分子对接技术筛选菲律宾蛤仔抗炎肽

Screening of anti-inflammatory peptides from Ruditapes philippinarumby molecular docking

基于分子对接技术筛选菲律宾蛤仔(Ruditapes philippinarum)来源的具有抗炎作用的活性肽。采用三氯乙酸-丙酮沉淀法提取菲律宾蛤仔蛋白质,通过双向凝胶电泳(2-dimensional electro phoresis,2-DE)技术和液质联用(LC-MS/MS)技术分离和鉴定其蛋白质,借助BIOPEP-UWM在线工具对主要蛋白进行虚拟酶解,使用PeptideRanker和ToxinPred等程序对肽段进行评分和理化性质分析;利用Discovery studio 2019和AutoDock Vina软件进行分子对接筛选潜在抗炎肽片段,探讨人工合成抗炎肽对LPS诱导的RAW264.7细胞炎症的改善作用。结果表明,原肌球蛋白和组蛋白为肌肉组织的主要蛋白质。选用原肌球蛋白作为底物,通过虚拟酶解和分子对接筛选出8种潜在的新颖活性肽。其中DQTF与TLR2和TLR4分别存在7个氨基酸残基和8个氨基酸残基结合位点,而GYTR与TLR2分子中的10个氨基酸残基和TLR4分子中的7个氨基酸残基紧密结合(结合能均低于-5 kcal/mol),表明其是潜在的抗炎肽。活性验证结果表明,2条合成寡肽均能显著增强RAW264.7细胞活力(P<0.001),并显著降低细胞产NO能力(P<0.05),表现出良好的抗炎活性。综上,基于虚拟酶解和分子对接方法可用于筛选菲律宾蛤仔来源的抗炎肽。

Screening of active peptides with anti-inflammatory effects of Ruditapes philippinarum based on molecular docking.In this study,the proteins of R.philippinarum were extracted by trichloroacetic acid-acetone precipitation and identified by 2-DE and LC-MS/MS.The main protein was enzymolized by BIOPEP-UWM and the PeptideRanker and ToxinPred programs were used to evaluate and predict the physicochemical properties of peptides.Molecular docking was performed using Discovery studio 2019 and AutoDock Vina to screen potential anti-inflammatory peptide fragments,the improvement of LPS-induced inflammation in RAW264.7 cells was verified by synthetic anti-inflammatory peptides.The results showed that tropomyosin and histone were the main proteins of muscle tissue.Eight potential unreported active peptides were screened to use tropomyosin as substrate by virtual enzymolysis.DQTF had seven amino acid residues and eight amino acid residues binding sites with TLR2 and TLR4 respectively,while GYTR was closely combined with ten amino acid residues in TLR2 molecule and seven amino acid residues in TLR4 molecule,with the binding energy lower than-5 kcal/mol,indicating that it was a potential anti-inflammatory peptide.The activity verification results showed that the two synthetic oligopeptides could significantly enhance the cell viability(P<0.001)and reduce the ability of NO production(P<0.05),showing great anti-inflammatory activity.In conclusion,virtual enzymolysis and molecular docking methods can be used to screen anti-inflammatory peptides derived from R.philippinarum.