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Functional Assays of Thiol Isomerase ERp5

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Functional Disulphide Bonds

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1967))

Abstract

Endoplasmic reticulum protein 5 (ERp5) is a member of the thiol isomerase family of enzymes, whose prototype member is protein disulphide isomerase (PDI). Thiol isomerases catalyze reduction/oxidation (redox) reactions which lead to the cleavage, formation, or isomerization of disulphide bonds in protein substrates. Thiol isomerase reactions on protein disulphides are important for the correct folding of proteins in the endoplasmic reticulum and for the regulation of various protein functions in the extracellular space. Apart from the disulphide reactions, thiol isomerases assist protein folding by chaperone activity.

The disulphide redox activity of ERp5 can be measured with functional assays involving artificial or natural substrates containing disulphide bonds. Herein we describe step-by-step assays of ERp5 reductase, isomerization, and de-nitrosylation activity. Disulphide reductase assays include insulin or di-eosin-GSSG as substrates whereas the isomerization assay includes RNase as substrate. The reduction of natural substrates, i.e., integrin αIIbβ3, can be detected using maleimide labels of free thiols and Western blotting. The biotin switch assay is used to measure the de-nitrosylation of S-nitrosylated substrates. These assays can measure the activity of purified ERp5 protein but can also be applied for the measurement of thiol isomerase activity in cellular samples.

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Acknowledgments

Funding was provided by the St George and Sutherland Medical Research Foundation (FP).

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Authors and Affiliations

  1. Haematology Research Group, Heart Research Institute, Newtown, NSW, Australia

    Alexander Dupuy & Freda Passam

  2. Cardiovascular Division, Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia

    Alexander Dupuy & Freda Passam

Authors
  1. Alexander Dupuy
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  2. Freda Passam
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Corresponding author

Correspondence to Freda Passam .

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Editors and Affiliations

  1. The Centenary Institute, NHMRC Clinical Trials Centre, Sydney Medical School, University of Sydney, Sydney, NSW, Australia

    Philip Hogg

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Dupuy, A., Passam, F. (2019). Functional Assays of Thiol Isomerase ERp5. In: Hogg, P. (eds) Functional Disulphide Bonds. Methods in Molecular Biology, vol 1967. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9187-7_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9187-7_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9186-0

  • Online ISBN: 978-1-4939-9187-7

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