Abstract
Newly synthesized thyroglobulin (Tg), the thyroid prohormone, forms detectable high molecular weight mixed disulfide adducts: until now, only Tg "adduct B" was identified as primarily engaging the endoplasmic reticulum oxidoreductases ERp57 and protein disulfide isomerase. Here, we demonstrate that the faster migrating Tg adduct C primarily engages the CaBP1/P5 oxidoreductase, whereas the slower migrating Tg adduct A primarily engages ERp72. Upon siRNA-mediated knockdown of CaBP1/P5 or ERp72, adducts C or A, respectively, are decreased. Within the three Tg adduct bands that do not exhibit a precursor-product relationship, Tg exhibits distinct oxidation patterns. We present evidence suggesting that disulfide maturation occurs within Tg monomers engaged in each of the adduct bands. Moreover, the same Tg substrate molecules can form simultaneous mixed disulfides with both CaBP1/P5 and protein disulfide isomerase, although these are generally viewed as components of distinct oxidoreductase-chaperone protein complexes. Such substrate-oxidoreductase combinations offer Tg the potential for simultaneous oxidative maturation along different parallel tracks leading to the native state.
Highlights
Secretory proteins acquire their native three-dimensional conformation through repeated brief interactions with endoplasmic reticulum (ER) chaperones and oxidoreductases
The transient nature of these adducts is unaffected by treatment with proteasome inhibitors, suggesting that they are not primarily misfolded forms destined for endoplasmic reticulum-associated degradation but rather intermolecular adducts captured in the process of Tg intramolecular disulfide maturation [30]
ER members of the protein disulfide isomerase (PDI) superfamily may have overlapping/ interchangeable activities, or may be dedicated to distinct enzyme-catalyzed reactions or distinct substrates (4 –7, 35, 36). Secretory proteins such as Tg are potential substrates for multiple ER oxidoreductases that participate in oxidative protein folding
Summary
Secretory proteins acquire their native three-dimensional conformation through repeated brief interactions with ER chaperones and oxidoreductases. The same Tg substrate molecules can form simultaneous mixed disulfides with both CaBP1/P5 and protein disulfide isomerase, these are generally viewed as components of distinct oxidoreductase-chaperone protein complexes Such substrate-oxidoreductase combinations offer Tg the potential for simultaneous oxidative maturation along different parallel tracks leading to the native state. For many secretory proteins, acquiring a three-dimensional globular structure is linked to formation and isomerization of disulfide bonds [3] These covalent interactions are catalyzed by a group of ER oxidoreductases: resident proteins that belong to the protein disulfide isomerase (PDI) family comprising Ͼ20 members (4 –7). We have definitively identified distinct mixed disulfide-linked adducts between newly synthesized Tg and ER oxidoreductases ERp72 and CaBP1/P5 and provide evidence to suggest that Tg oxidative maturation occurs within these distinct adduct bands, facilitating multidomain disulfide maturation within Tg monomers
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