Oxidative Protein Folding in Vitro: A Study of the Cooperation between Quiescin-Sulfhydryl Oxidase and Protein Disulfide Isomerase

Abstract

The flavin-dependent Quiescin-sulfhydryl oxidase (QSOX) inserts disulfide bridges into unfolded reduced proteins with the reduction of molecular oxygen to form hydrogen peroxide. This work investigates how QSOX and protein disulfide isomerase (PDI) cooperate in vitro to generate native pairings in two unfolded reduced proteins: ribonuclease A (RNase: 4 disulfide bonds and 105 disulfide isomers of the fully oxidized protein) and avian riboflavin binding protein (RfBP: 9 disulfide bonds and greater than 34 million corresponding disulfide pairings). Experiments combining avian or human QSOX with up to 200 μM of avian or human reduced PDI show that the isomerase is not a significant substrate of QSOX. Both reduced RNase and RfBP can be efficiently refolded in an aerobic solution containing micromolar concentrations of reduced PDI and nanomolar levels of QSOX without any added oxidized PDI or glutathione redox buffer. Refolding of RfBP is followed continuously using the complete quenching of the fluorescence of free riboflavin that occurs on binding to apo-RfBP. The rate of refolding is half-maximal at 30 μM reduced PDI when 1 μM of the reduced client protein is used in the presence of 30 nM QSOX. The use of high concentrations of PDI, in considerable excess over the folding protein client, reflect the concentration prevailing in the lumen of the endoplasmic reticulum and allow the redox poise of these in vitro experiments to be set with oxidized and reduced PDI. In the absence of either QSOX or redox buffer, the fastest refolding of RfBP is accomplished with excess reduced PDI and just enough oxidized PDI to generate 9-disulfides in the protein client. These in vitro experiments are discussed in terms of current models for oxidative folding in the endoplasmic reticulum.Two distinct flavin-linked sulfhydryl oxidase families (Ero1 (1,2) and Quiescin-sulfhydryl oxidase; QSOX 1 (3,4)), have been suggested to drive the generation of disulfide bonds during oxidative protein folding in higher eukaryotes (4-12). For Ero1, a series of pioneering investigations on yeast (13,14), and complementary studies on mammalian cells (15,16), have led to one model for oxidative folding shown in Figure 1A. Here, the immediate oxidant for an unfolded reduced protein client is oxidized protein disulfide isomerase (PDI). Reduced PDI is then reoxidized by the FAD-linked sulfhydryl oxidase Ero1 (13,17,18) with the eventual reduction of dioxygen to hydrogen peroxide (19). PDI intervenes again to catalyze the * Author for correspondence. Phone: (302) 831-2689. Fax: (302) 831-6335. cthorpe@udel.edu.. Figures S1-S4 show sequence comparisons of human and avian PDI; a comparison of the reductase and isomerase activities of PDI; the refolding of RfBP monitored by absorbance; and the effect of riboflavin on RfBP refolding. This material is available free of charge via the Internet at http://pubs.acs.org 1 Abbreviations: cCMP, cytidine 2',3' cyclic monophosphate; DTNB, 5,5′-dithiobis(2-nitrobenzoate); DTT, dithiothreitol; ER, endoplasmic retic...

The Emerging Role of QSOX1 in Cancer

“…Therefore, if the benefit that QSOX1 provides to tumor cells resides in the ECM, there is more QSOX1-related tumor biology to discover. Thankfully, early work on the enzymology was established by Colin Thorpe's group (71) and crystal structures are known for QSOX1, reported by the Fass group (6). If QSOX1 plays an important role in tumor cell invasion and subsequent metastasis, these early studies provide a firm foundation on which to continue studying not FIG.…”

“…As mentioned earlier, QSOX1 is considered as generating disulfide bonds indiscriminately in proteins as they are produced in the ER, while PDI corrects and re-shuffles mispaired disulfides resulting in proper disulfide pairings. While QSOX1 and PDI cooperate to fold a wide range of proteins, PDI is not a substrate of QSOX1 despite the fact that PDI contains multiple CxxC motifs with free thiols in reduced form (71). Neither QSOX1 nor PDI are efficient in folding proteins independent of each other.…”

ER membrane-localized oxidoreductase Ero1 is required for disulfide bond formation in the rice endosperm

“…6), indicating that the alternative electron transfer pathways for disulfide bond formation operate in the subaleurone cells. A possible pathway may involve quiescin-sulfhydryl oxidase, which has been identified in higher eukaryotes, and can catalyze the direct oxidation of a wide range of unfolded proteins without additional partners in vitro (29).…”

Distinct Roles of Protein Disulfide Isomerase and P5 Sulfhydryl Oxidoreductases in Multiple Pathways for Oxidation of Structurally Diverse Storage Proteins in Rice

“…RNase A refolding assays were essentially conducted by the method of Lyles and Gilbert (1991) in buffer (0.1 M Tris·HCl, pH 8.0, 4.5 mM cyclic CMP [cCMP], 2 mM EDTA, 1 mM GSH, 0.2 mM GSSG, 10 mM reduced, denatured RNase A [rRNase], and 1 mM PDIL) at 378C for 13 min for PDIL1;1 and 30 min for PDIL2;3. Kinetic assays and effects of GSSG concentrations on rRNase refolding activities were conducted by following the method of Rancy and Thorpe (2008). Briefly, rRNase was incubated at 288C in buffer (0.1 M Tris·HCl, pH 8.0, 2 mM EDTA, 1 mM GSH, 0.2 mM or otherwise indicated concentrations of GSSG, 10 mM rRNase, and 1 mM PDIL).…”