Functional Assays of Thiol Isomerase ERp5.

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 αIIbß3, 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.