Hiram F. Gilbert

Thiol/disulfide exchange equilibria and disulfide bond stability

Publisher Summary Disulfide-bond formation is a versatile oxidation used biologically in diverse processes, such as enzyme catalysis, protection against oxidative damage, stabilization of extracellular proteins, and regulation of biological activity. Because disulfide formation is a reversible process, disulfide-bond stability often plays an important role in the biological utility of disulfide bonds. In turn, the ability to form and break a specific disulfide bond under appropriate biological conditions depends on the nature of the oxidant or reductant, the disulfide stability, the kinetics of the forward and reverse reactions, and the nature and redox state of the environment in which the reaction occurs. The stability of disulfide bonds in small molecules and proteins spans an enormous range, a factor of approximately 1011, corresponding to a free-energy difference of about 15 kcal/mol or a redox potential difference of 0.33 V. This chapter outlines the importance of reversible thiol/disulfide exchange and discusses some practical considerations in measuring disulfide bond stability.

PROTEIN DISULFIDE ISOMERASE AND ASSISTED PROTEIN FOLDING

The folding protein (and protein folder) is beset with a number of problems in translating the simple instructions encoded by DNA into the complex, three-dimensional structure of a correctly folded protein. Nature has solved these problems in an adequate, if not elegant, fashion, but we are just beginning to understand the variety of strategies that cells use to ensure efficient protein folding. This minireview will focus on several of the more general principles of assisted folding using the folding catalyst, protein disulfide isomerase (PDI), as an example. This remarkable resident of the endoplasmic reticulum (ER) inserts disulfides into folding proteins and provides a mechanism to correct errors in disulfide pairing when they occur. At high concentrations, it functions as an ATP-independent chaperone that inhibits aggregation; yet, at lower concentrations, it can also participate in an unusual interaction with substrate that leads to PDI-facilitated aggregation (anti-chaperone behavior).

Redox control of enzyme activities by thiol/disulfide exchange

Publisher Summary The process of thiol/disulfide exchange provides a mechanism for the equilibration of the sulfhydryl oxidation state of proteins with the thiol/ disulfide status of the surrounding environment. If this process occurs in vivo and results in changes in the activities of certain enzymes, changes in enzyme activity could be coupled to changes in the redox potential of the cell. This chapter examines the possibility of metabolic regulation by thiol/disulfide exchange with respect to each of the six criteria: (1) the intracellular thiol/disulfide status should vary in vivo in response to some metabolic signal; (2) the oxidation of protein sulfhydryl groups by thiol/disulfide exchange should activate some enzymes, inactivate others, and not affect the activities of others; (3) the thiol/disulfide redox potential of a regulated protein should be near the observed thiol/disulfide ratio in vivo ; (4) thiol/disulfide exchange reactions must be kinetically competent under physiological conditions; (5) for regulated enzymes, the oxidized and reduced forms of the enzyme should both be observable in vivo ; and (6) the response of particular enzyme activities to oxidation by thiol/ disulfide exchange should be consistent with the metabolic function of the enzyme.

In Vivo and in Vitro Function of theEscherichia coli Periplasmic Cysteine Oxidoreductase DsbG

We have characterized in vivo andin vitro the recently identified DsbG fromEscherichia coli. In addition to sharing sequence homology with the thiol disulfide exchange protein DsbC, DsbG likewise was shown to form a stable periplasmic dimer, and it displays an equilibrium constant with glutathione comparable with DsbA and DsbC. DsbG was found to be expressed at approximately 25% the level of DsbC. In contrast to earlier results (Andersen, C. L., Matthey-Dupraz, A., Missiakas, D., and Raina, S. (1997) Mol. Microbiol. 26, 121–132), we showed that dsbG is not essential for growth and thatdsbG null mutants display no defect in folding of multiple disulfide-containing heterologous proteins. Overexpression of DsbG, however, was able to restore the ability ofdsbC mutants to express heterologous multidisulfide proteins, namely bovine pancreatic trypsin inhibitor, a protein with three disulfides, and to a lesser extent, mouse urokinase (12 disulfides). As in DsbC, the putative active site thiols in DsbG are completely reduced in vivo in adsbD-dependent fashion, as would be expected if DsbG is acting as a disulfide isomerase or reductase. However, the latter is not likely because DsbG could not catalyze insulin reductionin vitro. Overall, our results indicate that DsbG functions primarily as a periplasmic disulfide isomerase with a narrower substrate specificity than DsbC.

