title: An in vivo platform for identifying inhibitors of protein aggregation.
abstract: Protein aggregation underlies an array of human diseases, yet only one small-molecule therapeutic targeting this process has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, that is capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid β) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split β-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of islet amyloid polypeptide aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation.
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.|School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Janet C
authors.initials: JC
authors.lastname: Saunders
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.|School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Lydia M
authors.initials: LM
authors.lastname: Young
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.|School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Mahood
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.|School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Jackson
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.|School of Chemistry, University of Leeds, LS2 9JT, UK.
authors.forename: Charlotte H
authors.initials: CH
authors.lastname: Revill
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.|School of Chemistry, University of Leeds, LS2 9JT, UK.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Foster
authors.affiliation: Avacta Analytical plc, Wetherby, LS23 7FZ, UK.
authors.forename: D Alastair
authors.initials: DA
authors.lastname: Smith
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.|School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.|School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Brockwell
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.|School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
mesh_terms: D016229:Amyloid beta-Peptides / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D015153:Blotting, Western; D003474:Curcumin / Q000494:pharmacology; D004298:Dopamine / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D046529:Microscopy, Electron, Transmission; D066263:Protein Aggregation, Pathological; D011485:Protein Binding / Q000187:drug effects; D021241:Spectrometry, Mass, Electrospray Ionization; D001618:beta-Lactamases / Q000737:chemistry
chemical_list: D016229:Amyloid beta-Peptides; D001618:beta-Lactamases; D003474:Curcumin; D004298:Dopamine