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Investigating the Effects of O-GlcNAc Modifications in Parkinson’s Disease Using Semisynthetic α-Synuclein

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Expressed Protein Ligation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2133))

Abstract

α-Synuclein is a small aggregation-prone protein associated with Parkinson’s disease (PD). The protein’s biochemical and biophysical properties can be heavily influenced by various types of posttranslational modification (PTMs) such as phosphorylation, ubiquitination, and glycosylation. To understand the site-specific effects of various PTMs have on the protein and its aggregation, obtaining a homogeneous sample of the protein of interest with the specific modification of interest is key. Expressed protein ligation (EPL) has emerged as robust tool for building synthetic proteins bearing site-specific modifications. Here, we outline our approach for building α-synuclein with site specific O-GlcNAc modifications, an intracellular subtype of glycosylation that has been linked to the inhibition of protein aggregation. More specifically, we provide specific protocols for the synthesis of α-synuclein bearing an O-GlcNAc modification at threonine 72, termed α-synuclein(gT72). However, this general approach utilizing two recombinant fragments and one synthetic peptide is applicable to other sites and types of modifications and should be transferable to various other protein targets, including aggregation prone proteins like tau and TDP-43.

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Acknowledgments

This research was supported by the National Institutes of Health (Grant R01GM114537 to M.R.P.).

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Authors and Affiliations

  1. Department of Chemistry, University of Southern California, Los Angeles, CA, USA

    Ana Galesic & Matthew R. Pratt

  2. Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA

    Matthew R. Pratt

Authors
  1. Ana Galesic
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  2. Matthew R. Pratt
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Correspondence to Matthew R. Pratt .

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Editors and Affiliations

  1. ProteoDesign, Barcelona, Barcelona, Spain

    Miquel Vila-Perelló

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Galesic, A., Pratt, M.R. (2020). Investigating the Effects of O-GlcNAc Modifications in Parkinson’s Disease Using Semisynthetic α-Synuclein. In: Vila-Perelló, M. (eds) Expressed Protein Ligation. Methods in Molecular Biology, vol 2133. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0434-2_15

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  • DOI: https://doi.org/10.1007/978-1-0716-0434-2_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0433-5

  • Online ISBN: 978-1-0716-0434-2

  • eBook Packages: Springer Protocols

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