双功能抑制剂SK10对Cu~(2+)存在下Aβ聚集的抑制和解聚作用
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摘要
由于β-淀粉样蛋白(amyloidβ-protein,Aβ)的聚集是阿尔茨海默症(Alzheimer’s disease,AD)发病机制中的关键过程,而金属离子Cu~(2+)存在下的Aβ的聚集速率加快,且Cu~(2+)本身会促进活性氧(ROS)的产生,增加神经毒性,因此开发既能螯合Cu~(2+)又能抑制Aβ聚集的双功能抑制剂非常重要.本文将金属螯合三肽(SSH)和Aβ聚集七肽抑制剂相组合,设计合成了新的双功能Aβ聚集十肽抑制剂SSHLVFFARK-通过硫代黄素T(ThT)荧光实验、聚集动力学实验、原子力显微镜检测(AFM)、等温滴定量热和MTT细胞毒性测定等研究了SK10对Cu~(2+)存在下Aβ聚集的抑制和解聚作用以及细胞毒性的抑制作用.实验结果表明:SK10不仅可以抑制Cu~(2+)存在下的Aβ40聚集(ThT荧光强度降低了60%),随着SK10浓度的提高使Aβ40纤维逐渐减少,还能降低Cu~(2+)存在下Aβ40聚集产生的细胞毒性,使细胞活性恢复至90%以上;同样,SK10对Cu~(2+)存在下的Aβ42聚集也有抑制作用.由于SK10对Cu~(2+)具有较强的特异性亲和力,能够螯合复合物中的Cu~(2+),因此能抑制由Cu~(2+)催化产生的活性氧(ROS)对细胞的毒性.进一步研究表明SK10还可解聚形成的聚集体,使已形成的聚集体消失,缓解其产生的细胞毒性,使细胞活性提高到90%.以上研究结果不但体现了SK10的药用潜力,也为今后设计和开发金属螯合Aβ聚集双功能抑制剂提供了思路.
The aggregation of amyloid β-protein(Aβ) is an important process in the pathogenesis of Alzheimer's disease(AD). The aggregation rate of Aβ in the presence of metal ions such as Cu~(2+) gets accelerated,and Cu~(2+)itself promotes the production of reactive oxygen species(ROS) and increases neurotoxicity. Therefore,it is important to develop dual-function inhibitors that simultaneously chelate copper ions and inhibit Aβ aggregation. In this study,we designed a novel bifunctional decapeptide SSHLVFFARK-NH2(SK10) by combining a metal chelating tripeptide(SSH) and an Aβ aggregation inhibitor Ac-LVFFARK-NH2(LK7). We conducted thioflavin T(ThT) fluorescence and aggregation kinetics experiments and performed atomic force microscopy(AFM),isothermal titration calorimetry,and MTT cytotoxicity assays to investigate the inhibition and depolymerization of SK10 on the aggregation and cytotoxicity of Aβ in the presence of Cu~(2+). The experimental results show that SK10 can inhibit Aβ40 aggregation in the presence of Cu~(2+)(Th T fluorescence decreased by 60%) and the Aβ40 fiber gradually decreased with increase in the SK10 concentration and reduce the cytotoxicity induced by Aβ40 aggregation in the presence of Cu~(2+) such that the cellular activity was restored to >90%. Similarly, SK10 inhibited the aggregation of Aβ42 in the presence of Cu~(2+). Because SK10 has a specific affinity for Cu~(2+)(Kd=0.03 μmol/L) and is capable of sequestering Cu~(2+) from Cu~(2+)-Aβ40 species,it can inhibit the toxicity of ROS catalyzed by copper ions with respect to cells. Additional studies revealed that the formed Cu~(2+)-Aβ40 aggregates can be depolymerized by SK10,whereby the formed Cu~(2+)-Aβ40 aggregates disappear and the cell viability is increased to 90%. The above results reflect both the medicinal potential of SK10 and provide ideas for the design and development of bifunctional inhibitors that can chelate metal ions and inhibit Aβ aggregation.
