with three repetitive zincbinding domains[J].J Biol Chem,1988,263(31):15857-15859
[5]Tse-Dinh YC.Zinc(II)coordination in Escherichia coli DNA topoisomerase I is required for cleavable complex formation with DNA[J].J Biol Chem,1991,266(22):14317-14320
[6]Lu J,Wang W,Tan G,et al.Escherichia coli topoisomerase I is an iron and zinc binding protein[J].Biometals,2011,24(4):729-736
[7]Wang W,Su X,Wang X,et al.Iron inhibits Escherichia coli topoisomerase I activity by targeting the first two zinc-binding sites in the C-terminal domain[J].Protein Sci,2014,23(11):1619-1628
[8]Rouault TA.Mammalian iron-sulphur proteins:novel insights into biogenesis and function[J].Nat Rev Mol Cell Biol,2015,16(1):45-55
[9]Cowart RE,Singleton FL,Hind JS.A comparison of bathophenanthrolinedisulfonic acid and ferrozine as chelators of iron(II)in reduction reactions[J].Anal Biochem,1993,211(1):151-155
[10]Hansen SB,Radic Z,Talley TT,et al.Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein[J].J Biol Chem,2002,277(44):41299-41302
[11]Zhang Z,Cheng B,Tse-Dinh YC.Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I[J].Proc Natl Acad Sci U S A,2011,108(17):6939-6944
[12]Ahumada A,Tse-Dinh YC.The Zn(II)binding motifs of E.coli DNA topoisomerase I is part of a high-affinity DNA binding domain[J].Biochem Biophys Res Commun,1998,251(2):509-514
[13]Ahumada A,Tse-Dinh YC.The role of the Zn(II)binding domain in the mechanism of E.coli DNA topoisomerase I[J].BMC Biochem,2002,3:13
[14]Cheng B,Zhu CX,Ji C,et al.Direct interaction between Escherichia coli RNA polymerase and the zinc ribbon domains of DNA topoisomerase I[J].J Biol Chem,2003,278(33):30705-30710
[15]Ren B,Duan X,Ding H.Redox control of the DNA damageinducible protein Din G helicase activity via its iron-sulfur cluster[J].J Biol Chem,2009,284(8):4829-4835
[16]Wiley SE,Murphy AN,Ross SA,et al.Mito NEET is an ironcontaining outer mitochondrial membrane protein that regulates oxidative capacity[J].Proc Natl Acad Sci U S A,2007,104(13):5318-5323
[17]Landry AP,Ding H.Redox control of human mitochondrial outer membrane protein Mito NEET[2Fe-2S]clusters by biological thiols and hydrogen peroxide[J].J Biol Chem,2014,289(7):4307-4315
[18]Liu F,Rehmani I,Esaki S,et al.Pirin is an iron-dependent redox regulator of NF-kappa B[J].Proc Natl Acad Sci U S A,2013,110(24):9722-9727
[19]Dixon SJ,Lemberg KM,Lamprecht MR,et al.Ferroptosis:an iron-dependent form of nonapoptotic cell death[J].Cell,2012,149(5):1060-1072
[20]Dixon SJ,Stockwell BR.The role of iron and reactive oxygen species in cell death[J].Nat Chem Biol,2014,10(1):9-17
[21]Yang WS,Stockwell BR.Ferroptosis:death by lipid peroxidation[J].Trends Cell Biol,2016,26(3):165-176
[22]Xie Y,Hou W,Song X,et al.Ferroptosis:process and function[J].Cell Death Differ,2016,23(3):369-379
[23]Friedmann Angeli JP,Schneider M,Proneth B,et al.Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice[J].Nat Cell Biol,2014,16(12):1180-1191
[24]Matak P,Matak A,Moustafa S,et al.Disrupted iron homeostasis causes dopaminergic neurodegeneration in mice[J].Proc Natl Acad Sci U S A,2016,113(13):3428-3435
[25]Jha JC,Banal C,Chow BS,et al.Diabetes and kidney disease:Role of oxidative stress[J].Antioxid Redox Signal,2016,25(12):657-684
[26]Lu J,Holmgren A.The thioredoxin antioxidant system[J].Free Radic Biol Med,2014,66:75-87
[27]Bresciani G,da Cruz IB,Gonzalez-Gallego J.Manganese superoxide dismutase and oxidative stress modulation[J].Adv Clin Chem,2015,68:87-130
[28]Couto N,Wood J,Barber J.The role of glutathione reductase and related enzymes on cellular redox homeostasis network[J].Free Radic Biol Med,2016,95:27-42
[29]Dooley CT,Dore TM,Hanson GT,et al.Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators[J].J Biol Chem,2004,279(21):22284-22293
[30]Schmidt R,Schippers JH.ROS-mediated redox signaling during cell differentiation in plants[J].Biochim Biophys Acta,2015,1850(8):1497-1508
[31]Reczek CR,Chandel NS.ROS-dependent signal transduction[J].Curr Opin Cell Biol,2015,33:8-13