Active Sites Of Protein Disulfide Isomerase Research Articles

Kinetic-based trapping by intervening sequence variants of the active sites of protein-disulfide isomerase identifies platelet protein substrates

Thiol isomerases such as protein-disulfide isomerase (PDI) direct disulfide rearrangements required for proper folding of nascent proteins synthesized in the endoplasmic reticulum. Identifying PDI substrates is challenging because PDI catalyzes conformational changes that cannot be easily monitored (e.g. compared with proteolytic cleavage or amino acid phosphorylation); PDI has multiple substrates; and it can catalyze either oxidation, reduction, or isomerization of substrates. Kinetic-based substrate trapping wherein the active site motif CGHC is modified to CGHA to stabilize a PDI-substrate intermediate is effective in identifying some substrates. A limitation of this approach, however, is that it captures only substrates that are reduced by PDI, whereas many substrates are oxidized by PDI. By manipulating the highly conserved -GH- residues in the CGHC active site of PDI, we created PDI variants with a slowed reaction rate toward substrates. The prolonged intermediate state allowed us to identify protein substrates that have biased affinities for either oxidation or reduction by PDI. Because extracellular PDI is critical for thrombus formation but its extracellular substrates are not known, we evaluated the ability of these bidirectional trapping PDI variants to trap proteins released from platelets and on the platelet surface. Trapped proteins were identified by mass spectroscopy. Of the trapped substrate proteins identified by mass spectroscopy, five proteins, cathepsin G, glutaredoxin-1, thioredoxin, GP1b, and fibrinogen, showed a bias for oxidation, whereas annexin V, heparanase, ERp57, kallekrein-14, serpin B6, tetranectin, and collagen VI showed a bias for reduction. These bidirectional trapping variants will enable more comprehensive identification of thiol isomerase substrates and better elucidation of their cellular functions.

134 - Nitroarachidonic Acid (NO2AA) Inhibits Protein Disulfide Isomerase (PDI) Through Reversible Covalent Adduct Formation with Critical Cysteine Residues

Nitroarachidonic acid (NO 2 AA) is a nitroalkene exhibiting pleiotropic anti-inflammatory actions in many cell types. We have recently shown that NO 2 AA inhibits phagocytic NADPH oxidase 2 (NOX2) by preventing formation of the active complex in the membrane, without affecting the phosphorylation of cytosolic subunits. Recent work indicates the participation of Protein disulfide isomerase (PDI) activity on NOX2 active complex formation. Thus, the nitroalkene could affect PDI activity which might help to explain our previous observations on NOX2 inhibition. We determined that both the reductase and chaperone activities of PDI were inhibited by NO 2 AA in a dose and time- dependent manner, being a reversible effect. The mechanism of inhibition did not involve the release of nitric oxide ( ● NO). Since nitroalkenes are potent electrophiles and PDI has critical cysteine residues for its activity, the formation of a reversible covalent adduct between NO 2 AA and PDI may occur. We demonstrated the reversible covalent modification of PDI by NO 2 AA using the modified PEG switch assay as well as LC-MS/MS. Trypsinization of modified PDI confirmed that Cys residues present in the active site of PDI were key targets accounting for nitroalkene modification. Future work will be directed to determine if these modifications participates in the reported NO 2 AA modulation of NOX2.