The aggregation of amyloid β-protein(Aβ) is an important process in the pathogenesis of Alzheimer's disease(AD). The aggregation rate of Aβ in the presence of metal ions such as Cu~(2+) gets accelerated,and Cu~(2+)itself promotes the production of reactive oxygen species(ROS) and increases neurotoxicity. Therefore,it is important to develop dual-function inhibitors that simultaneously chelate copper ions and inhibit Aβ aggregation. In this study,we designed a novel bifunctional decapeptide SSHLVFFARK-NH2(SK10) by combining a metal chelating tripeptide(SSH) and an Aβ aggregation inhibitor Ac-LVFFARK-NH2(LK7). We conducted thioflavin T(ThT) fluorescence and aggregation kinetics experiments and performed atomic force microscopy(AFM),isothermal titration calorimetry,and MTT cytotoxicity assays to investigate the inhibition and depolymerization of SK10 on the aggregation and cytotoxicity of Aβ in the presence of Cu~(2+). The experimental results show that SK10 can inhibit Aβ40 aggregation in the presence of Cu~(2+)(Th T fluorescence decreased by 60%) and the Aβ40 fiber gradually decreased with increase in the SK10 concentration and reduce the cytotoxicity induced by Aβ40 aggregation in the presence of Cu~(2+) such that the cellular activity was restored to >90%. Similarly, SK10 inhibited the aggregation of Aβ42 in the presence of Cu~(2+). Because SK10 has a specific affinity for Cu~(2+)(Kd=0.03 μmol/L) and is capable of sequestering Cu~(2+) from Cu~(2+)-Aβ40 species,it can inhibit the toxicity of ROS catalyzed by copper ions with respect to cells. Additional studies revealed that the formed Cu~(2+)-Aβ40 aggregates can be depolymerized by SK10,whereby the formed Cu~(2+)-Aβ40 aggregates disappear and the cell viability is increased to 90%. The above results reflect both the medicinal potential of SK10 and provide ideas for the design and development of bifunctional inhibitors that can chelate metal ions and inhibit Aβ aggregation.
引文
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[15]Miyamoto T,Fukino Y,Kamino S,et al.Enhanced stability of Cu2+-ATCUN complexes under physiologically relevant conditions by insertion of structurally bulky and hydrophobic amino acid residues into the AT-CUN motif[J].Dalton Transactions,2016,45(23):9436-9445.
[16]And C H,Sarkar B.Amino terminal Cu(Ⅱ)-and Ni(Ⅱ)-binding(ATCUN)motif of proteins and peptides:Metal binding,DNA cleavage,and other properties[J].Cheminform,1997,28(27):123-130.
[17]Ghosh A,Pradhan N,Bera S,et al.Inhibition and degradation of amyloid beta(A?40)fibrillation by designed small peptide:A combined spectroscopy,microscopy,and cell toxicity study[J].ACS Chemical Neuroscience,2017,8(4):718-722.
[18]Rafferty J,Nagaraj H,Mccloskey A P,et al.Peptide therapeutics and the pharmaceutical industry:Barriers encountered translating from the laboratory to patients[J].Current Medicinal Chemistry,2016,23(37):4231-4259.
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[20]Adessi C,Frossard M J,Boissard C,et al.Pharmacological profiles of peptide drug candidates for the treatment of Alzheimer’s disease[J].Journal of Biological Chemistry,2003,278(16):13905-13911.
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[22]Lowe T L,Strzelec A,Kiessling L L,et al.Structurefunction relationships for inhibitors of beta-amyloid toxicity containing the recognition sequence KLVFF[J].Biochemistry,2001,40(26):7882-7889.
[23]Xiong N,Dong X Y,Zheng J,et al.Design of LVFFARK and LVFFARK-functionalized nanoparticles for inhibiting amyloid?-protein fibrillation and cytotoxicity[J].Acs Appl Mater Interfaces,2015,7(10):5650-5662.
