Protein Disulfide Isomerase Binding Research Articles

An αIIbβ3 monoclonal antibody traps a semiextended conformation and allosterically inhibits large ligand binding

AbstractMonoclonal antibodies (mAbs) have provided valuable information regarding the structure and function of platelet αIIbβ3. Protein disulfide isomerase (PDI) has been implicated in αIIbβ3 activation and binds to thrombin-activated αIIbβ3. Using human platelets as the immunogen, we identified a new mAb (R21D10) that inhibits the binding of PDI to platelets activated with thrombin receptor–activating peptide (T6). R21D10 also partially inhibited T6-induced fibrinogen and PAC-1 binding to platelets, as well as T6- and adenosine 5'-diphosphate–induced platelet aggregation. Mutual competition experiments showed that R21D10 does not inhibit the binding of mAbs 10E5 (anti-αIIb cap domain) or 7E3 (anti-β3 β-I domain), and immunoblot studies indicated that R21D10 binds to β3. The dissociation of αIIbβ3 by EDTA had a minimal effect on R21D10 binding. Cryogenic electron microscopy of the αIIbβ3-R21D10 Fab complex revealed that R21D10 binds to the β3 integrin-epidermal growth factor 1 (I-EGF1) domain and traps an intermediate conformation of αIIbβ3 with semiextended leg domains. The binding of R21D10 produces a major structural change in the β3 I-EGF2 domain associated with a new interaction between the β3 I-EGF2 and αIIb thigh domains, which may prevent the swing-out motion of the β3 hybrid domain required for high-affinity ligand binding and protect αIIbβ3 from EDTA-induced dissociation. R21D10 partially reversed the ligand binding priming effect of eptifibatide, suggesting that it could convert the swung-out conformation into a semiextended conformation. We concluded that R21D10 inhibits ligand binding to αIIbβ3 via a unique allosteric mechanism, which may or may not be related to its inhibition of PDI binding.

Studies of Protein Disulfide Isomerase (PDI) Binding to Platelets and Production of a Murine Monoclonal Antibody (mAb) to Integrin αIIbβ3 That Inhibits PDI Binding and Platelet Aggregation

