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Artificial proteins target SUMO

SUMOylation is the covalent attachment of SUMO-1, SUMO-2, SUMO-3, or combinations thereof to target proteins to control protein function and localization. Hughes et al. screened a library of artificial proteins called Affimers to identify those that bound to SUMO-1 or SUMO-2 (which is nearly identical to SUMO-3) and incorporated a negative selection step to remove SUMO-2–binding Affimers that also bound to SUMO-1. The authors identified Affimers that recognized SUMO-1, SUMO-2 and SUMO-3 (SUMO-2/3), or all three isoforms. Biochemical and cellular assays showed that these SUMO-specific Affimers (S-Affs) did not interfere with SUMO conjugation or deconjugation but inhibited a cellular stress response that required SUMO-mediated protein-protein interactions. In addition to generating S-Affs that will be useful tools for studying SUMO-dependent cellular processes, this study also shows the applicability of this technology for generating reagents that interfere with specific protein-protein interactions for basic research and potentially for clinical development (see the Protocol by Tang et al.).

Abstract

Because protein-protein interactions underpin most biological processes, developing tools that target them to understand their function or to inform the development of therapeutics is an important task. SUMOylation is the posttranslational covalent attachment of proteins in the SUMO family (SUMO-1, SUMO-2, or SUMO-3), and it regulates numerous cellular pathways. SUMOylated proteins are recognized by proteins with SUMO-interaction motifs (SIMs) that facilitate noncovalent interactions with SUMO. We describe the use of the Affimer system of peptide display for the rapid isolation of synthetic binding proteins that inhibit SUMO-dependent protein-protein interactions mediated by SIMs both in vitro and in cells. Crucially, these synthetic proteins did not prevent SUMO conjugation either in vitro or in cell-based systems, enabling the specific analysis of SUMO-mediated protein-protein interactions. Furthermore, through structural analysis and molecular modeling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO-1–mediated interactions or interactions mediated by either SUMO-2 or SUMO-3. Not only will these reagents enable investigation of the biological roles of SUMOylation, but the Affimer technology used to generate these synthetic binding proteins could also be exploited to design or validate reagents or therapeutics that target other protein-protein interactions.

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Supplementary Material

Summary

Fig. S1. Structures of proteins used in this study.
Fig. S2. Identification of isoform-specific S-Affs.
Fig. S3. Cell viability after expression of S-Affs.
Fig. S4. S-Affs interact with SUMOylated cellular proteins.
Table S1. Crystallographic data collection, processing, and refinement statistics for the SUMO-1:S1S2D5, SUMO-2:S1S2D5, and SUMO-2:S2B3 complexes.
Table S2. Key hydrogen bonds predicted from MD simulations of the SUMO-1:S1S2D5, SUMO-2:S1S2D5, SUMO-1:S2B3, SUMO-1-Alt:S2B3, and SUMO-2:S2B3 complexes.
References (60)

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Science Signaling
Volume 10 | Issue 505
November 2017

Submission history

Received: 13 September 2016
Accepted: 28 September 2017

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Acknowledgments

We thank I. Manfield (University of Leeds) for the technical assistance; J. Henley (University of Bristol), R. Hay and E. Jaffray (University of Dundee) for the reagents; and A. Berndt (University of Leeds) for the advice and critical reading of the manuscript. Funding: This work was funded, in part, by a Leeds Cancer Research UK Development Fund and the Wellcome Trust (Institutional Strategic Support Fund) (to D.J.H.), the Wellcome Trust (089330), the Biotechnology and Biological Sciences Research Council (BB/K00306/1 and BB/M006557/1) (to A.W.), and the University of Leeds through the Biomedical Health Research Centre support for the Leeds BioScreening Technology Group (to M.J.M. and D.C.T.). Author contributions: D.J.H., D.C.T., and A.W. conceived the study. D.J.H., M.J.M., D.C.T., and A.W. designed the experiments. D.J.H., C.T., A.A.S.T., N.P., C.H.T., U.M., K.Z.Z., G.H., J.D., E.F., and D.C.T. conducted the experiments. D.J.H., C.T., A.A.S.T., C.H.T., T.G., T.A.E., J.D., E.F., D.C.T., and A.W. analyzed the data. D.J.H., D.C.T., and A.W. wrote the paper with input from all the authors. Competing interests: The Affimer technology is under a patent application filed by the University of Leeds and are commercialized through a licensing agreement by Avacta Life Sciences. The University of Leeds receives royalties on sales of reagents through Avacta Life Sciences, which are distributed according to university policy. Data and materials availability: Structures deposited in PDB (www.pdb.org) are referred to as Adhirons and have been assigned the following identification numbers: 5ELJ (SUMO-1:S1S2D5*), 5EQL (SUMO-2:S1S2D5*), and 5ELU (SUMO-2:S2B3). *S1S2D5 is referred to as S2D5 in the PDB. Material is available upon request.

Authors

Affiliations

School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews KY16 9ST, UK.
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
BioScreening Technology Group, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Natalie Penswick
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
BioScreening Technology Group, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Chi H. Trinh
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
BioScreening Technology Group, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
BioScreening Technology Group, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Thomas A. Edwards
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Jianxin Duan
Schrödinger GmbH, Mannheim, Germany.
Schrödinger Inc., Cambridge, MA 02142, USA.
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
BioScreening Technology Group, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
BioScreening Technology Group, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.

Funding Information

Wellcome Trust: award304312, 089330
Wellcome Trust: award304321, ISSF
Biotechnology and Biological Sciences Research Council: award304313, BB/K000306/1 and BB/M006557/1
University Of Leeds: award304314, Biomedical Health Research Centre
University Of Leeds: award304319, Biomedical Health Research Center
University Of Leeds: award304320, Cancer Research UK Development Fund

Notes

*
Corresponding author. Email: [email protected] (D.J.H.); [email protected] (D.C.T.); [email protected] (A.W.)

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