Insulin-degrading enzyme modulates the natriuretic peptide-mediated signaling response

Abstract

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.

FIGURE 1.

Differential responses to NPs upon IDE knockdown in human NPR-A- and NPR-B-expressing 293 cells. Intracellular cGMP content was determined in 293-neo cells stably expressing human NPR-A (A–C) or NPR-B (D–F) lentivirally infected with control (●) or IDE siRNA (○) at the indicated times of treatment with 100 nm ANP, BNP, and CNP. Data are means ± S.E. for three independent experiments with two replications per condition.

FIGURE 2.

IDE treatment changes the ability of NPs to stimulate cGMP signaling. 100 nm of each peptide was incubated with 2 nm human recombinant IDE for 5 min in a total volume of 0.1 ml. Proteolysis was terminated by addition of 0.1 ml of 0.1 m HCl, and bioactivity was evaluated by measuring the ability of neutralized extracts to elevate cGMP concentration in 293-neo cells stably expressing human NPR-A (A) or NPR-B (B). Data are means ± S.E. for three independent experiments with two replications per condition. Statistical significance reflects comparison with the activity of the peptide with no IDE. *, p < 0.05.

FIGURE 3.

Mass spectrometry analyses of NP degradation by human IDE. A, representative ESI-FTICR-MS spectrum of ANP before (lower panel) and after IDE digestion (upper two panels). ANP and IDE were mixed in a 50:1 molar ratio. After 1-s and 5-min incubations, EDTA (80 mm) and trifluoroacetic acid (0.03%) were added to stop the reactions. B, representative ESI-ECD mass of 2274 Da. C, primary sequence and IDE cleavage sites of seven natriuretic peptides. Initial cleavage sites and secondary cutting sites are shown as big and small arrows, respectively. The gray shaded amino acids indicate residues conserved in all NPs. Similarly, the cleavage sites are also shown on the tertiary structure of ANP (PDB:1ANP).

FIGURE 4.

Kinetics of hydrolysis of NPs by IDE and structural analysis of ANP- and BNP-bound IDE. A, sequence alignment and kinetic parameters of NPs with the upper part containing naturally occurring NPs, and the middle and lower parts containing variants of BNP and ANP, respectively. The region within the loop is enclosed within the gray shaded box. Unless otherwise noted, all NPs are of human origin. B, global view of the structure of ANP-bound IDE-CF-E111Q monomer. IDE-N and IDE-C are colored green and yellow, respectively. ANP is in a sphere representation with atoms colored red for oxygen, blue for nitrogen, and white for carbon. C, detailed interaction of the N terminus of ANP (left) and BNP (right) with the exosite of IDE. D, model for the interaction of IDE and ANP (ANP is based on a solution structure from PDB:1ANP). The IDE monomer is shown as a light green surface representation and the catalytic chamber is shown in dark green (red sphere for catalytic zinc). The backbone of ANP is shown as a ribbon with the N and C termini depicted in gray and green, respectively, and the disulfide-linked ring shown in red.

FIGURE 5.

Model for the cleavage of ANP by IDE. A, biased stochastic mode for the cleavage of ANP by IDE. ANP cleavage by IDE has revealed a novel mechanism for substrate hydrolysis by IDE, involving a biased stochastic mode, as opposed to the previously proposed sequential mode. B, four modes of substrate cleavage by IDE exemplified by the cleavages of insulin, Aβ, bradykinin, and ANP. See “Results” for details about these four modes.

FIGURE 6.

IDE-mediated degradation of fsANP in the absence and presence of ANP and CNP. MALDI-TOF mass spectra of (A) ANP, (B) fsANP, (C) CNP, and a 1:1 molar ratio of (D) ANP + fsANP and (E) CNP + fsANP. Each spectrum depicts the peptide(s) alone (bottom panel) or after incubation with IDE (top panel).

FIGURE 7.

Monomeric IDE mutant R767A retains its ability to switch NP substrate selectivity. A, cartoon diagram depicting IDE dimer arrangement revealed in IDE crystal structures. Arg-767 located at the dimer interface is highlighted. Native-PAGE analysis (B) and size exclusion chromatography (C) of IDE-R767A are shown. D, enzymatic activity of IDE-R767A using bradykinin mimetic substrate V. E, IDE-R767A mediated degradation of ANP, fsANP, and a 1:1 mixture of both NPs. The MALDI-TOF spectra depict the peptides alone (left panels) or after incubation with IDE-R767A (right panels).