L.W. and J.W. contributed equally Introduction: The platelet integrin αIIbβ3 plays a pivotal role in both hemostasis and thrombosis, and is an established target for antiplatelet therapy. Upon activation, αIIbβ3 undergoes a conformational change from a low-affinity, bent-closed conformation to a high-affinity, extended-open conformation, resulting in the binding of fibrinogen and von Willebrand factor and platelet aggregation. αIIbβ3 activation is regulated by protein disulfide isomerase (PDI), an enzyme that catalyzes disulfide bond formation, reduction, and isomerization. One of us (LW) previously showed that PDI binds to thrombin-activated platelets, but the αIIb or β3 conformation(s) and domain(s) to which PDI binds is unknown. Methods: PDI was produced in E. coli and purified and labeled with Alexa-488. Human platelets and Alexa-488-PDI were stimulated with a variety of agonists either in the absence or presence of unlabeled PDI (background control) and then analyzed by flow cytometry. For monoclonal antibody (mAb) production, BALB/cAnN mice were immunized with human platelets and their spleen cells isolated and fused with a non-secretory myeloma cell line. Hybridomas were isolated by colony picking in semi-solid media and culture supernatants were assayed for their ability to inhibit PDI binding to platelets activated with the PAR1 thrombin receptor activating peptide SFLLRN (T6). One mAb that inhibited PDI binding (21D10) was assessed for its effects on platelet aggregation. The epitope of 21D10 on αIIbβ3 was explored by comparing the binding properties of 21D10 to that of previously characterized anti-αIIbβ3 mAbs. Using cryo-electron microscopy (cryo-EM) single particle analysis, we determined the structure of the Fab fragment of 21D10 in complex with full-length αIIbβ3 purified from human platelets. Results: T6, the PAR4 thrombin receptor activating peptide AYPGKF, and collagen-related peptide induced PDI binding to washed platelets [mean fluorescence intensity (MFI) of 547 ± 21 compared with 229 ± 12 for background control, p < 0.001; 576 ± 111 vs 215 ± 27 for background control, p < 0.05; and 934 ± 99 vs 167 ± 14 for background control, p < 0.001, respectively]. The reducing agent dithiothreitol (DTT) also enhanced PDI binding (MFI of 595 ± 147 vs 178 ± 24 for background control, p < 0.05). mAb 21D10 and its Fab fragment (40 μg/ml) inhibited PDI binding to platelets [MFI of 357 ± 25 vs 567 ± 60 for buffer control, p < 0.01 (figure); and 286 ± 38 vs 507 ± 86 for buffer control; p < 0.05)], fibrinogen binding (6,493 ± 815 vs 15,058 ± 1,935 for buffer control, p < 0.001; 1,714 ± 276 vs 14,917 ± 2,129 for buffer control, p < 0.0001), and maximum platelet aggregation (mean ± SD of 32 ± 8 vs 67 ± 2% for buffer control, p < 0.01; and 30 ± 8 vs 69 ± 3% for buffer control; p < 0.01). For comparison, the PDI inhibitor rutin at 45 μM produced similar inhibition. 21D10 did not inhibit the binding of mAbs 10E5 or 7E3, both of which inhibit fibrinogen binding to αIIbβ3, to platelets, and vice versa. EDTA treatment of platelets at either 22°C, pH 7.4, or 37°C, pH 8.0, had minimal effect on 21D10 binding, while both conditions inhibited 7E3 binding and the latter inhibited 10E5 binding. 21D10 reacted with the nonreduced but not reduced β3 subunit by immunoblotting. With chymotrypsin digested platelets, both 21D10 and the mAb 7H2, which binds to the PSI domain of β3, bound to bands extending down to 66 kDa, but 21D10 also bound to the bands below 66 kDa, whereas 7H2 did not. The cryo-EM structure of the 21D10 Fab-αIIbβ3 complex revealed that 21D10 binds to two different αIIbβ3 conformations that differ in the orientation of the leg domains (in one the legs are nearly bent and in the other the legs are partially extended). In both conformations, 21D10 binds to the PSI, I-EGF-1 and I-EGF-2 domains of β3 (figure). Conclusion: We have shown that in addition to thrombin, T6, a PAR4 activating peptide, collagen-related peptide, and DTT can induce PDI binding to platelets. We produced a mAb to human platelets (21D10) that can inhibit T6-induced PDI binding to platelets. Immunoblotting and cryo-EM analysis shows that 21D10 binds to the PSI, I-EGF1, and I-EGF2 regions of β3. If 21D10 prevents PDI binding by steric hindrance, it is likely that PDI binds to β3 at a site distant from the RGD ligand binding pocket. The functional effects of 21D10 may reflect its inhibition of PDI to platelets, but it may also reflect inhibition of other related proteins, in particular, ERp57.

Naringin binds to protein disulfide isomerase to inhibit its activity and modulate the blood coagulation rates: Implications in controlling thrombosis

Currently used antithrombotic drugs are beset with several drawbacks which necessitates the need for new and cheaper alternatives. Protein disulfide isomerase (PDI) is secreted in the blood plasma in cellular stress conditions and initiates the thrombus formation. A screening of library of natural compounds revealed that naringin had a high binding affinity for the PDI (−8.2 kcal/mol). Recombinant PDI was purified using the affinity chromatography. Incubation of purified PDI (3 μM) with naringin (0–100 μM, pH 7.4, 25 °C) partially modulated its conformation. Consequently, the fluorescence emission spectra of the PDI binding to naringin were assessed using the Stern-Volmer equation, which indicated an association constant of 2.78 × 104 M−1 suggesting an appreciable affinity for the naringin, with a unique binding site. An insulin turbidity assay showed that PDI activity is decreased in the presence of naringin indicating inhibition. Molecular dynamic simulation studies showed the changes in the PDI structure on binding to the naringin. Incubation of naringin (80 μM) in fresh human plasma along with exogenous PDI (175 nM) showed a significant delay in the intrinsic and extrinsic coagulation pathways. We show that naringin is able to modulate the PDI conformation and activity resulting in altered blood coagulation rates.

A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of proinflammatory interleukin 12 family cytokines

Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and β subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate β subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Here, we site-specifically introduce photocrosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone-client complexes for mass spectrometry. Our analysis reveals that a large set of endoplasmic reticulum chaperones interacts with IL-12α and IL-23α. Among these chaperones, we focus on protein disulfide isomerase (PDI) family members and reveal IL-12 family subunits to be clients of several incompletely characterized PDIs. We find that different PDIs show selectivity for different cysteines in IL-12α and IL-23α. Despite this, PDI binding generally stabilizes unassembled IL-12α and IL-23α against degradation. In contrast, α:β assembly appears robust, and only multiple simultaneous PDI depletions reduce IL-12 secretion. Our comprehensive analysis of the IL-12/IL-23 chaperone machinery reveals a hitherto uncharacterized role for several PDIs in this process. This extends our understanding of how cells accomplish the task of specific protein assembly reactions for signaling processes. Furthermore, our findings show that cytokine secretion can be modulated by targeting specific endoplasmic reticulum chaperones.

