Folding and Maturation of Tyrosinase-related Protein-1 Are Regulated by the Post-translational Formation of Disulfide Bonds and by N-Glycan Processing

Abstract

In this study we have explored the endoplasmic reticulum associated events accompanying the maturation of the tyrosinase-related protein-1 (TRP-1) nascent chain synthesized in mouse melanoma cells. We show that TRP-1 folding process occurs much more rapidly than for tyrosinase, a highly homologous protein, being completed post-translationally by the formation of critical disulfide bonds. In cells pretreated with dithiothreitol (DTT), unfolded TRP-1 is retained in the endoplasmic reticulum by a prolonged interaction with calnexin and BiP before being targeted for degradation. The TRP-1 chain was able to fold into DTT-resistant conformations both in the presence or absence of ␣-glucosidase inhibitors, but folding occurred through different pathways. During the normal folding pathway, TRP-1 interacts with calnexin. In the presence of ␣-glucosidase inhibitors, the interaction with calnexin is prevented, with TRP-1 folding being assisted by BiP. In this case, the process has similar kinetics to that of untreated TRP-1 and yields a compact form insensitive to DTT as well. However, this form has different thermal denaturation properties than the native conformation. We conclude that disulfide bridge burring is crucial for the TRP-1 export. This suggests that although various folding pathways may complete this process, the native form may be acquired only through the normal unperturbed pathway.

Homocysteine Increases the Expression of Vascular Endothelial Growth Factor by a Mechanism Involving Endoplasmic Reticulum Stress and Transcription Factor ATF4

“…We suggest that the reductive potential of these compounds interferes with thiol oxidation and protein disulfide formation in the ER, thus inhibiting correct protein folding. Such is the case for DTT (46,47). The amino acid L-cysteine had no significant effect on VEGF expression.…”

Tyrp1 and Oculocutaneous Albinism Type 3

“…Tyrp1 in humans (36) and in mice (44) is synthesized as a precursor polypeptide of approximately 55-69 kDa in the endoplasmic reticulum (ER). This nascent polypeptide undergoes rapid post-translation folding via di-sulfide bonds into its initial native conformation assisted by molecular chaperones like calnexin, a lectin chaperone, and BiP, the immunoglobulin heavy chain binding protein (both of which are resident ER chaperones) (45,46). Formation of disulfide bonds during the folding of Tyrp1 is crucial for its transport through both secretory pathways in the ER and its subsequent processing/maturation in the Golgi apparatus.…”

Conserved Intramolecular Disulfide Bond Is Critical to Trafficking and Fate of ATP-binding Cassette (ABC) Transporters ABCB6 and Sulfonylurea Receptor 1 (SUR1)/ABCC8

“…Typically, disulfide bonds are maintained by the oxidizing environment of the ER and protein-disulfide isomerases. If the necessary disulfide bonds are not formed, the proteins might be retained in the ER and then degraded (7). Among the ABC transporters, it was discovered that ABCG2 contains an intramolecular disulfide bond, which is critical for protein stability (8).…”