The Second CGHC Motif of Protein Disulfide Isomerase Mediates Thrombosis

Protein disulfide isomerase (PDI) catalyzes thiol-disulfide exchange reactions that are critical for protein function. PDI has two distinct CGHC redox-active sites, but nothing is known about which site is important in physiologic reactions including thrombosis. To study the role of PDI and its active sites in thrombosis we used two genetically-modified models, Mx1-Cre/PDIfl/fl mice with blood cells and vessel wall cells lacking PDI, and transgenic PDI(ss-oo) mice with PDI lacking the second CGHC active site. Using laser-induced cremaster arteriole injury fibrin deposition was decreased in both mouse models. Both mouse models showed attenuation of platelet accumulation using laser arterial injury and FeCl3-induced mesenteric artery injury. The defects in fibrin deposition and platelet accumulation were rescued by infusion of recombinant PDI(oo-ss) containing only a functional second active site, confirming the second active site of PDI was critical for both fibrin formation and platelet accumulation. In vitro studies showed that platelet aggregation depended on the second CGHC motif of PDI, and that binding of PDI protein to platelets depended on the presence of the αIIbβ3 integrin. Studies on platelet secretion revealed that P-selectin expression and ATP release were dependent on a functional second active site of PDI. Treatment of the platelets with eptifibatide did not affect P-selectin expression. Together these findings suggest the second active of PDI catalyzes a reaction in αIIbβ3 supporting platelet aggregation and a reaction in a non-αIIbβ3 substrate supporting secretion. In vivo studies on coagulation showed that PDI infusion rescued fibrin formation in eptifibatide-treated mice in which platelet accumulation was completely blocked, suggesting that PDI directly contributes to activation of coagulation. In summary, our results indicate that the second CGHC active site of PDI has a role in platelet function and coagulation. Targeting this site may have a dual inhibitory effect on thrombosis by decreasing both platelet and fibrin accumulation. DisclosuresNo relevant conflicts of interest to declare.

Alkylation of protein disulfide isomerase by the episulfonium ion derived from the glutathione conjugate of 1,2-dichloroethane and mass spectrometric characterization of the adducts

The reactivity of the episulfonium ion derived from S-(2-chloroethyl)glutathione (CEG), the glutathione conjugate of 1,2-dichloroethane, with the catalytic sites of protein disulfide isomerase (PDI) was investigated. The two cysteine residues of the two active sites of PDI are expected to be the major targets of alkylation. PDI was incubated with equimolar to 100-fold excess CEG. The activity of PDI was irreversibly inhibited with a concurrent loss of two thiols; however, PDI oxidative refolding activity was not completely inhibited. With mass spectrometry, sequencing PDI identified one alkylation event on each of the N-terminal cysteine residues in the two active site peptides. PDI appears robust and able to maintain some activity by steric constraint. We have established that the episulfonium ion of CEG can adduct PDI and may have important toxicologic significance for 1,2-dichloroethane toxicity.

Protein disulfide isomerase, a multifunctional protein chaperone, shows copper-binding activity

Protein disulfide isomerase (PDI) is a 55 kDa multifunctional protein of the endoplasmic reticulum (ER) involved in protein folding and isomerization. In addition to the chaperone and catalytic functions, PDI is a major calcium-binding protein of the ER. Although the active site of PDI has a similar motif CXX C to the Cu-binding motif in Wilson and Menkes proteins and in other copper chaperones, there has been no report on any metal-binding capability of PDI other than calcium binding. We present evidence that PDI is a copper-binding protein. In the absence of reducing agent freshly reduced PDI can bind a maximum of 4 mol of Cu(II) and convert to Cu(I). These bound Cu(I) are surface exposed as they can be competed readily by BCS reagent, a Cu(I) specific chelator. However, when the binding is performed using the mixture of Cu(II) and 1 mM DTT, the total number of Cu(I) bound increases to 10 mol/mol, and it is slower to react with BCS, indicating a more protected environment. In both cases, the copper-bound forms of PDI exist as tetramers while apo-protein is a monomer. These findings suggest that PDI plays a role in intracellular copper disposition.