Rutin Increases Shear-Dependent TGF-β1 Activation By Interacting with Putative PDI Binding Sites: Role of Oxidation in Shear-Dependent TGF-β1 Activation

TGF-β1 is a very potent cytokine that, when activated, exerts both physiological and pathological functions, but the mechanism of activation remains unclear. Platelets contain 40-100-fold more TGF-β1 than other cell types and release TGF-β1 in an inactive latent complex comprised of mature TGF-β1 bound with latency-associated peptide and latent-TGF-β1 binding protein 1. We previously demonstrated that shear force activates latent TGF-β1 released from platelets in vitro, and that thiol-disulfide exchange contributes to this process. We also showed that protein disulfide isomerase (PDI) regulates shear-mediated TGF-β1 activation differentially, depending on whether PDI remains in its oxidized or reduced state. To test whether oxidation is required for latent TGF-β1 activation, we subjected platelet releasates to physiological shear/stirring for 2 hours in a closed chamber with room air or argon gas. We found ~10-fold increase in TGF-β1 activation compared to unsheared samples in room air, while the samples sheared under argon gas resulted in ~25% lower TGF-β1 activation (21 ± 1.0 % in room air vs. 16 ± 2.0 % of total TGF-β1 in argon gas, p=0.03). We also found that adding H2O2 to the platelet releasate resulted in increased shear-dependent TGF-β1 activation (active TGF-β1 was 16 ± 2.0 % in sheared samples and 22 ± 2.2 % of total TGF-β1 in sheared with H2O2 samples, p=0.03), indicating that oxidation during shear contributes to TGF-β1 activation. Since PDI can catalyze both oxidation and reduction of substrates, and because we previously showed that the reduced form of PDI inhibited shear-dependent TGF-β1 activation, we hypothesized that blocking the reduction function of PDI may increase shear-dependent TGF-β1 activation. To test this hypothesis, we assessed the effect of quercetin-3-rutinoside (rutin), a specific PDI inhibitor identified by high throughput screening, on shear-induced TGF-β1 activation. Rutin increased shear-induced TGF-β1 activation in a dose-dependent manner in that > 2.0 fold higher TGF-β1 activity was observed with 25 uM rutin, a concentration that blocked 55 to 80% of insulin reduction by PDI, confirming that rutin increases shear-dependent TGF-β1 activation by blocking the reduction function of PDI. We then assessed how rutin blocks PDI reduction function using PyMol modeling to identify the most likely interactions between rutin and PDI domains. After adjusting for the possible rotations of likely bond orientations, and considering minimum steric forces, the best model identified strong pi-pi interactions between Trp396 of the b' domain of PDI with the quercetin moiety of rutin (Figure-1). This interaction may stabilize the active site sulfhydryls of PDI so they maintain an inactive conformation, which buries the active site sulfhydryls, causing a loss in their solvent accessibility and thus inhibiting PDI's reductase function and increasing TGF-β1 activation. We conclude that thiol isomerases differentially regulate shear-dependent TGF-β1 activation. Thus, thiol-disulfide exchange regulator, such as PDI may be a potential target for suppressing or enhancing TGF-β1 activation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quercetin-3-Rutinoside Blocks the Disassembly of Cholera Toxin by Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is mainly located in the endoplasmic reticulum (ER) but is also secreted into the bloodstream where its oxidoreductase activity is involved with thrombus formation. Quercetin-3-rutinoside (Q3R) blocks this activity, but its inhibitory mechanism against PDI is not fully understood. Here, we examined the potential inhibitory effect of Q3R on another process that requires PDI: disassembly of the multimeric cholera toxin (CT). In the ER, PDI physically displaces the reduced CTA1 subunit from its non-covalent assembly in the CT holotoxin. This is followed by CTA1 dislocation from the ER to the cytosol where the toxin interacts with its G protein target for a cytopathic effect. Q3R blocked the conformational change in PDI that accompanies its binding to CTA1, which, in turn, prevented PDI from displacing CTA1 from its holotoxin and generated a toxin-resistant phenotype. Other steps of the CT intoxication process were not affected by Q3R, including PDI binding to CTA1 and CT reduction by PDI. Additional experiments with the B chain of ricin toxin found that Q3R could also disrupt PDI function through the loss of substrate binding. Q3R can thus inhibit PDI function through distinct mechanisms in a substrate-dependent manner.