Reduction-Reoxidation Cycles Contribute to Catalysis of disulfide Isomerization by Protein-disulfide Isomerase

Protein-disulfide isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding. This process can be error-prone in its early stages, and any incorrect disulfides that form must be rearranged to their native configuration. When the second cysteine (CGHC) in the PDI active site is mutated to Ser, the isomerase activity drops by 7–8-fold, and a covalent intermediate with the substrate accumulates. This led to the proposal that the second active site cysteine provides an escape mechanism, preventing PDI from becoming trapped with substrates that isomerize slowly (Walker, K. W., and Gilbert, H. F. (1997) J. Biol. Chem. 272, 8845–8848). Escape also reduces the substrate, and if it is invoked frequently, disulfide isomerization will involve cycles of reduction and reoxidation in preference to intramolecular isomerization of the PDI-bound substrate. Using a gel-shift assay that adds a polyethylene glycol-conjugated maleimide of 5 kDa for each sulfhydryl group, we find that PDI reduction and oxidation are kinetically competent and essential for isomerization. Oxidants inhibit isomerization and oxidize PDI when a redox buffer is not present to maintain the PDI redox state. Reductants also inhibit isomerization as they deplete oxidized PDI. These rapid cycles of PDI oxidation and reduction suggest that PDI catalyzes isomerization by trial and error, reducing disulfides and oxidizing them in a different configuration. Disulfide reduction-reoxidation may set up critical folding intermediates for intramolecular isomerization, or it may serve as the only isomerization mechanism. In the absence of a redox buffer, these steady-state reduction-oxidation cycles can balance the redox state of PDI and support effective catalysis of disulfide isomerization.

Sulfhydryl Oxidase from Egg White A FACILE CATALYST FOR DISULFIDE BOND FORMATION IN PROTEINS AND PEPTIDES

Both metalloprotein and flavin-linked sulfhydryl oxidases catalyze the oxidation of thiols to disulfides with the reduction of oxygen to hydrogen peroxide. Despite earlier suggestions for a role in protein disulfide bond formation, these enzymes have received comparatively little general attention. Chicken egg white sulfhydryl oxidase utilizes an internal redox-active cystine bridge and a FAD moiety in the oxidation of a range of small molecular weight thiols such as glutathione, cysteine, and dithiothreitol. The oxidase is shown here to exhibit a high catalytic activity toward a range of reduced peptides and proteins including insulin A and B chains, lysozyme, ovalbumin, riboflavin-binding protein, and RNase. Catalytic efficiencies are up to 100-fold higher than for reduced glutathione, with typical K m values of about 110–330 μm/protein thiol, compared with 20 mm for glutathione. RNase activity is not significantly recovered when the cysteine residues are rapidly oxidized by sulfhydryl oxidase, but activity is efficiently restored when protein disulfide isomerase is also present. Sulfhydryl oxidase can also oxidize reduced protein disulfide isomerase directly. These data show that sulfhydryl oxidase and protein disulfide isomerase can cooperate in vitro in the generation and rearrangement of native disulfide pairings. A possible role for the oxidase in the protein secretory pathway in vivo is discussed.

