The irreversible binding of amyloid peptide substrates to insulin ...

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Jul 3, 2008 - thermolysin, neprilysin and other members of the clan. In recent years, several unique features of IDE have attracted the attention of.
[Prion 2:2, 51-56; April/May/June 2008]; ©2008 Landes Bioscience

Commentary & View

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The irreversible binding of amyloid peptide substrates to insulin-degrading enzyme

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A biological perspective

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Matías B. de Tullio, Laura Morelli and Eduardo M. Castaño* Fundación Instituto Leloir-Instituto de Investigaciones Bioquímicas de Buenos Aires; CONICET; Buenos Aires Argentina

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Abbreviations: Aβ, amyloid β; ABAD, amyloid β-binding alcohol dehydrogenase; ABri, amyloid Bri; AD, Alzheimer’s disease; ADan, amyloid Dan; AICD, amyloid precursor protein intracellular domain; ANF, atrial natriuretic factor; DTT, dithiothreitol, endo-Lys C, endoproteinase-lysine C; gE, glycoprotein E; IDE, insulin-degrading enzyme; IDE-AβSCx, IDE-Aβ stable complex; IR, insulin receptor; MALDITOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; PI3K, phosphatidyl inositol 3-kinase; rIDE, recombinant IDE; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; VZV, varicella zoster virus

researchers in the amyloid field and, particularly, in the field of cerebral amyloidoses associated with neurodegeneration. IDE is capable of degrading in vitro several peptides with amyloidogenic potential in vivo such as calcitonin (thyroid amyloid), atrial natriuretic factor (ANF) (atrial amyloidosis), amylin (pancreatic islet amyloidosis associated with diabetes type 2), ABri and ADan (familial British and Danish dementias, respectively) (reviewed in refs. 1 and 2). More significantly, its genetic targeting in mice has demonstrated a pivotal role of IDE in the degradation of amyloid β (Aβ) of Alzheimer’s disease (AD), the ~5 kDa intracellular domain of the amyloid precursor protein (AICD) and insulin. In effect, IDE K.O. mice show elevated steady-state levels of Aβ and AICD in their brains as well as hyperinsulinemia and glucose intolerance.3,4 This physiological relevance of IDE may have its counterpart under pathological conditions such as AD, in which Aβ accumulates into oligomers and fibrils that are toxic to neurons. In support of this contention, evidence is growing that relates abnormal insulin metabolism and an increased risk for AD (reviewed in ref. 5). IDE activity was found to be lower in brain homogenates and cortical microvessels loaded with Aβ in AD patients as compared with age-matched controls.6,7 It has been proposed that oxidation may inactivate IDE by its reaction with the lipid peroxidation product 4-hydroxy-2-nonenal and the concomitant formation of protein adducts that have been detected in AD brains.8,9 More recently, lymphoblasts from affected members of AD families linked with the region 10q23-24 harboring the IDE gene showed lower IDE activity as compared with unaffected members,10 suggesting that the genetic linkage of AD in some chromosome 10-linked AD families may be related to a systemic defect in IDE activity. The recently solved crystal structure of an inactive mutant of human IDE (E111Q) bound to insulin B chain, Aβ1-40, amylin and glucagon has shown that backbone conformation of the substrate is a major determinant of recognition.11 This involves the binding of β strands of substrate conformers which are poorly populated

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Insulin-degrading enzyme (IDE) is a conserved Zn2+metalloendopeptidase involved in insulin degradation and in the maintenance of brain steady-state levels of amyloid β peptide (Aβ) of Alzheimer’s disease (AD). Our recent demonstration that IDE and Aβ are capable of forming a stoichiometric and extremely stable complex raises several intriguing possibilities regarding the role of this unique protein-peptide interaction in physiological and pathological conditions. These include a protective cellular function of IDE as a “dead-end chaperone” alternative to its proteolytic activity and the potential impact of the irreversible binding of Aβ to IDE upon its role as a varicella zoster virus receptor. In a pathological context, the implications for insulin signaling and its relationship to AD pathogenesis are discussed. Moreover, our findings warrant further research regarding a possible general and novel interaction between amyloidogenic peptides and other Zn2+metallopeptidases with an IDE-like fold and a substrate conformation-dependent recognition mechanism.

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Key words: amyloid, insulin-degrading enzyme, peptides, alzheimer’s disease, irreversible binding, metalloproteases

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Insulin-Degrading Enzyme, Amyloid Peptides and Alzheimer’s Disease

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Insulin-degrading enzyme (IDE) is a highly conserved and ubiquitous Zn2+ metallo peptidase involved in the degradation of insulin in vivo. It belongs to the M16 family of metallopeptidases defined by its “inverted” sequence at the catalytic site (His-X-X-Glu-His) as compared to the classical sequence (His-Glu-X-X-His) found in thermolysin, neprilysin and other members of the clan. In recent years, several unique features of IDE have attracted the attention of *Correspondence to: Eduardo M. Castaño; Fundación Instituto Leloir; 435 Av. Patricias Argentinas; Ciudad de Buenos Aires C1405BWE Argentina; Tel.: +54.11.5238.7500; Fax: +54.11.5238.7501; Email: [email protected] Submitted: 07/03/08; Accepted: 07/31/08 Previously published online as a Prion E-publication: http://www.landesbioscience.com/journals/prion/article/6710 www.landesbioscience.com