The b′ domain of protein disulfide isomerase cooperates with the a and a′ domains to functionally interact with platelets

Essentials Protein disulfide isomerase (PDI) interacts with the αIIbβ3 integrin on platelets We generated PDI domain fragments and full-length PDI containing point mutations PDI interacts with αIIbβ3 through the b' domain, with the a and a' domains contributing This is the first report demonstrating PDI binding to a native protein on intact cells SUMMARY: Background Protein disulfide isomerase (PDI) is an oxidoreductase consisting of four domains arranged in the order a-b-b'-a' with an x-linker between the b' and a' domains. PDI binds to αIIb β3 integrin on activated platelets, and potentiates activation of this integrin through the C-terminal CGHC active-site motif. How PDI binds to platelet αIIb β3 is unknown. Objective and methods We used PDI domain fragments and full-length PDI containing point mutations to study inhibition of Alexa488-labeled PDI binding to thrombin-activated platelets. The effect of the PDI variants on platelet aggregation was tested. Results Only PDI fragments containing the b' domain bound to activated platelets. A double mutant of the b' domain had decreased binding, confirming the essential role of the b' domain. Addition of mutations in the a and a' domains further decreased binding, suggesting that these domains contribute to the interaction of PDI with platelets. The ability of the b' domain to interact directly with αIIb β3 was demonstrated with surface plasmon resonance, with contributions from the a and a' domains. The abb'x PDI fragment that binds to platelets but lacks the critical C-terminal active site inhibited platelet aggregation and invivo thrombosis. Moreover, site mutations in the a, b' and a' domains that resulted in partial binding to platelets partially recovered aggregation of PDI-null platelets. PDI mutants that did not bind showed no recovery. Conclusion PDI functionally interacts with αIIb β3 on platelets through the substrate-binding b' domain, with the a and a' domains contributing to efficient binding.

Complement C5 but not C3 is expendable for tissue factor activation by cofactor-independent antiphospholipid antibodies

AbstractThe complement and coagulation cascades interact at multiple levels in thrombosis and inflammatory diseases. In venous thrombosis, complement factor 3 (C3) is crucial for platelet and tissue factor (TF) procoagulant activation dependent on protein disulfide isomerase (PDI). Furthermore, C5 selectively contributes to the exposure of leukocyte procoagulant phosphatidylserine (PS), which is a prerequisite for rapid activation of monocyte TF and fibrin formation in thrombosis. Here, we show that monoclonal cofactor-independent antiphospholipid antibodies (aPLs) rapidly activate TF on myelomonocytic cells. TF activation is blocked by PDI inhibitor and an anti-TF antibody interfering with PDI binding to TF, and requires C3 but unexpectedly not C5. Other prothrombotic, complement-fixing antibodies, for example, antithymocyte globulin, typically induce TF activation dependent on C5b-7–mediated PS exposure on the outer membrane of monocytes. We show that aPLs directly induce procoagulant PS exposure independent of C5. Accordingly, mice deficient in C3, but not mice deficient in C5, are protected from in vivo thrombus formation induced by cofactor-independent aPLs. Only immunoglobulin G (IgG) fractions with cofactor-independent anticardiolipin reactivity from patients with antiphospholipid syndrome (APS) induce complement-independent monocyte PS exposure and PDI-dependent TF activation. Neither a human monoclonal aPL directed against β2-glycoprotein I (β2GPI) nor patient IgG with selective reactivity to β2GPI rapidly activated monocyte TF. These results indicate that inhibitors of PDI and TF, but not necessarily clinically available drugs targeting C5, have therapeutic benefit in preventing thrombosis associated with APS caused by pathogenic aPLs primarily reactive with lipid, independent of β2GPI.

PDI Inhibition Blocks Thrombin Generation in Humans By Interfering with Platelet Factor V Activation

