Oxidative protein folding: from thiol-disulfide exchange reactions to the redox poise of the endoplasmic reticulum

Abstract

This review examines oxidative protein folding within the mammalian endoplasmic reticulum (ER) from an enzymological perspective. In protein disulfide isomerase-first (PDI-first) pathways of oxidative protein folding, PDI is the immediate oxidant of reduced client proteins and then addresses disulfide mispairings in a second isomerization phase. In PDI-second pathways the initial oxidation is PDI-independent. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Strategies and challenges are discussed for determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDI(red):PDI(ox). The preponderance of evidence suggests that the mammalian ER is more reducing than first envisaged. The average redox state of major PDI-family members is largely to almost totally reduced. These observations are consistent with model studies showing that oxidative protein folding proceeds most efficiently at a reducing redox poise consistent with a stoichiometric insertion of disulfides into client proteins. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, this review concludes with an elaboration of a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC-MS-MS methods. Consortia of oxidoreductases that are in redox equilibrium can then be identified and compared to the glutathione redox poise of the ER to gain a more detailed understanding of the factors that influence oxidative protein folding within the secretory compartment.

Keywords: Disulfide exchange; Endoplasmic reticulum; Ero1; Glutathione; Oxidative protein folding; Peroxiredoxin; Protein disulfide isomerase; Quiescin sulfhydryl oxidase; Ratiometric mass spectrometry; Redox potential.

Figure 1. Mechanistic aspects of thiol-disulfide exchange reactions

Panel I illustrates reduction of a CxxC disulfide motif in A_ox by an unstructured reduced peptide dithiol (B_red; blue). Generation of the mixed-disulfide intermediate A-B involves an in-line transition state as depicted in the detail shown in Panel II.

Figure 2

Structure of human PDI. The coordinates for the oxidized protein are from Wang et al. [28]. Redox-active CxxC motifs are found in both a and a′ domains. The N-terminal cysteine sulfur atom of each motif (orange) is solvent accessible and engages in mixed disulfides with redox partners and proteins undergoing disulfide editing. The C-terminal cysteine by contrast is largely buried from solvent (yellow). The b and b′ domains are redox-inactive. Protein clients of PDI can occupy the central cavity with significant hydrophobic interactions with the b′ domain.

Figure 3

An example of a PDI-first pathway in oxidative protein folding. In the first phase the unfolded reduced protein is oxidized by PDI_ox. Here, reduced PDI is regenerated by the FAD-linked sulfhydryl oxidase, Ero1, with the formation of hydrogen peroxide. PDI_red is then involved in correction of mispaired disulfides prior to the iterative emergence of the native protein fold.

Figure 4

Some enzymatic and non-enzymatic oxidants for reduced PDI.

Figure 5

PDI-second pathways of oxidative folding. The initial oxidation of reduced proteins is PDI-independent so that PDI is only engaged in the second phase of oxidative protein folding (Panel A). One facile direct oxidant of reduced conformationally-mobile proteins is Quiescin-sulfhydryl oxidase. Panel B shows the structure of the open form of QSOX from Trypanosoma brucei (3QCP; [52]).

Figure 6

Reduction of PDI by GSH. The model and rate constants of Darby and Creighton [57] were used for the a domain of human PDI reduced with glutathione (Panel A). Panel B shows the time course for oxidized, reduced and mixed disulfide forms of the single CxxC motif using 100 μM PDI and 5 mM GSH [59]. Under these conditions the half-time for equilibration is 0.52 sec; t_1/2 values for 2.5 mM and 10 mM GSH are 1.12 and 0.25 sec respectively.

Figure 7

A potential pathway for redox cycling in the ER.

Figure 8

Electron micrograph of a plasma cell from guinea pig bone marrow showing an extensive reticular network of ER. Reproduced from [103].

Figure 9

The use of NEM to trap intraluminal redox state within the ER. The highlighted equilibrium depicts the reduction of one PDI CxxC motif by GSH. Approximately one proton is released at neutral pH values (see the text).

Figure 10

The rate of oxidative refolding of riboflavin binding protein in the presence of PDI redox buffers of defined concentration. Refolding was followed continuously by the quenching of riboflavin binding on association with folded oxidized RfBP in the absence of small molecule redox species and without other enzymatic catalysts of disulfide bond generation (panel A). Rates of refolding are plotted as a function of the percentage of PDI_red in the PDI redox buffer (panel B). The concentration of reduced RfBP was 1 μM and the aggregate concentration of reduced and oxidized forms comprising the PDI redox buffer was 30-fold higher (see the Text). Data redrawn from Rancy and Thorpe [49].

Figure 11

Schematic diagram of equilibrating and non-equilibrating redox species in the ER. Redoxactive proteins are distinguished by their shapes; the state of their redox active disulfides is shown by the blue and red shading. Some species are envisaged as directly in equilibrium with the glutathione redox pool and would have a range of redox states depending on their standard redox potentials. Other proteins are represented as interacting indirectly with the glutathione pool or with consortia of other proteins insulated from small molecular weight redox buffers.

Figure 12

A suggested protocol for a more global analysis of the redox state of thiol-disulfide oxidoreductases within the Er (see the Text).