【作者】 孟杰；

【导师】 董晓燕；

【作者基本信息】 天津大学 ， 生物化工， 2018， 硕士

【摘要】 淀粉样β蛋白（amyloid-βpeptide,Aβ）的聚集是阿尔兹海默症（Alzheimer’s disease,AD）的主要特征之一,Aβ单体能聚集形成具有细胞毒性的寡聚体、原纤维和纤维。此外,金属离子(特别是Cu²⁺)与Aβ相互作用会形成具有更强细胞毒性的聚集体,还会诱导活性氧自由基（reactive oxygen species,ROS）的生成,进一步加剧细胞毒性。因此,既能螯合金属离子又能抑制Aβ聚集的双功能抑制剂是有潜力的AD治疗药。同时为了减小副作用,开发能选择性螯合Cu²⁺,而不是螯合其它人体需要的金属离子(如K⁺和Ca²⁺等)也是非常必要的。本研究首先将精蛋白(Human protamine HP2,HP2_1-15)中与Cu²⁺结合的位点三肽（RTH）作为金属螯合剂,考察了其对Cu²⁺的选择性螯合能力以及ROS生成的抑制作用。实验结果表明,相比于其它金属离子(如K⁺,Ca²⁺,Mg²⁺和Zn²⁺)RTH对Cu²⁺具有显著的选择螯合性。RTH对Cu²⁺的结合力（K_d=0.01μM）远高于Aβ₄₀与Cu²⁺（K_d=2.79μM）的结合力,不仅能抑制Cu²⁺对Aβ₄₀聚集的加速作用,而且能抑制Cu²⁺或Aβ₄₀-Cu²⁺复合物催化的ROS生成。然后将RTH与Aβ聚集抑制剂LK7（Ac-LVFFARK-NH₂）相连接,得到双功能十肽抑制剂RTHLVFFARK-NH₂（RK10）。研究表明,RK10不仅能抑制Cu²⁺存在下Aβ的纤维化(当Aβ₄₀:Cu²⁺:RK10为1:0.4:4时,可使ThT荧光降至22%),还能改变Aβ₄₀聚集体的形态,不再形成纤维而是形成球形聚集体;RK10对Cu²⁺或Aβ-Cu²⁺复合物催化的ROS生成也有显著的抑制作用,当RK10浓度为Cu²⁺的四倍时,催化活性几乎完全被抑制;同时,RK10也可有效抑制Cu²⁺介导的Aβ细胞毒性,使细胞活性从46%(Aβ₄₀-Cu²⁺实验组)增至86%;等温滴定量热实验证明RK10同样能选择性螯合Cu²⁺,其螯合Cu²⁺的解离常数为0.02μM,因此足够剥离掉Aβ₄₀-Cu²⁺复合物中的Cu²⁺,同时由于RK10的双功能设计,其螯合Cu²⁺后,还能抑制Aβ₄₀聚集,进而改变Cu²⁺存在条件下Aβ₄₀的聚集路径,减轻细胞毒性。本研究设计合成的双功能抑制剂（RK10）,可高选择性螯合Cu²⁺,抑制Cu²⁺诱导的Aβ₄₀聚集,减轻Cu²⁺带来的氧化压力以及Aβ₄₀聚集产生的细胞毒性,这对今后多功能Aβ聚集抑制剂的开发和AD的治疗都大有裨益。更多还原

【Abstract】 The accumulation of amyloid-β（Aβ）peptide has been considered as one of the hallmarks of Alzheimer’s disease（AD）.In addition,the interactions of metal ions,especially Cu²⁺,with Aβproduce toxic aggregates and reactive oxygen species（ROS）further promoting neurotoxicity.Thus,bifunctional agents with the abilities to chelate metal ions and inhibit Aβaggregation are promising therapeutic agents for AD.Particularly,to reduce side effects,it is necessary to develop molecules with the ability to specifically chelate Cu²⁺rather than systemic metal ions.Tripeptide RTH was chosen as the Cu²⁺chelator,which is the binding site of Human protamine HP2(HP2_1-15)and Cu²⁺.The ability of RTH to selectively chelate Cu²⁺and prevent ROS production was explored.The results indicate that RTH has excellent selectivity for Cu²⁺over other biologically relevant metal ions(e.g.,K⁺,Ca²⁺,Mg²⁺,and Zn²⁺).The binding affinity of RTH to Cu²⁺（K_d=0.01μM）is far greater than Aβ₄₀（K_d=2.79μM）。RTH could inhibit Aβ₄₀ rapid aggregation induced by Cu²⁺and eliminate ROS generation catalyzed by Cu²⁺or Aβ₄₀-Cu²⁺complex.Next a bifunctional decapeptide RTHLVFFARK-NH₂（RK10）was designed based on RTH and an Aβaggregation inhibitor Ac-LVFFARK-NH₂（LK7）.The results investigate RK10 could inhibit Aβfibrillogenesis in the presense of Cu²⁺(the ThT fluorescence of Aβ₄₀-Cu²⁺system reduced to²2%by adding of RK10 when the molar ratio of Aβ₄₀:Cu²⁺:RK10 was 1:0.4:4).RK10 was capable of changing the morphology of Aβ₄₀ aggregates in the presense of Cu²⁺from mature fibrils to spherical aggregates.RK10 could prevent the production of reactive oxygen species（ROS）catalyzed by Cu²⁺or Aβ-Cu²⁺complex,and the catalytic activity of Cu²⁺was completely suppressed when four times of RK10 was added.Furthermore,RK10 was effective in inhibiting Cu²⁺-mediated Aβcytotoxicity.Results shown that the cell viability was increased from 46%(Aβ₄₀-Cu²⁺-treated group)to 86%by adding RK10.Isothermal titration calorimetry（ITC）measurements indicate that RK10 could also selectively chelate Cu²⁺.Moreover,RK10 chelates Cu²⁺with a dissociation constant of 0.02μM,making it has the ability to sequester Cu²⁺from Aβ₄₀-Cu²⁺complex.For the bifunctional design,RK10 could suppress Aβ₄₀ aggregation after chelating Cu²⁺,and then change the Aβ₄₀ aggregation pathway in the presense of Cu²⁺,reduce the cytotoxicity.We designed the bifunctional peptide inhibitor RK10,which could chelate Cu²⁺with high selectivity,suppress Aβaggregation,reduce oxidative stress induced by Cu²⁺and the cytotoxicity from Aβ₄₀ aggregation.Hence,our work will benefit in the future design of multifunctional inhibitors for AD treatment.更多还原