Protein disulfide isomerase (PDI) is a ubiquitously expressed oxidoreductase that serves an essential role in protein folding in the endoplasmic reticulum by reshuffling disulfide bonds within nascent proteins. PDI can be released from vascular cells, including platelets, and inhibition or platelet-specific deletion of PDI blocks thrombus formation in vivo. However, the specific function of PDI in thrombus formation is poorly understood. Unlike the role of proteases in blood coagulation, which have been studied in depth, little is known about PDI substrates in the vasculature. Several platelet and endothelial integrins have been identified as putative substrates for PDI, but whether coagulation factors are directly targeted by extracellular PDI has not been established. We now identify platelet factor V as a principal coagulation substrate of extracellular PDI. We developed an unbiased strategy to identify novel substrates of PDI in washed platelets using PDI variants capable of trapping substrates: FLAG-tagged PDI mutants modified by a substitution of arginine or proline for histidine (CGHC → CGRC; CGHC → CGPC) in the catalytic motif of both the a and a’ domains. Whereas the AGHA-PDI variant which has no catalytic activity serves as a control. The CGRC-PDI variant co-precipitated with platelet factor V in a redox-sensitive manner while there was no platelet factor V detected with the AGHA-PDI variant, thus confirming that binding of PDI to platelet-derived factor V occurs through disulfide bond exchange. Platelet factor V associates with multimerin-1 through a disulfide bond. Trapping PDI mutants also bind to multimerin-1 in a reaction requiring disulfide bond exchange. To evaluate the effect of PDI inhibition on the activation of platelet factor V, washed platelets from healthy donors were stimulated with 0.1 U/mL of thrombin in the presence of varying concentrations of isoquercetin (0 to 50 µM), which has previously been shown to inhibit PDI function. We observed a dose-dependent reduction of factor Va following platelet activation despite the fact that isoquercetin did not inhibit platelet release of PF4 or block Xa or thrombin enzymatic activity directly. We next performed a clinical study designed to determine whether oral isoquercetin inhibits thrombin generation in human subjects via its ability to inhibit platelet Va generation. Plasma samples collected from healthy participants before and 4 hours after ingestion of 1000 mg of isoquercetin (N=17). In plasma samples, post-isoquercetin platelet-dependent thrombin generation decreased by 51% compared with pre-ingestion controls (P=0.0004). Furthermore, we observed an overall 26% reduction in FVa in non-FV depleted plasma (P<0.001), which corresponded with a 53% decrease in FVa generated from platelets (P<0.001). These data confirm a significant effect of PDI inhibition on the generation of FVa following platelet activation. Considering that isoquercetin reduces platelet FVa generation and similarly inhibits platelet-dependent thrombin generation in a PDI-dependent manner, we investigated whether the addition of FVa in vitro restored platelet-dependent thrombin generation. The pre-incubation of 7 µg/mL FVa prior to stimulation with low dose thrombin restored platelet-dependent thrombin generation to within 80% baseline of pre-treatment levels. We conclude that platelet factor V is an essential substrate in mediating PDI-dependent thrombin generation on platelets and propose that PDI cleaves a disulfide bond that links platelet factor V to multimerin-1, thereby releasing platelet factor V for activation and subsequent thrombin generation. DisclosuresZwicker:Quercegen Pharma: Research Funding.

Structure-function analyses of microsomal triglyceride transfer protein missense mutations in abetalipoproteinemia and hypobetalipoproteinemia subjects

We describe two new hypolipidemic patients with very low plasma triglyceride and apolipoprotein B (apoB) levels with plasma lipid profiles similar to abetalipoproteinemia (ABL) patients. In these patients, we identified two previously uncharacterized missense mutations in the microsomal triglyceride transfer protein (MTP) gene, R46G and D361Y, and studied their functional effects. We also characterized three missense mutations (H297Q, D384A, and G661A) reported earlier in a familial hypobetalipoproteinemia patient. R46G had no effect on MTP expression or function and supported apoB secretion. H297Q, D384A, and G661A mutants also supported apoB secretion similarly to WT MTP. Contrary to these four missense mutations, D361Y was unable to support apoB secretion. Functional analysis revealed that this mutant was unable to bind protein disulfide isomerase (PDI) or transfer lipids. The negative charge at residue 361 was critical for MTP function as D361E was able to support apoB secretion and transfer lipids. D361Y most likely disrupts the tightly packed middle α-helical region of MTP, mitigates PDI binding, abolishes lipid transfer activity, and causes ABL. On the other hand, the hypolipidemia in the other two patients was not due to MTP dysfunction. Thus, in this study of five missense mutations spread throughout MTP's three structural domains found in three hypolipidemic patients, we found that four of the mutations did not affect MTP function. Thus, novel mutations that cause severe hypolipidemia probably exist in other genes in these patients, and their recognition may identify novel proteins involved in the synthesis and/or catabolism of plasma lipoproteins.

The Involvement of His50 during Protein Disulfide Isomerase Binding Is Essential for Inhibiting α-Syn Fibril Formation.

