An scFv intrabody against the nonamyloid component of alpha-synuclein reduces intracellular aggregation and toxicity |
| |
Authors: | Lynch Sandra M Zhou Chun Messer Anne |
| |
Institution: | 1 Division of Genetic Disorders, Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA 2 Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY 12201, USA |
| |
Abstract: | Prevention of abnormal misfolding and aggregation of α synuclein (syn) protein in vulnerable neurons should be viable therapeutic strategies for reducing pathogenesis in Parkinson's disease. The nonamyloid component (NAC) region of α-syn shows strong tendencies to form β-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 naïve 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 α-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-α-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 α-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. |
| |
Keywords: | syn synuclein NAC nonamyloid component scFv single-chain variable fragment PD Parkinson's disease htt huntingtin SAPE streptavidin-R-phycoerythrin CDR complementarity-determining region NLS nuclear localization signal DAPI 4&prime 6-diamidino-2-phenylindole VH heavy chain variable fragment VL light chain variable fragment FACS flow assisted cytometric sorting MTT 3-(4 5-dimethyl thiazolyl-2)-2 5 diphenyl tetrazolium bromide LDH lactate dehydrogenase PE phycoerythrin |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|