Abstract
A Disintegrin and Metalloprotease 17 (ADAM17) can cause the fast release of growth factors and inflammatory mediators from the cell surface. Its activity has to be turned on which occurs by various stimuli. The active form can be inactivated by a structural change in its ectodomain, related to the pattern of the formed disulphide bridges. The switch-off is executed by protein disulfide isomerases (PDIs) that catalyze an isomerization of two disulfide bridges and thereby cause a disulfide switch. We demonstrate that the integrity of the CGHC-motif within the active site of PDIs is indispensable. In particular, no major variation is apparent in the activities of the two catalytic domains of PDIA6. The affinities between PDIA1, PDIA3, PDIA6 and the targeted domain of ADAM17 are all in the nanomolar range and display no significant differences. The redundancy between PDIs and their disulfide switch activity in ectodomains of transmembrane proteins found in vitro appears to be a basic characteristic. However, different PDIs might be required in vivo for disulfide switches in different tissues and under different cellular and physiological situations.
Highlights
Post-translational protein modifications are typical responses to changing environmental parameters and enable organisms to maintain their metabolic and physiological homeostasis
Since PDIA6 tends to degrade during storage at or above 4 °C, the integrity of the protein was routinely verified by SDS-PAGE upon incubation at 37 °C (Fig. 1B)
The low activity of PDIA6 when performing the thiol switch within the isolated open membrane-proximal domain (opMPD) suggests either that PDIA6 has no high affinity binding site to the isolated opMPD and/or that PDIA6 is not the principal specific isomerase that catalyzes the inactivation of A Disintegrin and Metalloprotease 17 (ADAM17)
Summary
Post-translational protein modifications are typical responses to changing environmental parameters and enable organisms to maintain their metabolic and physiological homeostasis. We have used recombinant purified proteins to analyze the principal requirements of PDIs when catalyzing the disulfide switch. PDIA6 displays only low isomerization activity for the opMPD-to-clMPD switch (Fig. 1A).
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