cells Review Mechanisms of Disulfide Bond Formation in Nascent Polypeptides Entering the Secretory Pathway Philip J. Robinson and Neil J. Bulleid * Institute of Molecular, Cell and Systems Biology, College of Medical Veterinary and Life Sciences, Davidson Building, University of Glasgow, Glasgow G12 8QQ, UK; [email protected] * Correspondence: [email protected]; Tel.: +44-141-330-3870 Received: 27 July 2020; Accepted: 28 August 2020; Published: 29 August 2020 Abstract: Disulfide bonds are an abundant feature of proteins across all domains of life that are important for structure, stability, and function. In eukaryotic cells, a major site of disulfide bond formation is the endoplasmic reticulum (ER). How cysteines correctly pair during polypeptide folding to form the native disulfide bond pattern is a complex problem that is not fully understood. In this paper, the evidence for different folding mechanisms involved in ER-localised disulfide bond formation is reviewed with emphasis on events that occur during ER entry. Disulfide formation in nascent polypeptides is discussed with focus on (i) its mechanistic relationship with conformational folding, (ii) evidence for its occurrence at the co-translational stage during ER entry, and (iii) the role of protein disulfide isomerase (PDI) family members. This review highlights the complex array of cellular processes that influence disulfide bond formation and identifies key questions that need to be addressed to further understand this fundamental process. Keywords: disulfide formation; protein folding; protein secretion; protein synthesis; PDI; ER 1. Introduction Cell compartmentalisation during the evolution of eukaryotic cells resulted in membrane-bound organelles with specialised functions. The endoplasmic reticulum (ER) is an organelle involved in the sorting and targeting of newly synthesised proteins. It is estimated that approximately 30–40% of genes encode for proteins that are targeted to the ER [1,2], most of which enter co-translationally via the sec translocon. As the entry point to the secretory pathway, the ER lumen is the point at which nascent secretory proteins fold into their native three-dimensional (3D) structures. Approximately 80% of secretory proteins contain disulfide bonds [3], the majority of which form during the folding process. Disulfide bond formation in proteins occurs exclusively between cysteine sidechains via the oxidation of thiol groups (Figure1A). The cytosol is an unfavourable environment for disulfide formation because it contains robust NADPH-dependent reducing pathways to maintain proteins in a reduced form [4]. In contrast, the ER contains distinct enzymatic pathways involving the protein disulfide isomerase (PDI) family of proteins that channel oxidising equivalents to specific substrates [5,6]. Reductive pathways are also present in the ER, which are required to correct non-native disulfide bonds during folding or to reduce disulfide bonds before degradation [7]. Hence a balanced redox poise is required in the ER to enable disulfide formation, reduction, and isomerisation to take place in nascent proteins. Maintenance of this redox balance is considered to be aided by millimolar concentrations of glutathione [8]. This ubiquitous tripeptide molecule, composed of cysteine, glycine, and glutamic acid, can exist in both oxidised (GSSG) and reduced forms (GSH). In the cytosol, the activity of glutathione reductase maintains a high ratio of GSH to GSSG [9]. Glutathione reductase is absent in the ER and, instead, the presence of oxidative pathways lowers the GSH-to-GSSG ratio [10]. The presence of this Cells 2020, 9, 1994; doi:10.3390/cells9091994 www.mdpi.com/journal/cells Cells 2020, 9, x FOR PEER REVIEW 2 of 13 GSH‐to‐GSSG ratio [10]. The presence of this pool of glutathione is likely to contribute to mechanisms that counter oxidative and reductive stress in the ER. The mechanisms of protein folding were mainly studied through in vitro unfolding and refolding experiments using full‐length proteins [11,12]. To extrapolate these findings to an ER setting, we must consider the vectorial nature of protein synthesis, ER translocation, and the specialisedCells 2020, 9, 1994 folding conditions of the ER. In this review, we discuss the mechanisms of disulphide2 of 13 formation during folding in this context. Firstly, we discuss the biophysics of disulphide bonding in relation to folding and how this correlates with proposed folding mechanisms. Then, we highlight howpool these of glutathione mechanisms is likely apply to to contribute nascent polypeptides to mechanisms as they that counteremerge into oxidative the ER and lumen. reductive Finally, stress we in lookthe ER. at how and when ER resident factors interact with nascent polypeptides undergoing