[24]Bush A I,Tanzi R E.Therapeutics for Alzheimer’s disease based on the metal hypothesis[J].NeurotheraPeutics,2008,5(3):421-432.
[25]Mlynarz P,Valensin D,Kociolek K,et al.Impact of the peptide sequence on the coordination abilities of albumin-like tripeptides towards Cu2+,Ni2+and Zn2+ions.potential albumin-like peptide chelators[J].New Journal of Chemistry,2002,26(2):264-268.
[26]Wang Q,Shah N,Zhao J,et al.Structural,morphological,and kinetic studies of beta-amyloid peptide aggregation on self-assembled monolayers[J].Physical Chemistry Chemical Physics,2011,13(33):15200-15210.
[27]Cabaleiro-Lago C,Szczepankiewicz O,Linse S.The effect of nanoparticles on amyloid aggregation depends on the protein stability and intrinsic aggregation rate[J].Langmuir,2012,28(3):1852-1857.
[28]Guilloreau L,Combalbert S,Sournia-Saquet A,et al.Redox chemistry of copper-amyloid-?:The generation of hydroxyl radical in the presence of ascorbate is linked to redox-potentials and aggregation state[J].ChembioChem,2007,8(11):1317-1325.
[29]Lincoln K M,Richardson T E,Rutter L,et al.An nheterocyclic amine chelate capable of antioxidant capacity and amyloid disaggregation[J].ACS Chemical Neuroscience,2012,3(11):919-927.
[30]Sarell C J,Wilkinson S R,Viles J H.Substoichiometric levels of Cu2+ions accelerate the kinetics of fiber formation and promote cell toxicity of amyloid-beta from Alzheimer’s disease[J].Journal of Biological Chemistry,2010,285(53):41533-41540.
[31]Pedersen J T,Ostergaard J,Rozlosnik N,et al.Cu(Ⅱ)mediates kinetically distinct,non-amyloidogenic aggregation of amyloid-beta peptides[J].Journal of Biological Chemistry,2011,286(30):26952-26963.
[32]Jensen M,Canning A,Chiha S,et al.Inhibition of Cu-amyloid-beta by using bifunctional peptides with beta-sheet breaker and chelator moieties[J].ChemistryA European Journal,2012,18(16):4836-4839.
[33]Meng J,Zhang H,Dong X,et al.Rthlvffark-NH2:Apotent and selective modulator on Cu2+-mediated amyloid-?protein aggregation and cytotoxicity[J].Journal of Inorganic Biochemistry,2018,181:56-64.
[34]Hu X,Zhang Q,Wang W,et al.Tripeptide GGH as the inhibitor of copper-amyloid-beta-mediated redox reaction and toxicity[J].ACS Chemical Neuroscience,2016,7(9):1255-1263.
[35]Smith D G,Cappai R,Barnham K J.The redox chemistry of the Alzheimer’s disease amyloid beta peptide[J].Biochimica Et Biophysica Acta,2007,1768(8):1976-1990.
[36]Huang X D,Cuajungco M P,Atwood C S,et al.Cu(Ⅱ)potentiation of Alzheimer a beta neurotoxicitycorrelation with cell-free hydrogen peroxide production and metal reduction[J].Journal of Biological Chemistry,1999,274(52):37111-37116.
[37]Jarrett J T,Berger E P,Lansbury P T J.The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation:Implications for the pathogenesis of Alzheimer’s disease[J].Biochemistry,1993,32(18):4693-4697.
[2]Mattson M P.Pathways towards and away from Alzheimer’s disease[J].Nature,2004,430(7000):631.
[3]Huang Y,Mucke L.Alzheimer mechanisms and therapeutic strategies[J].Cell,2012,148(6):1204-1222.
[4]Tiraboschi P,Hansen L A,Thal L J,et al.The importance of neuritic plaques and tangles to the development and evolution of AD[J].Neurology,2004,62(11):1984-1989.
[5]Citron M.Alzheimer’s disease:Strategies for disease modification[J].Nature Reviews Drug Discovery,2010,9(5):387.
