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首页> 外文期刊>Journal of Molecular Biology >An scFv intrabody against the nonamyloid component of alpha-synuclein reduces intracellular aggregation and toxicity
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An scFv intrabody against the nonamyloid component of alpha-synuclein reduces intracellular aggregation and toxicity

机译：抗α-突触核蛋白的非淀粉样成分的scFv体内抗体可减少细胞内聚集和毒性

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Prevention of abnormal misfolding and aggregation of alpha synuclein (syn) protein in vulnerable neurons should be viable therapeutic strategies for reducing pathogenesis in Parkinson's disease. The nonamyloid component (NAC) region of alpha-syn shows strong tendencies to form beta-sheet structures, and deletion of this region has been shown to reduce aggregation and toxicity in vitro and in vivo. The binding of a molecular species to this region may mimic the effects of such deletions. Single-chain variable fragment (ScFv) antibodies retain the binding specificity of antibodies and, when genetically manipulated to create high-diversity libraries, allow in vitro selection against peptides. Accordingly, we used a yeast surface display library of an entire naive repertoire of human scFv antibodies to select for binding to a NAC peptide. Candidate scFv antibodies (after transfer to mammalian expression vectors) were screened for viability in a neuronal cell line by transient cotransfection with A53T mutant alpha-syn. This provided a ranking of the protective efficacies of the initial panel of intracellular antibodies (intrabodies). High steady-state expression levels and apparent conformational epitope binding appeared more important than in vitro affinity in these assays. None of the scFv antibodies selected matched the sequences of previously reported anti-alpha-syn scFv antibodies. A stable cell line expressing the most effective intrabody, NAC32, showed highly significant reductions in abnormal aggregation in two separate models. Recently, intrabodies have shown promising antiaggregation and neuroprotective effects against misfolded mutant huntingtin protein. The NAC32 study extends such work significantly by utilizing information about the pathogenic capacity of a specific alpha-syn region to offer a new generation of in vitro-derived antibody fragments, both for further engineering as direct therapeutics and as a tool for rational drug design for Parkinson's disease. (c) 2007 Elsevier Ltd. All rights reserved.
机译：预防易损神经元中的异常折叠错误和α突触核蛋白（syn）蛋白聚集是减少帕金森病发病机理的可行治疗策略。 α-syn的非淀粉样成分（NAC）区显示出形成β-折叠结构的强烈趋势，该区域的缺失已显示出在体外和体内均可减少聚集和毒性。分子种类与该区域的结合可以模拟这种缺失的作用。单链可变片段（ScFv）抗体保留了抗体的结合特异性，并且在进行基因操作以创建高多样性文库时，可以进行针对肽的体外选择。因此，我们使用了完整的人类scFv抗体原始库的酵母表面展示文库来选择与NAC肽的结合。通过与A53T突变体α-syn瞬时共转染，筛选了候选scFv抗体（转移至哺乳动物表达载体后）在神经元细胞系中的生存力。这提供了细胞内抗体（体内）初始面板的保护效果的等级。在这些测定中，高稳态表达水平和明显的构象表位结合似乎比体外亲和力更重要。所选的scFv抗体均未与先前报道的抗α-synscFv抗体的序列匹配。表达最有效的体内抗体NAC32的稳定细胞系在两个单独的模型中显示出异常聚集的显着降低。最近，体内抗体显示出对错误折叠的亨廷顿蛋白错误折叠的抗聚集和神经保护作用。 NAC32研究通过利用有关特定α-syn区域的致病能力的信息显着扩展了此类工作，从而提供了新一代的体外衍生抗体片段，既可以作为直接疗法进行进一步工程设计，也可以作为合理药物设计的工具用于帕金森氏病。 （c）2007 Elsevier Ltd.保留所有权利。

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