An increased level of protein disulfide isomerase (PDI) is a protective response to various neurodegenerative disorders, including Parkinson's disease. Interaction of PDI with α-synuclein (α-Syn) has been shown to inhibit its aggregation. Here, we report the residue-specific mapping of binding of PDI to α-Syn. We demonstrate that α-Syn N-terminal residues V3-S9 and L38-V40 bind more strongly to PDI than residues V49-V52 do, as do C-terminal residues E123-M127 and D135-E137. In addition, we show that residue H50 is key in preventing aggregation. These findings improve our understanding of PDI-protected aggregation of wild-type α-Syn and its H50Q familial mutant.

Abstract 192: Characterization of Microsomal Triglyceride Transfer Protein Missense Mutations Found in Abetalipoproteinemia and Hybobetalipoproteinemia Subjects

We describe two new hypolipidemic patients with very low plasma triglyceride and apolipoprotein B (apoB) levels and lipid malabsorption with plasma lipid profiles similar to abetalipoproteinemia (ABL) patients. In these patients, we identified two previously uncharacterized missense mutations in the microsomal triglyceride transfer protein (MTP) gene, R46G and D361Y, and studied their effects on function. We also characterized three missense mutations (H297Q, D384A, and G661A) reported earlier in a familial hypobetalipoproteinemia patient. R46G had no effect on MTP expression or function and supported apoB secretion. Similarly, H297Q, D384A, and G661A mutants supported apoB secretion similarly to WT MTP. Contrary to these four missense mutations, D361Y was unable to support apoB secretion. Functional analysis revealed that this mutant was unable to bind protein disulfide isomerase (PDI) or transfer lipids. The negative charge at residue 361 was critical for MTP function as D361E was able to support apoB secretion and transfer lipids. D361Y most likely disrupts the tightly packed middle α-helical region of MTP, mitigates PDI binding, abolishes lipid transfer activity, and causes ABL. On the other hand, the hypolipidemia in the other two patients was not due to MTP dysfunction. Thus, in this study of five missense mutations spread throughout MTP’s three structural domains found in three hypolipidemic patients, we found that four of the mutations did not affect MTP function. Thus, there probably exist novel mutations in other genes that cause severe hypolipidemia and their recognition may identify novel proteins involved in the synthesis and/or catabolism of plasma lipoproteins.

Structural basis of redox-dependent substrate binding of protein disulfide isomerase.

Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b′ and a′ domains provide substrate binding sites and undergo an open–closed domain rearrangement depending on the redox states of the a′ domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b′–a′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a′ domain partially masks the binding surface of the b′ domain, causing steric hindrance against the peptide in the reduced form of the b′–a′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI.

Abstract 547: A Novel Abetalipoproteinemia Missense Mutation Highlights the Importance of N-terminal β-barrel in Microsomal Triglyceride Transfer Protein Function

Background: The use of microsomal triglyceride transfer protein (MTP) inhibitors is limited to severe hyperlipidemias due to associated hepatosteatosis. Comprehensive knowledge about the structure-function of MTP might help design new molecules that avoid steatosis. Characterization of mutations in MTP causing abetalipoproteinemia (ABL) have revealed that the central α-helical and C-terminal β-sheet domains are important for protein disulfide isomerase (PDI) binding and lipid transfer activity. Our aim was to identify and characterize ABL mutations in the N-terminal region to understand the function of this domain. Methods and Results: We identified a novel missense mutation (D169V) in a 4-month old Turkish male with severe signs of ABL. To study the effect of this mutation on MTP function, we created mutants via site directed mutagenesis. Although D169V was expressed in the endoplasmic reticulum and interacted with apoB17, it was unable to bind PDI, transfer lipids, and support apoB secretion. Computational modeling suggested that D169 could form an internal salt bridge with K187 and K189. Mutagenesis of these lysines to leucines abolished PDI heterodimerization, lipid transfer, and apoB secretion, without affecting apoB binding. Further, mutants with preserved charges (D169E, K187R, K189R) rescued these activities. Conclusions: D169V is detrimental because it disrupts an internal salt bridge leading to loss of PDI binding and lipid transfer activities; however, it does not affect apoB-binding. We propose that future inhibitors should avoid this region of the N-terminal domain.

Protein disulfide isomerase as a novel target for cyclopentenone prostaglandins: implications for hypoxic ischemic injury.