[6]Golde T E,Eckman C B,Younkin S G.Biochemical detection of abeta isoforms:Implications for pathogenesis,diagnosis,and treatment of Alzheimer’s disease[J].Biochimica Et Biophysica Acta,2000,1502(1):172-187.
[7]Vivekanandan S,Brender J R,Lee S Y,et al.A partially folded structure of amyloid-beta(1-40)in an aqueous environment[J].Biochemical&Biophysical Research Communications,2011,411(2):312-316.
[8]Haass C,Selkoe D J.Soluble protein oligomers in neurodegeneration:Lessons from the Alzheimer’s amyloid beta-peptide[J].Nature Reviews Molecular Cell Biology,2007,8(2):101.
[9]Dong J,Atwood C S,Anderson V E,et al.Metal binding and oxidation of amyloid-beta within isolated senile plaque cores:Raman microscopic evidence[J].Biochemistry,2003,42(10):2768.
[10]Atwood C S,Moir R D,Huang Xudong,et al.Dramatic aggregation of Alzheimer A?by Cu(Ⅱ)is induced by conditions representing physiological acidosis[J].The Journal of Biological Chemistry,1998,273(21):12817-12842.
[11]董晓燕,齐凤娟,张焕,等.Cu2+诱导的Aβ40种子的特性及其对Aβ40聚集的影响[J].天津大学学报:自然科学与工程技术版,2018,51(12):1287-1295.Dong Xiaoyan,Qi Fengjuan,Zhang Huan,et al.Characteristics of Cu2+-mediated Aβ40 seeds and their effects on Aβ40 aggregation[J].Journal of Tianjin University:Science and Technology,2018,51(12):1287-1295(in Chinese).
[12]Zafrilla P,Mulero J,Xandri J M,et al.Oxidative stress in Alzheimer patients in different stages of the disease[J].Current Medicinal Chemistry,2006,13(9):1075-1083.
[13]Lee J Y,Friedman J E,Angel I,et al.The lipophilic metal chelator DP-109 reduces amyloid pathology in brains of human beta-amyloid precursor protein transgenic mice[J].Neurobiology of Aging,2004,25(10):1315-1321.
[14]Cahoon L.The curious case of clioquinol[J].Nature Medicine,2009,15(4):356-359.
[15]Miyamoto T,Fukino Y,Kamino S,et al.Enhanced stability of Cu2+-ATCUN complexes under physiologically relevant conditions by insertion of structurally bulky and hydrophobic amino acid residues into the AT-CUN motif[J].Dalton Transactions,2016,45(23):9436-9445.
[16]And C H,Sarkar B.Amino terminal Cu(Ⅱ)-and Ni(Ⅱ)-binding(ATCUN)motif of proteins and peptides:Metal binding,DNA cleavage,and other properties[J].Cheminform,1997,28(27):123-130.
[17]Ghosh A,Pradhan N,Bera S,et al.Inhibition and degradation of amyloid beta(A?40)fibrillation by designed small peptide:A combined spectroscopy,microscopy,and cell toxicity study[J].ACS Chemical Neuroscience,2017,8(4):718-722.
[18]Rafferty J,Nagaraj H,Mccloskey A P,et al.Peptide therapeutics and the pharmaceutical industry:Barriers encountered translating from the laboratory to patients[J].Current Medicinal Chemistry,2016,23(37):4231-4259.
[19]Soto C,Sigurdsson E M,Morelli L,et al.?-sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis:Implications for Alzheimer’s therapy[J].Nature Medicine,1998,4(7):822-826.
[20]Adessi C,Frossard M J,Boissard C,et al.Pharmacological profiles of peptide drug candidates for the treatment of Alzheimer’s disease[J].Journal of Biological Chemistry,2003,278(16):13905-13911.
[21]Permanne B,Adessi C,Saborio G P,et al.Reduction of amyloid load and cerebral damage in a transgenic mouse model of Alzheimer’s disease by treatment with a beta-sheet breaker peptide[J].Faseb Journal Official Publication of the Federation of American Societies for Experimental Biology,2002,16(8):860-862.