Studies on the function of yeast protein disulfide isomerase in renaturation of proteins

Renaturation of two enzymes lacking disulfide bonds, citrate synthase (CS), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and another protein containing disulfide bonds, lysozyme (LZM), were studied in order to dissect the possible chaperone function from the isomerase function of yeast protein disulfide isomerase (PDI). Our findings suggest no independent chaperone activity of yeast PDI with respect to the two enzymes lacking disulfide bonds, GAPDH and CS, since neither of these enzymes required PDI for renaturation. In contrast, a high level of renaturation of LZM was observed in the presence of PDI. Renaturation of LZM involved formation and rearrangement of disulfide bonds. Additional studies using LZM as a substrate were done to examine the role of cysteine residues in the two active sites of PDI. Studies with a series of cysteine to serine mutants and truncation mutants of yeast PDI revealed that the two active sites of PDI were not equal in activity. An intramolecular disulfide bond in at least one active site of PDI was required for the oxidation of reduced LZM. The first cysteine in each active site was necessary for disulfide bond rearrangement, i.e., isomerization, in LZM, while the second cysteine was not.

Mutagenic studies on human protein disulfide isomerase by complementation of Escherichia coli dsbA and dsbC mutants

Protein disulfide isomerase (PDI) exhibits both an oxido-reductase and an isomerase activity on proteins containing cysteine residues. These activities arise from two active sites, both of which contain pairs of redox active cysteines. We have developed two simple in vivo assays for these activities of PDI, based on the demonstration that PDI can complement both a dsbA mutation and a dsbC mutation when expressed to the periplasm of Escherichia coli. We constructed a variety of mutants in and around the active sites of PDI and analysed them using these complementation assays. Our analysis showed that the active site amino acid residues have a major role in determining the activities exhibited by PDI, particularly the N-terminal cysteine of the N-terminal active site. The roles of the histidine residue at position 38 and the glutamic acid residue at position 30 were also studied using these assays. The results show that these two in vivo assays should be useful for rapid screening of mutants in PDI prior to purification and detailed biochemical analysis.

Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell

We investigated the effect of protein disulfide isomerase (PDI) on in vivo protein folding of human lysozyme (h-LZM) in a specially constructed yeast coexpression system. Coexpression with PDI increased the amounts of intracellular h-LZM with the native conformation, leading to an increase in h-LZM secretion. The results indicated that PDI is a real catalyst of protein folding in the cell. The secretion of h-LZM increased even when both active sites of PDI were disrupted, suggesting that the effect of PDI resulted from a function other than the formation of disulfide bonds. This is the first finding that PDI without isomerase activity accelerates protein folding in vivo.

The Relative Protein Disulphide Isomerase (PDI) Activities of Gonadotropins, Thioredoxin and PDI

The three-dimensional configuration assumed by a polypeptide chain occurs spontaneously, with or without the participation of molecular chaperones, and is due to the combined interactions of the amino acids in the chain. In the case of disulphide-bond containing proteins, one or more enzymes catalyse the random cleavage and correct reformation of nascent protein disulphide bonds, e.g. protein disulfide-isomerase (PDI). PDI was first discovered in the ninteen sixties and was purified from bovine liver by Anfinsen and coworkers. The PDI active site contains reducible disulphide groups that participate in the reactivation of denatured protein. Generally, its activity is measured with respect to the catalysis of correct disulphide formation, and therefore reactivation, of reduced RNAase. Alternatively oxidized denatured (‘scrambled’) RNAase can be used as substrate and disulphide isomerization leading to reactivation measured. That thioredoxin (TRX), a ubiquitous, monomeric, multifunctional protein may have a role analogous to that of PDI was first suggested because its active site includes an amino acid sequence (Trp-Cys-Gly-Pro-Cys) similar to that in the active site of PDI (Trp-Cys-Gly-His-Cys). These vicinyl Cys residues of TRX and PDI can undergo reversible oxidation to form disulphide bonds. Recently Boniface and Reichert [ l ] pointed out that the Bsubunits of gonadotropic hormones also contain similar vicinyl Cys residues. Bovine, ovine or porcine LH-8 have the sequence His-Cys-Gly-Pro-Cys at positions 89-93 and bovine, ovine, and porcine FSH-8 have a similar sequence (His-Cys-Gly-Lys-Cys) in the same position. They found experimentally that purified ovine FSH and bovine LH preparations were about 60 and 300 times as active as thioredoxin, respectively. We used the assay of Pigiet et al. [2] with reduced, denatured RNAase as substrate to compare the activities of gonadotropins @LH and pFSH), PDI and thioredoxin.

