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Rapid selection of cyclic peptides that reduce α-synuclein toxicity in yeast and animal models

Nature Chemical Biology volume 5, pages 655–663 (2009)Cite this article

Abstract

Phage display has demonstrated the utility of cyclic peptides as general protein ligands but cannot access proteins inside eukaryotic cells. Expanding a new chemical genetics tool, we describe the first expressed library of head-to-tail cyclic peptides in yeast (Saccharomyces cerevisiae). We applied the library to selections in a yeast model of α-synuclein toxicity that recapitulates much of the cellular pathology of Parkinson's disease. From a pool of 5 million transformants, we isolated two related cyclic peptide constructs that specifically reduced the toxicity of human α-synuclein. These expressed cyclic peptide constructs also prevented dopaminergic neuron loss in an established Caenorhabditis elegans Parkinson's model. This work highlights the speed and efficiency of using libraries of expressed cyclic peptides for forward chemical genetics in cellular models of human disease.

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Figure 1: A cyclic peptide library that expresses and processes in yeast.
Figure 2: Identification of two CPs that selectively reduce α-synuclein toxicity in yeast.
Figure 3: Rapid generation of structure–activity relationship data and rapid minimization using point mutagenesis.
Figure 4: Selected CPs operate downstream of known vesicle trafficking defects.
Figure 5: Selected CPs reduce α-syn-mediated toxicity in C. elegans dopaminergic neurons.

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Acknowledgements

We thank S. Benkovic (Pennsylvania State University) for plasmids, E. Spooner for mass spectrometry assistance and N. Azubuine for preparation of media. This work was supported by a National Research Service Award fellowship from the US National Institute of Neurological Disorders and Stroke (NINDS) and National Institute on Aging (J.A.K.), an R21 grant from NINDS (S.L. and J.A.K.) and the Morris K. Udall Centers of Excellence for Parkinson's Disease Research (S.L.) Parkinson's disease research in the Caldwell Lab (G.A.C, K.A.C., S.H.) was supported by the American Parkinson Disease Association, Michael J. Fox Foundation and US National Institute of Environmental Health Sciences.

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Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    Joshua A Kritzer, Sandro Santagata & Susan Lindquist

  2. Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama, USA

    Shusei Hamamichi, Kim A Caldwell & Guy A Caldwell

  3. Integrated Imaging Center and Department of Biology, Johns Hopkins University, Baltimore, Maryland, USA

    J Michael McCaffery

  4. Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA

    Sandro Santagata

  5. Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, USA

    Todd A Naumann

  6. Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge Massachusetts, USA.,

    Susan Lindquist

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Contributions

J.A.K. conceived the overall strategy, tested the construct in yeast, designed and constructed the library, performed the selections and all subsequent experiments in yeast, constructed the metazoan CP construct and worm expression plasmids and wrote the manuscript. S.H. constructed the worm lines, designed and performed the worm experiments, analyzed worm data and wrote the manuscript. J.M.M. processed and imaged yeast by electron microscopy. S.S. contributed to the design of the metazoan CP construct. T.A.N. designed and tested the HPQ yeast CP construct. K.A.C. analyzed worm data and wrote the manuscript. G.A.C. designed the worm experiments, analyzed worm data and wrote the manuscript. S.L. suggested the approach, provided advice and suggestions throughout and supervised the project.

Corresponding author

Correspondence to Susan Lindquist.

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Supplementary Figures 1–6, Supplementary Tables 1–2, Supplementary Results and Supplementary Methods (PDF 296 kb)

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Kritzer, J., Hamamichi, S., McCaffery, J. et al. Rapid selection of cyclic peptides that reduce α-synuclein toxicity in yeast and animal models. Nat Chem Biol 5, 655–663 (2009). https://doi.org/10.1038/nchembio.193

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