[22]Lowe T L,Strzelec A,Kiessling L L,et al.Structurefunction relationships for inhibitors of beta-amyloid toxicity containing the recognition sequence KLVFF[J].Biochemistry,2001,40(26):7882-7889.
[23]Xiong N,Dong X Y,Zheng J,et al.Design of LVFFARK and LVFFARK-functionalized nanoparticles for inhibiting amyloid?-protein fibrillation and cytotoxicity[J].Acs Appl Mater Interfaces,2015,7(10):5650-5662.
[24]Bush A I,Tanzi R E.Therapeutics for Alzheimer’s disease based on the metal hypothesis[J].NeurotheraPeutics,2008,5(3):421-432.
[25]Mlynarz P,Valensin D,Kociolek K,et al.Impact of the peptide sequence on the coordination abilities of albumin-like tripeptides towards Cu2+,Ni2+and Zn2+ions.potential albumin-like peptide chelators[J].New Journal of Chemistry,2002,26(2):264-268.
[26]Wang Q,Shah N,Zhao J,et al.Structural,morphological,and kinetic studies of beta-amyloid peptide aggregation on self-assembled monolayers[J].Physical Chemistry Chemical Physics,2011,13(33):15200-15210.
[27]Cabaleiro-Lago C,Szczepankiewicz O,Linse S.The effect of nanoparticles on amyloid aggregation depends on the protein stability and intrinsic aggregation rate[J].Langmuir,2012,28(3):1852-1857.
[28]Guilloreau L,Combalbert S,Sournia-Saquet A,et al.Redox chemistry of copper-amyloid-?:The generation of hydroxyl radical in the presence of ascorbate is linked to redox-potentials and aggregation state[J].ChembioChem,2007,8(11):1317-1325.
[29]Lincoln K M,Richardson T E,Rutter L,et al.An nheterocyclic amine chelate capable of antioxidant capacity and amyloid disaggregation[J].ACS Chemical Neuroscience,2012,3(11):919-927.
[30]Sarell C J,Wilkinson S R,Viles J H.Substoichiometric levels of Cu2+ions accelerate the kinetics of fiber formation and promote cell toxicity of amyloid-beta from Alzheimer’s disease[J].Journal of Biological Chemistry,2010,285(53):41533-41540.
[31]Pedersen J T,Ostergaard J,Rozlosnik N,et al.Cu(Ⅱ)mediates kinetically distinct,non-amyloidogenic aggregation of amyloid-beta peptides[J].Journal of Biological Chemistry,2011,286(30):26952-26963.
[32]Jensen M,Canning A,Chiha S,et al.Inhibition of Cu-amyloid-beta by using bifunctional peptides with beta-sheet breaker and chelator moieties[J].ChemistryA European Journal,2012,18(16):4836-4839.
[33]Meng J,Zhang H,Dong X,et al.Rthlvffark-NH2:Apotent and selective modulator on Cu2+-mediated amyloid-?protein aggregation and cytotoxicity[J].Journal of Inorganic Biochemistry,2018,181:56-64.
[34]Hu X,Zhang Q,Wang W,et al.Tripeptide GGH as the inhibitor of copper-amyloid-beta-mediated redox reaction and toxicity[J].ACS Chemical Neuroscience,2016,7(9):1255-1263.
[35]Smith D G,Cappai R,Barnham K J.The redox chemistry of the Alzheimer’s disease amyloid beta peptide[J].Biochimica Et Biophysica Acta,2007,1768(8):1976-1990.
[36]Huang X D,Cuajungco M P,Atwood C S,et al.Cu(Ⅱ)potentiation of Alzheimer a beta neurotoxicitycorrelation with cell-free hydrogen peroxide production and metal reduction[J].Journal of Biological Chemistry,1999,274(52):37111-37116.
[37]Jarrett J T,Berger E P,Lansbury P T J.The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation:Implications for the pathogenesis of Alzheimer’s disease[J].Biochemistry,1993,32(18):4693-4697.
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