folding. Figure 1. Disulfide bond formation and conformational folding. (A) Schematic diagram of a nascent Figurepolypeptide, 1. Disulphide with the bond molecular formation structure and conformational of cysteine sidechains folding. (A highlighted) Schematic diagram before (i )of and a nascent after ( ii) polypeptide,disulfide bond with formation. the molecular (B) Mechanistic structure of schemecysteine to sidechains describe foldinghighlighted coupled before disulfide (i) and after formation. (ii) disulphideAt the pre-folding bond formation. stage, the nascent (B) Mechanistic polypeptide scheme exists asto a dynamicdescribe randomfolding coil,coupled in which disulphide disulfide formation.interchange At can the takepre‐folding place to stage, form the transient nascent disulfide polypeptide bonded exists species as a dynamic (TDBS). random If folding coil, follows in which the disulphidefolded precursor interchange mechanism can take (red place arrows), to form then transient formation disulphide of the nascent bonded tertiary species structure (TDBS). If occurs folding first followsand promotes the folded native precursor disulfide mechanism formation. (red Ifarrows), folding then follows formation the quasi-stochastic of the nascent tertiary mechanism structure (blue occursarrows), first then and disulfide promotes formation native occurs disulphide first and formation. drives the If formation folding follows of the native the quasi tertiary‐stochastic structure. mechanismAt the post-folding (blue arrows), stage, the then native disulphide fold is complete,formation andoccurs disulfide first and bonds drives are buriedthe formation and protected. of the native tertiary structure. At the post‐folding stage, the native fold is complete, and disulphide bonds areThe buried mechanisms and protected. of protein folding were mainly studied through in vitro unfolding and refolding experiments using full-length proteins [11,12]. To extrapolate these findings to an ER setting, we must 2.consider Mechanisms the vectorial of Disulphide nature Bond of protein Formation synthesis, during ER Protein translocation, Folding and the specialised folding conditionsCysteine of theresidues ER. In in this proteins review, are we frequently discuss the found mechanisms in the interior of disulfide of the protein formation fold, during where folding they arein this sterically context. hindered Firstly, wefrom discuss undergoing the biophysics disulphide of disulfidebond formation, bonding reduction, in relation or to rearrangement. folding and how Thisthis correlatesconstraint with means proposed that disulphide folding mechanisms. formation is more Then, likely we highlight to occur howat earlier these stages mechanisms in the folding apply to process.nascent polypeptidesThe relationship as they between emerge intofolding the ERand lumen. disulphide Finally, formation we look at was how first and whenexperimentally ER resident demonstratedfactors interact almost with nascent 50 years polypeptides ago by the refolding undergoing of ribonuclease folding. [13]. This experiment showed that refolding from a reduced, denatured precursor results in a single native disulphide pattern, despite over2. Mechanisms 100 disulphide of Disulfide bond configurations Bond Formation being duringpossible. Protein Hence, Folding the correct disulphide bond pattern must Cysteinebe encoded residues in the inamino proteins‐acid are sequence frequently and foundmust form in the during interior the of folding the protein process. fold, Since where then, they manyare sterically in vitro hinderedrefolding experiments from undergoing showed disulfide that disulphide bond formation, bonds are reduction, required to or enable rearrangement. specific proteinsThis constraint to refold means [14]. This that led disulfide to a consensus formation that isdisulphide more likely bonds to form occur first at earlierbefore folding stages inand the subsequentlyfolding process. drive The the relationship formation of between structure folding [15]. Recent and disulfide studies showed formation that was this first is not experimentally always the case,demonstrated and disulphide almost bonding 50 years can ago also by thefollow refolding nascent of structure ribonuclease formation [13]. This [16–18]. experiment showed that refoldingTo understand from a reduced, the mechanisms denatured precursor of disulphide results formation in a single during native disulfidefolding, pattern,it is important despite overto consider100 disulfide the physical bond configurations requirements for being cysteine possible. coupling. Hence, Paired the cysteines correct disulfide are often bond separated pattern in the must amino‐acid sequence and must come into close proximity and in the correct