Mixed-Disulfide Folding Intermediates between Thyroglobulin and Endoplasmic Reticulum Resident Oxidoreductases ERp57 and Protein Disulfide Isomerase

Abstract

We present the first identification of transient folding intermediates of endogenous thyroglobulin (Tg; a large homodimeric secretory glycoprotein of thyrocytes), which include mixed disulfides with endogenous oxidoreductases servicing Tg folding needs. Formation of disulfide-linked Tg adducts with endoplasmic reticulum (ER) oxidoreductases begins cotranslationally. Inhibition of ER glucosidase activity blocked formation of a subgroup of Tg adducts containing ERp57 while causing increased Tg adduct formation with protein disulfide isomerase (PDI), delayed adduct resolution, perturbed oxidative folding of Tg monomers, impaired Tg dimerization, increased Tg association with BiP/GRP78 and GRP94, activation of the unfolded protein response, increased ER-associated degradation of a subpopulation of Tg, partial Tg escape from ER quality control with increased secretion of free monomers, and decreased overall Tg secretion. These data point towards mixed disulfides with the ERp57 oxidoreductase in conjunction with calreticulin/calnexin chaperones acting as normal early Tg folding intermediates that can be "substituted" by PDI adducts only at the expense of lower folding efficiency with resultant ER stress.Membrane and secretory proteins are cotranslationally translocated in the lumen of the endoplasmic reticulum (ER), where they acquire their three-dimensional structure (including the formation and isomerization of disulfide bonds), typically culminating in oligomeric assembly. This is a complex task, both facilitated and monitored by ER folding enzymes and molecular chaperones. Glycoproteins are an important subset of exportable proteins, and those bearing Asn-linked oligosaccharides fold preferentially with the aid of calreticulin (CRT) and calnexin (CNX), both of which possess a lectin-like binding site that prefers association with monoglucosylated oligosaccharide processing intermediates (4). CRT and CNX might directly influence protein folding (32), but an additional critical function of these proteins is to bring newly synthesized exportable glycoproteins in close proximity with ERp57 (47), an oxidoreductase that works in a complex with CRT/CNX and promotes proper disulfide bond formation (21,46,54).Another molecular chaperone is BiP (GRP78), which binds to unfolded polypeptides, helps to prevent protein aggregation through noncovalent associations regulated by its ATPase domain (9), and works cooperatively with protein disulfide isomerase (PDI) to promote oxidative protein folding (36). Indeed, recently the concept of two distinct chaperone-oxidoreductase complexes, one comprising CRT/CNX/ERp57 and the other including BiP/PDI (37), has emerged. This fits well with earlier proposals of a reticular-like matrix in the ER lumen in which different chaperone systems are organized (25,51). In this view, PDI plays a role in the BiP system analogous to that of ERp57 in the CRT/CNX system. However, while the absence of the CRT contribution to the ERp57 system can be functionally compensated for by the presence of CNX, the...

Increased hexosamine biosynthetic pathway flux dedifferentiates INS-1E cells and murine islets by an extracellular signal-regulated kinase (ERK)1/2-mediated signal transmission pathway

“…Instead, prolonged exposure of beta cells to high glucose or glucosamine induces ER stress. Following the stress, ERK is activated through inositol-requiring 1 (IRE1)-dependent mechanisms [27]. This long term ERK activation may inhibit proinsulin transcription by the already described, mainly post-translational mechanisms.…”

The Cholinesterase-like Domain, Essential in Thyroglobulin Trafficking for Thyroid Hormone Synthesis, Is Required for Protein Dimerization

“…At the zero chase time, a series of high molecular weight intermediates termed the "A, B, and C" bands ( Fig. 5A, left) were identified as Tg adducts with resident oxidoreductases of the ER lumen (43). (These adducts appear as a smear when the cells are lysed in the absence of N-ethylmaleimide or a similar alkylating treatment (Fig.…”

“…To examine the relationship of the intersubunit disulfide bond of Tg-CD with its dimerization, we employed sucrose velocity gradient centrifugation. Gradients were loaded from the top and finally collected from the bottom as described previously (43). In the first three gradients shown in Fig.…”

Maturation of Thyroglobulin Protein Region I

“…Tg oxidative maturation includes mixed disulfide complexes called bands A, B, and C (17) that engage ER oxidoreductases, such as Tg-ERp57 and Tg-protein-disulfide isomerase (Tg-PDI) adducts (15). After dissociation from ER oxidoreductases, Tg monomer folding proceeds during its residence in the ER, en route to final formation of roughly 60 intrachain disulfide bonds (16).…”

“…The normal folding process for Tg begins with transient formation of mixed disulfide bonds (14) with endogenous ER oxidoreductases in complexes called "A," "B," and "C," as visualized by nonreducing SDS-PAGE (15). Concomitant with resolution of these complexes, and thereafter, there is extensive intramolecular disulfide bond maturation within internal repeat domains of the molecule (16).…”