Involvement of two sulfur atoms of protein disulfide isomerase and one sulfur atom of the DsbA/PpfA protein in the oxidation of mutant human lysozyme.

Protein disulfide isomerase (PDI) and the DsbA/PpfA protein catalyze the oxidation of mutant human lysozyme, L79CC81A, which has two native disulfide bonds, Cys6-Cys128 and Cys30-Cys116, a non-native Cys79-Cys95, and 2 free cysteine residues at positions 65 and 77. Oxidation of L79CC81A (R-form) yielded two isomers, L79CC81A-a (A-form) with tandem-linked Cys65-Cys77 and Cys79-Cys95, and L79CC81A-b (B-form) with cross-linked Cys65-Cys79 and Cys77-Cys95 (Kanaya, E., Ishihara, K., Tsunasawa, S., Nokihara, K., and Kikuchi, M. (1993) Biochem. J. 292, 469-476). PDI mainly enhanced the formation of the A- form in the absence of oxidized glutathione (GSSG); however, as the concentration of GSSG increased, it markedly accelerated the formation of the B-form. In contrast, the DspA/PpfA protein mainly enhanced the formation of the A-form, regardless of the presence or absence of GSSG. These results and the presumed spatial locations of Cys65, Cys77, and Cys79-Cys95 in the R-form suggest that 1 of the half-cystine residues in the active site of PDI and the DsbA/PpfA protein can react with 1 of the 2 free Cys residues of the R-form. The dependence on GSSG of the B-form formation with PDI can be explained by the formation of two transient intermolecular disulfide bonds between PDI and the R-form and the attack of GSSG by the resultant thiolate anion of Cys79 or Cys95. The independence of the reaction with the DsbA/PpfA protein from GSSG can be explained by the formation of one transient intermolecular disulfide bond. The possible formation of the two transient intermolecular disulfide bonds involving two sulfur atoms of PDI and 2 cysteine or half-cystine residues of the substrate could explain the high isomerase activity of PDI.

Mimicking the active site of protein disulfide-isomerase by substitution of proline 34 in Escherichia coli thioredoxin

To mimic the active sites (Trp-Cys-Gly-His-Cys) contained in two thioredoxin-like domains of the eukaryotic enzyme protein disulfide-isomerase (PDI, EC 5.3.4.1), the Pro-34 residue of Escherichia coli thioredoxin (Trx) was replaced by His using site-directed mutagenesis. The mutant P34H Trx was isolated in high yield and was stable. The equilibrium between Trx and NADPH in the thioredoxin reductase (TR)-catalyzed reaction revealed that the redox potential (E'o) or P34H Trx at pH 7.0 was -235 mV as compared with -270 mV for wild type (wt) Trx. The higher E'o value made P34H Trx more similar to PDI and contributed to prominent changes in Trx functions, e.g. improved activity with TR and slower reduction of protein disulfides. Compared to wt Trx, the P34H oxidized Trx was about twice as good a substrate for TR from E. coli and four times as efficient with calf thymus TR. A novel fluorimetric assay permitted direct recording of the reaction between insulin disulfide(s) and reduced Trx. At pH 8 and 15 degrees C, second-order rate constants for wt Trx of 2 x 10(4) M-1 s-1 and for P34H Trx of 3 x 10(3) M-1 s-1 were obtained, and a different equilibrium was observed consistent with differences in E'o values. Also when the reduction mechanism of insulin was examined using NADPH and TR, P34H Trx behaved differently from wt Trx or PDI. P34H Trx may be useful as an analogue of PDI for disulfide formation in vivo and in vitro.