Disulfide bonds in ER protein folding and homeostasis

Abstract

Proteins that are expressed outside the cell must be synthesized, folded, and assembled in a way that ensures they can function in their designate location. Accordingly, these proteins are primarily synthesized in the endoplasmic reticulum (ER), which has developed a chemical environment more similar to that outside the cell. This organelle is equipped with a variety of molecular chaperones and folding enzymes that both assist the folding process, while at the same time exerting tight quality control measures that are largely absent outside the cell. A major post-translational modification of ER-synthesized proteins is disulfide bridge formation, which is catalyzed by the family of protein disulfide isomerases. As this covalent modification provides unique structural advantages to extracellular proteins, multiple pathways to disulfide bond formation have evolved. However, the advantages that disulfide bonds impart to these proteins come at a high cost to the cell. Very recent reports have shed light on how the cell can deal with or even exploit the side reactions of disulfide bond formation to maintain homeostasis of the ER and its folding machinery.

Figure 1. Folding reactions occurring in the ER and features recognized by ER quality control components

Folding and oligomerization take place in the ER and features that occur on non-native structures are identified by various chaperones and folding enzymes, which serve to prevent incompletely folded or assembled proteins from moving further along the secretory pathway. (A) Exposed hydrophobic regions (yellow) that will eventually be buried upon folding or oligomerization are often recognized by the Hsp70 chaperone BiP (red). (B) The lectins calreticulin (blue) and calnexin bind to sugar moieties possessing one terminal glucose residue (grey hexagon), which can be found on incompletely folded glycoproteins. (C) PDIs (green) form mixed disulfide bonds with free thiol groups to catalyze disulfide bond formation, reduction, or isomerization.

Figure 2. Different pathways to disulfide bridge formation in the ER

(A) For some proteins, correct disulfide bonds can form co-translationally due to independent domain-like structures in the protein. (B) In other cases, the ER chaperone machinery, for simplicity only BiP (red) and PDI (green) are shown, inhibits premature folding and disulfide bridge formation of a protein with disulfide bonds that have to form between non-adjacent cysteines. Once the complete polypeptide chain has emerged from the translocon and is released from the chaperones, folding and disulfide bridge formation can proceed. (C) If non-native disulfide bonds between adjacent cysteines form co-translationally, these must be isomerized by a PDI (green) to allow the correct pair to form. Once folded, proteins can proceed to the Golgi and further along the secretory pathway (A–C). (D) In some cases, incorrect disulfide bonds are formed and terminal misfolding occurs, which renders the protein a substrate for ERAD. Retrotranslocation and proteolysis by the proteasome (blue) is likely preceded by reduction of the wrong disulfide bonds (reductase shown in light green) or even native ones in correctly folded regions of the protein [91].

Figure 3. An overview over the oxidative folding and quality control mechanisms in the ER

Proteins enter the ER co-translationally as unfolded polypeptide chains. (A) If they are non-glycosylated or the first N-linked glycan occurs fairly far into the sequence, they can bind BiP (red) along with PDI (green oval). Further components like the ERdj proteins are omitted for simplicity. (B) If the substrate is glycosylated, they interact with the calreticulin/calnexin system. Calreticulin (dark blue) interacts with the PDI family member ERp57 (dark green hexagon), which contributes to the oxidative folding of glycoproteins. If folding is successful, the proteins can leave the calreticulin/calnexin cycle, if not, UGT1 (light blue), which can cooperate with Sep15, another member of the PDI family (light green circle), re-glucosylate the substrate and reenter the protein into the calreticulin/calnexin cycle. (C) In both cases, once folding is nearly complete, any free thiols remaining can covalently engage ERp44 (yellow circle), which inhibits premature transport to the Golgi until these thiols are buried or part of a disulfide bond. (D) PDIs can be oxidized by Ero1 (dark brown), which generates H₂O₂. This can be used indirectly to oxidize Prx4 (one dimer of its decameric structure is shown in light brown), which in turn oxidizes PDI (green) generating two disulfide bonds per molecule of O₂. PDIs are further coupled to the GSH/GSSG system, which could regulate Ero1 activity or even oxidize substrates that do not require other PDI-associated functions.