Hormone Binding by Protein Disulfide Isomerase, a High Capacity Hormone Reservoir of the Endoplasmic Reticulum

Protein disulfide isomerase (PDI) is a folding assistant of the eukaryotic endoplasmic reticulum, but it also binds the hormones, estradiol, and 3,3′,5-triiodo-l-thyronine (T3). Hormone binding could be at discrete hormone binding sites, or it could be a nonphysiological consequence of binding site(s) that are involved in the interaction PDI with its peptide and protein substrates. Equilibrium dialysis, fluorescent hydrophobic probe binding (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS)), competition binding, and enzyme activity assays reveal that the hormone binding sites are distinct from the peptide/protein binding sites. PDI has one estradiol binding site with modest affinity (2.1 ± 0.5 μm). There are two binding sites with comparable affinity for T3 (4.3 ± 1.4 μm). One of these overlaps the estradiol site, whereas the other binds the hydrophobic probe, bis-ANS. Neither estradiol nor T3 inhibit the catalytic or chaperone activity of PDI. Although the affinity of PDI for the hormones estradiol and T3 is modest, the high local concentration of PDI in the endoplasmic reticulum (>200 μm) would drive hormone binding and result in the association of a substantial fraction (>90%) of the hormones in the cell with PDI. High capacity, low affinity hormone sites may function to buffer hormone concentration in the cell and allow tight, specific binding to the true receptor while preserving a reasonable number of hormone molecules in the very small volume of the cellular environment.

Facilitated Protein Aggregation EFFECTS OF CALCIUM ON THE CHAPERONE AND ANTI-CHAPERONE ACTIVITY OF PROTEIN DISULFIDE-ISOMERASE

Protein disulfide-isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding in the eukaryotic endoplasmic reticulum. At high concentrations, it also serves as a chaperone and inhibits aggregation. However, at lower concentrations, PDI can display the unusual ability to facilitate aggregation, termed anti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771). Under reducing conditions (10 mM dithiothreitol) and at a low concentration (0.1-0.3 μM) relative to the unfolded protein substrate, PDI facilitates aggregation of alcohol dehydrogenase (11 μM) that has been denatured thermally or chemically. But at higher concentrations (>0.8 μM), PDI inhibits aggregation under the same conditions. With denatured citrate synthase, PDI does not facilitate aggregation, but higher concentrations do inhibit aggregation. Anti-chaperone behavior is associated with the appearance of both PDI and substrate proteins in insoluble complexes, while chaperone behavior results in the formation of large (>500 kDa) but soluble complexes that contain both proteins. Physiological concentrations of calcium and magnesium specifically increase the apparent rate of PDI-dependent aggregation and shift the chaperone activity to higher PDI concentrations. However, calcium has no effect on the Km or Vmax for PDI-catalyzed oxidative folding, suggesting that the interactions that lead to chaperone/anti-chaperone behavior are distinct from those required for catalytic activity. To account for this unusual behavior of a folding catalyst, a model with analogy to classic immunoprecipitation is proposed; multivalent interactions between PDI and a partially aggregated protein stimulate further aggregate formation by noncovalently cross-linking smaller aggregates. However, at high ratios of PDI to substrate, cross-linking may be inhibited by saturation of the sites with PDI. The effects of PDI concentration on substrate aggregation and the modulation of the behavior by physiological levels of calcium may have implications for the involvement of PDI in protein folding, aggregation, and retention in the endoplasmic reticulum.

Detection of oxidized and reduced glutathione with a recycling postcolumn reaction

A rapid, sensitive, and selective method for the quantitation of both oxidized (GSSG) and reduced (GSH) glutathione in biological materials is described. Oxidized and reduced glutathione are resolved by anion-exchange high-performance liquid chromatography and detected with an in-line, recycling postcolumn reaction. The recycling reaction specifically amplifies the response to oxidized and reduced glutathione 20-100 times over that obtained with a stoichiometric reaction, permitting the detection of 2 pmol glutathione. Oxidized and reduced glutathione levels were measured in rat liver and in dog heart mitochondria. Special precautions are necessary to avoid artifacts which lead to either underestimation or overestimation of GSSG levels. GSH/GSSG ratios of approximately 100-300 were observed in samples prepared from rapidly frozen rat liver. Somewhat higher GSH/GSSG ratios were observed in isolated dog heart mitochondria.

The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum

In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 ± 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization.