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Nuclear Receptors pp 107–124Cite as

Detection of ADP-Ribosylation of the Androgen Receptor Using the Recombinant Macrodomain AF1521 from Archaeoglobus fulgidus

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1966))

Abstract

ADP-ribosylation is a posttranslational modification generated by members of the superfamily of ADP-ribosyltransferases, known as the Parp enzymes. Depending on the superfamily member, Parp enzymes can mono- or poly-ADP-ribosylate a protein substrate. Parp superfamily members confer regulation to a variety of biological processes that include cell signaling, DNA repair, transcription, and stress responses. Here, we describe biochemical methods for detection of ADP-ribose conjugated to the androgen receptor (AR) using the archaeal macrodomain, AF1521, from Archaeoglobus fulgidus. The utility of AF1521 is based on its highly selective recognition of ADP-ribose conjugated to protein. AF1521 immobilized on beads can be used to enrich for ADP-ribosylated proteins, which in our application results in recovery of ADP-ribosylated AR from prostate cancer cell extracts. We engineered tandem AF1521 macrodomains and found this improves the recovery of ADP-ribosylated AR under native conditions, and it enabled development of an assay for detection of ADP-ribosylation on blots. Thus, AF1521 can be used to query ADP-ribosylation of protein under both native and denaturing conditions. Our assays should prove useful for understanding how ADP-ribosylation regulates AR function.

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Acknowledgment

This work was supported by NIH/NCI grant R01CA214872 to B.M.P.

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Authors and Affiliations

  1. Center for Cell Signaling, University of Virginia, Charlottesville, VA, USA

    Teddy Kamata, Chun-Song Yang, Kasey Jividen, Adam Spencer, Natalia Dworak, Luke T. Oostdyk & Bryce M. Paschal

  2. Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, USA

    Teddy Kamata, Luke T. Oostdyk & Bryce M. Paschal

Authors
  1. Teddy Kamata
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  2. Chun-Song Yang
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  3. Kasey Jividen
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  4. Adam Spencer
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  5. Natalia Dworak
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  6. Luke T. Oostdyk
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  7. Bryce M. Paschal
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Correspondence to Bryce M. Paschal .

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Editors and Affiliations

  1. School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO, USA

    Mostafa Z. Badr

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Kamata, T. et al. (2019). Detection of ADP-Ribosylation of the Androgen Receptor Using the Recombinant Macrodomain AF1521 from Archaeoglobus fulgidus. In: Badr, M. (eds) Nuclear Receptors. Methods in Molecular Biology, vol 1966. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9195-2_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9195-2_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9194-5

  • Online ISBN: 978-1-4939-9195-2

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