The aggregation of amyloid-beta （Aβ） peptide, has been demonstrated to be critical for the development of Alzheimer＇s disease （AD）. All aggregation inhibitors are thus considered to be drug candidates for AD the...The aggregation of amyloid-beta （Aβ） peptide, has been demonstrated to be critical for the development of Alzheimer＇s disease （AD）. All aggregation inhibitors are thus considered to be drug candidates for AD therapy. In the present study, we developed a novel screening tool based on the yeast two-hybrid system to screen Aβ aggregation inhibitors. The human Aβ42 peptide cDNA was cloned using assembly PCR and inserted into each of the yeast expression plasmids containing either the GAL4 activation domain （GAL4AD） or the DNA-binding domain （GAL4BD）. Co-transformation of the above plasmids led to the expression of the fusion proteins GAL4AD-Aβ42 and GAL4BD-Aβ42 in the AH 109 yeast strain. The self interaction of Aβ42 fragments reconstructed the GAL4 transcriptor and thus activated the GAL4 responsive transcription of four reporter genes including HIS3, ADE2, lacZ and MEL1. The expression of the reporter genes rendered the multiple auxotrophic yeast cells capable of growing on the synthetic SD media lacking adenine and histidine. Growth arrest was used as a marker for screening Aβ aggregation inhibitors in this system, and the evaluation of Rhodiola species revealed potential resources for the development of Aβ aggregation inhibitors.展开更多
Protein-protein interactions(PPIs)play vital roles in biological processes.However,the discovery of transient and biologically relevant PPIs remain a formidable challenge using conventional strategies such as co-immun...Protein-protein interactions(PPIs)play vital roles in biological processes.However,the discovery of transient and biologically relevant PPIs remain a formidable challenge using conventional strategies such as co-immunoprecipitation(CoIP),yeast two hybrids(Y2H)and Forster resonance energy transfer(FRET)[1].Genetically encoded photocrosslinkers have recently emerged as a powerful approach for probing intracellular PPIs with the capability for in situ studies,low perturbation to cells as well as the wide generality.This facile strategy also demonstrated an advantage of high spatiotemporal resolution,which offers a robust capture strategy for the discovery of transient and/or weak PPIs with lowered false-positive backgrounds[2].展开更多
文摘The aggregation of amyloid-beta （Aβ） peptide, has been demonstrated to be critical for the development of Alzheimer＇s disease （AD）. All aggregation inhibitors are thus considered to be drug candidates for AD therapy. In the present study, we developed a novel screening tool based on the yeast two-hybrid system to screen Aβ aggregation inhibitors. The human Aβ42 peptide cDNA was cloned using assembly PCR and inserted into each of the yeast expression plasmids containing either the GAL4 activation domain （GAL4AD） or the DNA-binding domain （GAL4BD）. Co-transformation of the above plasmids led to the expression of the fusion proteins GAL4AD-Aβ42 and GAL4BD-Aβ42 in the AH 109 yeast strain. The self interaction of Aβ42 fragments reconstructed the GAL4 transcriptor and thus activated the GAL4 responsive transcription of four reporter genes including HIS3, ADE2, lacZ and MEL1. The expression of the reporter genes rendered the multiple auxotrophic yeast cells capable of growing on the synthetic SD media lacking adenine and histidine. Growth arrest was used as a marker for screening Aβ aggregation inhibitors in this system, and the evaluation of Rhodiola species revealed potential resources for the development of Aβ aggregation inhibitors.
基金supported by the National Key Research and Development Program(2016YFA0501500)the National Natural Science Foundation of China(21225206,21432002,21521003)
文摘Protein-protein interactions(PPIs)play vital roles in biological processes.However,the discovery of transient and biologically relevant PPIs remain a formidable challenge using conventional strategies such as co-immunoprecipitation(CoIP),yeast two hybrids(Y2H)and Forster resonance energy transfer(FRET)[1].Genetically encoded photocrosslinkers have recently emerged as a powerful approach for probing intracellular PPIs with the capability for in situ studies,low perturbation to cells as well as the wide generality.This facile strategy also demonstrated an advantage of high spatiotemporal resolution,which offers a robust capture strategy for the discovery of transient and/or weak PPIs with lowered false-positive backgrounds[2].
