Luis A. Ralat

Molecular bases for the recognition of short peptide substrates and cysteine-directed modifications of human insulin-degrading enzyme

Insulin degrading enzyme (IDE) utilizes a large catalytic chamber to selectively bind and degrade peptide substrates such as insulin and amyloid beta (Abeta). Tight interactions with substrates occur at an exosite located approximately 30 A 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 1.9 A 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 K(m) 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 13 cysteines is insensitive to the inhibition by S-nitrosoglutathione, hydrogen peroxide, or N-ethylmaleimide. Specifically, cysteine 819 of human IDE is located inside the catalytic chamber pointing toward 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.

Insulin-degrading Enzyme Modulates the Natriuretic Peptide-mediated Signaling Response

Natriuretic peptides (NPs) are cyclic vasoactive peptide hormones with high therapeutic potential. Three distinct NPs (ANP, BNP, and CNP) can selectively activate natriuretic peptide receptors, NPR-A and NPR-B, raising the cyclic GMP (cGMP) levels. Insulin-degrading enzyme (IDE) was found to rapidly cleave ANP, but the functional consequences of such cleavages in the cellular environment and the molecular mechanism of recognition and cleavage remain unknown. Here, we show that reducing expression levels of IDE profoundly alters the response of NPR-A and NPR-B to the stimulation of ANP, BNP, and CNP in cultured cells. IDE rapidly cleaves ANP and CNP, thus inactivating their ability to raise intracellular cGMP. Conversely, reduced IDE expression enhances the stimulation of NPR-A and NPR-B by ANP and CNP, respectively. Instead of proteolytic inactivation, IDE cleavage can lead to hyperactivation of BNP toward NPR-A. Conversely, decreasing IDE expression reduces BNP-mediated signaling. Additionally, the cleavages of ANP and BNP by IDE render them active with NPR-B and a reduction of IDE expression diminishes the ability of ANP and BNP to stimulate NPR-B. Our kinetic and crystallographic analyses offer the molecular basis for the selective degradation of NPs and their variants by IDE. Furthermore, our studies reveal how IDE utilizes its catalytic chamber and exosite to engulf and bind up to two NPs leading to biased stochastic, non-sequential cleavages and the ability of IDE to switch its substrate selectivity. Thus, the evolutionarily conserved IDE may play a key role in modulating and reshaping the strength and duration of NP-mediated signaling.

Glutathione S-Transferase Pi Has at Least Three Distinguishable Xenobiotic Substrate Sites Close to Its Glutathione-binding Site

Benzyl isothiocyanate (BITC), present in cruciferous vegetables, is an efficient substrate of human glutathione S-transferase P1-1 (hGST P1-1). BITC also acts as an affinity label of hGST P1-1 in the absence of glutathione, yielding an enzyme inactive toward BITC as substrate. As monitored by using BITC as substrate, the dependence of k of inactivation (KI) of hGST P1-1 on [BITC] is hyperbolic, with KI = 66 ± 7 μm. The enzyme incorporates 2 mol of BITC/mol of enzyme subunit upon complete inactivation. S-Methylglutathione and 8-anilino-1-naphthalene sulfonate (ANS) each yield partial protection against inactivation and decrease reagent incorporation, whereas S-(N-benzylthiocarbamoyl)glutathione or S-methylglutathione + ANS protects completely. Mapping of proteolytic digests of modified enzyme by using mass spectrometry reveals that Tyr103 and Cys47 are modified equally. S-Methylglutathione reduces modification of Cys47, indicating this residue is at/near the glutathione binding region, whereas ANS decreases modification of Tyr103, suggesting this residue is at/near the BITC substrate site, which is also near the binding site of ANS. The Y103F and Y103S mutant enzymes were generated, expressed, and purified. Both mutants handle substrate 1-chloro-2,4-dinitrobenzene normally; however, Y103S exhibits a 30-fold increase in Km for BITC and binds ANS poorly, whereas Y103F has a normal Km for BITC and Kd for ANS. These results indicate that an aromatic residue at position 103 is essential for the binding of BITC and ANS. This study provides evidence for the existence of a novel xenobiotic substrate site in hGST P1-1, which can be occupied by benzyl isothiocyanate and is distinct from that of monobromobimane and 1-chloro-2,4 dinitrobenzene.

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin.

Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex [L.A. Ralat, Y. Manevich, A.B. Fisher, R.F. Colman, Biochemistry 45 (2006) 360-372]. 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 2.5mM 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 4M 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 1mol/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.

Protective Role of Cys-178 against the Inactivation and Oligomerization of Human Insulin-degrading Enzyme by Oxidation and Nitrosylation

Insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, hydrolyzes several physiologically relevant peptides, including insulin and amyloid-β (Aβ). Human IDE has 13 cysteines and is inhibited by hydrogen peroxide and S-nitrosoglutathione (GSNO), donors of reactive oxygen and nitrogen species, respectively. Here, we report that the oxidative burst of BV-2 microglial cells leads to oxidation or nitrosylation of secreted IDE, leading to the reduced activity. Hydrogen peroxide and GSNO treatment of IDE reduces the Vmax for Aβ degradation, increases IDE oligomerization, and decreases IDE thermostability. Additionally, this inhibitory response of IDE is substrate-dependent, biphasic for Aβ degradation but monophasic for a shorter bradykinin-mimetic substrate. Our mutational analysis of IDE and peptide mass fingerprinting of GSNO-treated IDE using Fourier transform-ion cyclotron resonance mass spectrometer reveal a surprising interplay of Cys-178 with Cys-110 and Cys-819 for catalytic activity and with Cys-789 and Cys-966 for oligomerization. Cys-110 is near the zinc-binding catalytic center and is normally buried. The oxidation and nitrosylation of Cys-819 allow Cys-110 to be oxidized or nitrosylated, leading to complete inactivation of IDE. Cys-789 is spatially adjacent to Cys-966, and their nitrosylation and oxidation together trigger the oligomerization and inhibition of IDE. Interestingly, the Cys-178 modification buffers the inhibition caused by Cys-819 modification and prevents the oxidation or nitrosylation of Cys-110. The Cys-178 modification can also prevent the oligomerization-mediated inhibition. Thus, IDE can be intricately regulated by reactive oxygen or nitrogen species. The structure of IDE reveals the molecular basis for the long distance interactions of these cysteines and how they regulate IDE function.

Structure, function, and regulation of insulin-degrading enzyme.

The short half-life of insulin in the human body (4-6 min) prompted the search and discovery of insulin-degrading enzyme (IDE), a 110-kDa metalloprotease that can rapidly degrade insulin into inactive fragments. Genetic and biochemical evidence accumulated in the last sixty years has implicated IDE as an important physiological contributor in the maintenance of insulin levels. Recent structural and biochemical analyses reveal the molecular basis of how IDE uses size and charge distribution of the catalytic chamber and structural flexibility of substrates to selectively recognize and degrade insulin, as well as the regulatory mechanisms of this enzyme. These studies provide a path for potential therapeutics in the control of insulin metabolism by the degradation of insulin.

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase π

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S‐transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S‐(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1‐chloro‐2,4‐dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzymes use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8‐anilino‐1‐naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S‐Methylglutathione and S‐(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl‐bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S‐methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S‐(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S‐transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.