An in vivo platform for identifying inhibitors of protein aggregation

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...

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Published inNature chemical biology Vol. 12; no. 2; pp. 94 - 101
Main Authors Saunders, Janet C, Young, Lydia M, Mahood, Rachel A, Jackson, Matthew P, Revill, Charlotte H, Foster, Richard J, Smith, D Alastair, Ashcroft, Alison E, Brockwell, David J, Radford, Sheena E
Format Journal Article
LanguageEnglish
Published United States Nature Publishing Group 01.02.2016
Subjects
Amyloid beta-Peptides - metabolism
beta-Lactamases - chemistry
Biological Assay - methods
Blotting, Western
Cellular biology
Curcumin - pharmacology
Dopamine - chemistry
Dopamine - pharmacology
Humans
Microscopy, Electron, Transmission
Protein Aggregation, Pathological
Protein Binding - drug effects
Protein folding
Scientific method
Spectrometry, Mass, Electrospray Ionization
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Summary: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.
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J.C.S. and L.M.Y. contributed equally to this work. J.C.S. designed the study, purified the β-lactamase constructs, performed the in vivo experiments, performed the β-lactamase activity assays in vitro, performed TEM, analyzed results and wrote the manuscript. L.M.Y. conceived, designed and performed mass spectrometry experiments, performed TEM and thioflavin T fluorometry and analyzed results. R.A.M purified Aβ40 and performed TEM and thioflavin T fluorometry. M.P.J performed Western blots, dot blots, thioflavin T and NIAD-4 fluorometry. C.H.R. and R.J.F. designed and prepared the small molecule screening library. R.J.F also performed all PAINS analyses. D.A.S. contributed to experiment design. A.E.A. conceived and designed mass spectrometry experiments. D.J.B. and S.E.R. conceived and designed the experiments and wrote the manuscript. All authors contributed to manuscript preparation.
Author Contributions
ISSN:1552-4450
1552-4469
DOI:10.1038/nchembio.1988