Cyclooxygenase-2 (COX-2) is an important contributor to ischemic brain injury. Identification of the downstream mediators of COX-2 toxicity may allow the development of targeted therapies. Of particular interest is the cyclopentenone family of prostaglandin metabolites. Cyclopentenone prostaglandins (CyPGs) are highly reactive molecules that form covalent bonds with cellular thiols. Protein disulfide isomerase (PDI) is an important molecule for the restoration of denatured proteins following ischemia. Because PDI has several thiols, including thiols within the active thioredoxin-like domain, we hypothesized that PDI is a target of CyPGs and that CyPG binding of PDI is detrimental. CyPG-PDI binding was detected invitro via immunoprecipitation and MS. CyPG-PDI binding decreased PDI enzymatic activity in recombinant PDI treated with CyPG, and PDI immunoprecipitated from neuronal culture treated with CyPG or anoxia. Toxic effects of binding were demonstrated in experiments showing that: (a) pharmacologic inhibition of PDI increased cell death in anoxic neurons, (b) PDI overexpression protected neurons exposed to anoxia and SH-SY5Y cells exposed to CyPG, and (c) PDI overexpression in SH-SY5Y cells attenuated ubiquitination of proteins and decreased activation of pro-apoptotic caspases. In conclusion, CyPG production and subsequent binding of PDI is a novel and potentially important mechanism of ischemic brain injury. We show that CyPGs bind to PDI, cyclopentenones inhibit PDI activity, and CyPG-PDI binding is associated with increased neuronal susceptibility to anoxia. Additional studies are necessary to determine the relative role of CyPG-dependent inhibition of PDI activity in ischemia and other neurodegenerative disorders.

Protein disulfide isomerase capture during thrombus formation in vivo depends on the presence of β3 integrins

AbstractExtracellular protein disulfide isomerase (PDI) is required for platelet thrombus formation and fibrin generation after arteriolar wall injury in live mice. PDI is secreted from platelets and endothelial cells on cellular activation, but the mechanism of capture of secreted PDI within the injured vasculature is unknown. We establish that, like the endothelial β3 integrin αVβ3, the platelet integrin αIIbβ3 binds PDI. PDI also binds to recombinant β3. Using intravital microscopy, we demonstrate that PDI accumulation at the site of laser-induced arteriolar wall injury is markedly reduced in β3-null (β3−/−) mice, and neither a platelet thrombus nor fibrin is generated at the vessel injury site. The absence of fibrin after vascular injury in β3−/− mice is because of the absence of extracellular PDI. To evaluate the relative importance of endothelial αVβ3 versus platelet αIIbβ3 or αVβ3, we performed reciprocal bone marrow transplants on wild-type and β3−/− mice. PDI accumulation and platelet thrombus formation were markedly decreased after vessel injury in wild-type mice transplanted with β3−/− bone marrow or in β3−/− mice transplanted with wild-type bone marrow. These results indicate that both endothelial and platelet β3 integrins contribute to extracellular PDI binding at the vascular injury site.

Protein Disulfide Isomerase Plays An Important Role in Beta2 Integrin-Mediated Neutrophil Recruitment During Vascular Inflammation

Abstract 17Cell surface protein disulfide isomerase (PDI), a prototypic thiol isomerase, plays an important role in regulating integrin-mediated cellular functions. Although surface PDI has been reported to regulate L-selectin shedding on neutrophils, its role in neutrophil recruitment during vascular inflammation has not been explored. In this paper, we present novel findings on how neutrophil PDI regulates neutrophil recruitment into the site of vascular inflammation. Using real-time microscopy with cell-impermeable inhibitors for PDI (bacitracin or function-blocking antibodies), we demonstrated that inhibition of PDI reduces adhesion of human blood neutrophils to TNF-alpha-stimulated human umbilical vein endothelial cells (HUVECs) under venous shear. There was no additive effect on neutrophil adhesion in a combination of blocking anti-PDI and anti-beta2 antibodies, suggesting that surface PDI regulates beta2 integrin-mediated neutrophil adhesion to TNF-alpha-activated HUVECs under shear. Further, bacitracin and blocking anti-PDI antibodies diminished alphaMbeta2 integrin-mediated neutrophil adhesion to intercellular adhesion molecule-1 (ICAM-1)-coated surfaces under static conditions. When PDI gene is knocked down in neutrophil-like HL60 cells by shRNA, alphaMbeta2 activation and cell adhesion to TNF-alpha-activated HUVECs under shear were significantly reduced. High resolution confocal microscopy and immunoprecipitation assay revealed that surface PDI interacts with alphaMbeta2 integrin and that such interaction was enhanced by neutrophil activation and inhibited by bacitracin, a thiol isomerase inhibitor. Exogenous PDI equivalently bound to CHO cells stably expressing alphaMbeta2 or alphaLbeta2 when those cells were treated with Mn2+, whereas PDI binding was significantly reduced when the active site CGHC residues on PDI were mutated. Using multi-channel fluorescence intravital microscopy, we demonstrated that inhibition of PDI by infusion of PDI inhibitors impairs stable adhesion of neutrophils to the TNF-alpha-inflamed cremaster muscle venule wall in living mice. These findings suggest the potentially fundamental but poorly understood role for surface PDI in the regulation of beta2 integrin function in neutrophils. Therefore, our results provide the first evidence that inhibition of PDI may be a therapeutic target for the neutrophil-mediated vascular inflammation. Disclosures:No relevant conflicts of interest to declare.

