Glutathione S-transferase pi (GST pi) has been shown to reactivate oxidized 1-cysteine peroxiredoxin (1-Cys Prx, Prx VI, Prdx6, and AOP2). We now demonstrate that a heterodimer complex is formed between 1-Cys Prx with a C-terminal His6 tag and GST pi upon incubation of the two proteins at pH 8.0 in buffer containing 20% 1,6-hexanediol to dissociate the homodimers, followed by dialysis against buffer containing 2.5 mM glutathione (GSH) but lacking 1,6-hexanediol. The heterodimer can be purified by chromatography on nickel-nitriloacetic acid agarose in the presence of GSH. N-Terminal sequencing showed that equimolar amounts of the two proteins are present in the isolated complex. In the heterodimer, 1-Cys Prx is fully active toward either H2O2 or phospholipid hydroperoxide, while the GST pi activity is approximately 25% of that of the GST pi homodimer. In contrast, the 1-Cys Prx homodimer lacks peroxidase activity even in the presence of free GSH. The heterodimer is also formed in the presence of S-methylglutathione, but no 1-Cys Prx activity is found under these conditions. The yield of heterodimer is decreased in the absence of 1,6-hexanediol or GSH. Rapid glutathionylation of 1-Cys Prx in the heterodimer is detected by immunoblotting. Subsequently, a disulfide-linked dimer is observed on SDS-PAGE, and the free cysteine content is decreased by 2 per heterodimer. The involvement of particular binding sites in heterodimer formation was tested by site-directed mutagenesis of the two proteins. For 1-Cys Prx, neither Cys47 nor Ser32 is required for heterodimer formation but Cys47 is essential for 1-Cys Prx activation. For GST pi, Cys47 and Tyr7 (at or near the GSH-binding site) are needed for heterodimer formation but three other cysteines are not. We conclude that reactivation of oxidized 1-Cys Prx by GST pi occurs by heterodimerization of 1-Cys Prx and GST pi harboring bound GSH, followed by glutathionylation of 1-Cys Prx and then formation of an intersubunit disulfide. Finally, the GSH-mediated reduction of the disulfide regenerates the reduced active-site sulfhydryl of 1-Cys Prx.
Insulin degrading enzyme (IDE) utilizes a large catalytic chamber to selectively bind and degrade peptide substrates such as insulin and amyloid β (Aβ). Tight interactions with substrates occur at an exosite located ~30Å away from the catalytic center that anchors the N-terminus of substrates to facilitate binding and subsequent cleavages at the catalytic site. However, IDE also degrades peptide substrates that are too short to occupy both the catalytic site and the exosite simultaneously. Here, we use kinins as a model system to address the kinetics and regulation of human IDE with short peptides. IDE specifically degrades bradykinin and kallidin at the Pro/Phe site. A 1.9Å crystal structure of bradykinin-bound IDE reveals the binding of bradykinin to the exosite, and not to the catalytic site. In agreement with observed high Km values, this suggests low affinity of bradykinin for IDE. This structure also provides the molecular basis on how the binding of short peptides at the exosite could regulate substrate recognition. We also found that human IDE is potently inhibited by physiologically relevant concentrations of S-nitrosylation and oxidation agents. Cysteine-directed modifications play a key role, since an IDE mutant devoid of all thirteen cysteines is insensitive to the inhibition by S-nitroso-glutathione, hydrogen peroxide, or N-ethylmaleimide. Specifically, cysteine 819 of human IDE is located inside the catalytic chamber pointing towards an extended hydrophobic pocket and is critical for the inactivation. Thiol-directed modification of this residue likely causes local structural perturbation to reduce substrate binding and catalysis.
Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the M w of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The M w of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5 mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4 M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the K m value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41-85, 115-124, 131-163, and 164-197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1 mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer. KeywordsGlutathione S-transferase pi; 1-Cys peroxiredoxin; Heterodimer Glutathione S-transferases (GSTs 1 ), which catalyze the nucleophilic attack by the thiol of glutathione on electrophilic substrates, constitute a family of enzymes important in the detoxification of xenobiotics, endogenous compounds, and the products of oxidative stress [1,2]. The pi isozyme (GST pi), crystallized as a homodimer with a subunit molecular * Corresponding author. Fax: +1 302 831 6335. rfcolman@chem.udel.edu (R.F. Colman). 1 Abbreviations used: GST, glutathione S-transferase; GST pi, pi-class glutathione S-transferase; 1-Cys Prx, 1-Cysteine peroxiredoxin; Ni-NTA, nickel-nitriloacetic acid agarose; TRIS, tris (hydroxymethyl) aminomethane; EDTA, disodium ethylenediamine tetraacetate; MES, 2-(N-morpholino) ethane-sulfonate; WT, wild-type; GSH, glutathione; CDNB, 1-chloro-2,4-dinitrobenzene; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript weight of 23,500, is of particular interest because it exhibits diverse roles in mammalian cells: it provides a defense against carcinogenesis, since it catalyzes the inactivation of known carcinogens [3]; it contributes significantly to the development of resistance to cancer chemotherapy, since GST pi levels increase in tumors and the enzyme metabolizes key anticancer drugs [4][5][6][7]; and it promotes the cellular response to oxidative stress, since GST pi has recently been reported to activate the anti-oxidant enzyme 1-Cys peroxiredoxin [8,9].1-Cys peroxiredoxin (1-Cys Prx, Prdx 6, Prx VI, and AOP2), a homodime...
The natriuretic peptides (NPs), 2 mainly atrial (ANP), Btype (BNP), and C-type natriuretic peptides (CNP), play key roles in many cardiovascular functions (1). Their diuretic, natriuretic, and vasodilatory properties have been developed as therapeutic strategies for human cardiovascular diseases (2). The actions of NPs are mediated through binding and signal transduction of three natriuretic peptide receptors (NPR). NPR-A and NPR-B have guanylyl cyclase activity that raises intracellular cGMP levels. Effects of these receptors are mediated by the preferential binding of ANP and BNP to NPR-A and by that of CNP to NPR-B. NPR-C is a non-guanylyl cyclase receptor. In addition to its role in the clearance of NPs, NPR-C also can transmit signals via heterotrimeric G protein, G i (3, 4).NPs have a short half-life and their circulation levels are tightly controlled (5, 6). In addition to the regulation of NPs by gene transcription, secretion, and NPR-C mediated clearance, NP maturation and breakdown by multiple proteases are also key in NP regulation. For example, active NPs are converted from pro-NPs by furin, corin, and likely, by other proteases (7,8). Active NPs are postulated to be proteolytically inactivated by a membrane-bound metalloprotease, neprilysin (NEP) (9 -12). However, growing evidence propose the role of other proteases in the clearance of NPs. Meprin A is shown to be involved in the initial N-terminal cleavage of BNP and meprin A and NEP are thought to work together in the clearance of BNP (13). In addition, insulin-degrading enzyme (IDE) and DPP-IV have been shown to cleave NPs in vitro (14 -16) However, the functional consequence in the cleavage of NPs by these two proteases in the cellular setting remains unknown.IDE is a ubiquitously expressed zinc-metalloprotease that is involved in the clearance of insulin and amyloid- (A), peptides implicated in the pathogenesis of diabetes and Alzheimer's disease, respectively (17-19). We have recently solved the structure of human IDE in complex with various substrates and elucidated the molecular basis for the recognition and selective degradation of substrates by IDE (20 -22). Our structures reveal that IDE uses a sizable catalytic chamber to entrap, unfold, and degrade insulin, A, and other substrates. IDE recognizes its substrates mainly based on their tertiary structures. The size, dipole moment, structure flexibility, and location of the N-terminal end of the substrates are key factors for the selectivity of IDE.IDE was shown to degrade ANP and BNP (15,16), however it was never established whether the cleavages of ANP and * This work was supported, in whole or in part, by National Institutes of Health Grants GM81539 (to W.-J. T.), F32 GM87093 (to L. A. R.), 5T32HL07237-33 (to T. F.), and R21HL093402-01 (to L. R. P.). The atomic coordinates and structure factors (codes 3N56 and 3N57) CNP, C-type natriuretic peptide; DNP, dendroaspis natriuretic peptide; fsANP, frameshift mutant atrial natriuretic peptide; NPR-A, natriuretic peptide receptor-A; NPR-B,...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.