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

Protein-disulfide isomerase (PDI) has been proposed to exhibit an "unfoldase" activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demonstrating that CTA1 will unfold spontaneously upon its separation from the holotoxin at physiological temperature. Thus, PDI may simply dislodge CTA1 from the CT holotoxin without unfolding the CTA1 subunit. To evaluate the role of PDI in CT disassembly and CTA1 unfolding, we utilized a real-time assay to monitor the PDI-mediated separation of CTA1 from the CT holotoxin and directly examined the impact of PDI binding on CTA1 structure by isotope-edited Fourier transform infrared spectroscopy. Our collective data demonstrate that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit, thus uncovering a new mechanism for CTA1 dissociation from its holotoxin.

Interaction and functional association of protein disulfide isomerase with αVβ3 integrin on endothelial cells

Adhesive properties of endothelial cells are influenced by the thioldisulfide balance. However, the molecular mechanism of this effect is unclear, although recent observations indicate that integrin receptors may be direct targets for redox modulation. The purpose of this study was to examine whether protein disulfide isomerase (PDI) is directly involved in this process. As manganese ions are known to affect the thioldisulfide balance and activate integrins to maximal affinity, we searched for PDI interactions with integrins, particularly with alpha(V)beta(3), in Mn(2+)-treated endothelial cells. By employing confocal microscopy, flow cytometry and coimmunoprecipitation experiments, we showed that exposure of endothelial cells to Mn(2+) resulted in: (a) the appearance of surface protein thiol groups, which can be found in PDI and alpha(V)beta(3), and both proteins colocalizing on the cellular surface; and (b) the formation of the PDI-alpha(V)beta(3) complex, which dissociates upon reduction. In addition, PDI in a complex with alpha(V)beta(3) induces conversion of the integrin to the ligand-competent high-affinity state, as evidenced by increased binding of vitronectin. The membrane-impermeable sulfhydryl blockers 3-N-maleimidylpropionyl biocytin 3-N-maleimidylpropionyl biocytin and p-chloromercuriphenyl sulfonate, as well as the PDI inhibitors bacitracin, MA3 018, and MA3 019, abolished the binding of vitronectin and LM609 to endothelial cells that is activated by Mn(2+). Consistently, LM609 almost completely blocked binding of vitronectin to such cells. The formation of the PDI-alpha(V)beta(3) stoichiometric complex was further demonstrated by surface plasmon resonance analysis, which showed that the initial reversible binding of PDI becomes irreversible in the presence of Mn(2+), probably mediated by disulfide bonds. Thus, we show that Mn(2+) simultaneously modulates the thiol isomerase activity of PDI that is bound to alpha(V)beta(3) and induces its transition to the ligand-competent state, suggesting an alternative mechanism of integrin regulation.

ERp57 binds competitively to protein disulfide isomerase and calreticulin

In this study, we screened for protein disulfide isomerase (PDI)-binding proteins in bovine liver microsomes under strict salt concentrations, using affinity column chromatography. One main band observed using SDS–PAGE was identified as ERp57 (one of the PDI family proteins) by LC-MS/MS analysis. The K D value of PDI binding to ERp57 was calculated as 5.46 × 10 −6 M with the BIACORE system. The interactions between PDI and ERp57 occurred specifically at their a and b domains, respectively. Interestingly, low concentrations of ERp57 enhanced the chaperone activity of PDI, while high concentrations interfered with chaperone activity. On the other hand, ERp57 did not affect the isomerase activity of PDI. Additionally, following pre-incubation of ERp57 with calreticulin (CRT), decreased interactions were observed between ERp57 and PDI, and vice versa. Based on the data, we propose that once ERp57 binds to PDI or CRT, the resultant complex inhibits further interactions. Therefore, ERp57 selectively forms a protein-folding complex with PDI or CRT in ER.