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Amyloid peptides bind irreversibly with insulin-degrading enzyme

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Our in vitro studies on the degradation of Aβ by IDE has led to the unexpected finding that Aβ, in addition to being a substrate, can remain bound to IDE with a stability consistent with an irreversible interaction.13 The resistance to dissociation included harsh treatments such as boiling in high concentration of SDS with reducing agents, or pre-incubation with formic acid or 6 M guanidine HCl at pH 3. These criteria are usually used to define a non-disulfide covalent binding between proteins, with the only exception of amyloid self-assembly. Without any evidence thus far that they are covalently bound, amyloid-assembled proteins may display such stability, especially when extracted from tissue and regardless of the primary structure of the protein involved. In these cases, although a substantial amount of amyloid subunits can be dissociated with strong denaturants (a property that has allowed protein identification), there is always a remnant that resists dissociation. Several proteins with pathogenic relevance in AD and related diseases are known to bind to Aβ in an SDS-resistant manner, including apolipoprotein E, apolipoprotein J, α2-macroglobulin, transthyretin, gelsolin and the α7-nicotinic acethylcholine receptor subunit.14-20 Aβ also binds in vivo with high affinity to the enzyme 17β-hydroxysteroid dehydrogenase type 10 (HSD10), also known as Aβ-binding alcohol dehydrogenase (ABAD). Such interaction takes place within the mitochondrial matrix, results in a distortion of the ABAD active site, and may mediate part of the cytotoxic effects of Aβ.21,22 Yet, the tight binding between IDE and Aβ seems to be unique in its extremely high stability and in that IDE is the first Aβ-degrading peptidase shown to have such property. The biochemical characterization of this IDE-Aβ stable complex (IDE-AβSCx) has revealed several important features that are pertinent to its specificity and possible biological significance. Firstly, it seems to derive from the interaction between an Aβ monomer (or a very low order oligomer in the case of shorter Aβ peptides used in our study) and a natively folded IDE dimer under physiological conditions, indicating that it is unlikely to be the product of non-specific protein-peptide aggregation. Secondly, the Aβ sequence Leu17-Asn27, comprising

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IDE and Aβ Form an Extremely Stable Complex

the key residues that drive peptide self-assembly and several interactions with the IDE binding site as mentioned above, was sufficient to form IDE-AβSCx. Thirdly, IDE-AβSCx was formed with a very slow kinetics (t1/2 ~ 45 min) under pseudo-first order conditions (100-fold molar excess of peptide), suggestive of a conformational rearrangement. In addition, such kinetic behavior ruled out the unlikely possibility that IDE-AβSCx was a spurious product of the treatment with detergents or solvents used for its detection by SDS-PAGE or reverse-phase chromatography. Finally, the competition experiments with insulin pre-incubation, and the fragments of IDE obtained after partial digestion together with sequence and MALDI-TOF analysis supported that at least part of the catalytic domain of IDE was involved in IDE-AβSCx formation. The fact that by inhibiting IDE activity with Zn2+ chelators, the yield of IDE-AβSCx was highly increased (5–10 fold) indicates that its generation is independent of catalysis and that there are no fragments of Aβ involved. An interesting question that remains to be tackled is whether Aβ behaves as a true “suicide” inhibitor of IDE. Two technical problems have hindered the answer to this question. In the first place, the typical kinetic experiments required to determine the apparent rate constant of inhibition are hampered by the partial loss of activity of IDE when incubated alone at 37°C. Secondly, the allosteric behavior of the IDE dimer activity as a function of substrate concentration may impose an overall effect on residual activity that masks the inhibition of one Aβ-bound monomer if the second monomer in the IDE dimer remains Aβ-free and available for substrate binding. It derives from our experiments that after incubation with Aβ, only a fraction of active IDE molecules end up as IDE-AβSCx and therefore, the product is expected to be heterogeneous, containing fully occupied and Aβ-free IDE dimers together with species in which one IDE monomer is occupied and the other one remains free. Defining whether IDE-AβSCx is formed with an active versus a partially or fully inactive IDE will certainly impact on the interpretations of a possible biological role of IDE-AβSCx, particularly under pathological conditions in which Aβ may be steadily or transiently overproduced. Despite these limitations, our finding that IDE-AβSCx is prevented from forming by preincubation with an excess of substrates and that once IDE-AβSCx is completed, Aβ cannot be displaced by high affinity substrates at high concentrations suggest that at least part of the IDE binding site is irreversibly blocked in IDE-AβSCx. As a summary of our findings, a proposed model of IDE-AβSCx formation is presented in Figure 1. Although the shortest Aβ that we used lacks Lys, Tyr or Cys residues that are commonly found cross-linked in oxidized proteins, a covalent binding in IDE-AβSCx cannot be ruled out. As an example of this possibility, a nucleophilic attack of the amide nitrogen of Gly67 at the carbonyl carbon of Ser65 results in the internal covalent crosslinking and cyclization of the backbone in the chromophore region of green fluorescent protein.23 An alternative explanation for the high stability of IDE-AβSCx is a non-covalent binding similar to the cooperative interaction of amyloid self-assembly. Homologous amyloid formation is by far more favorable than heterologous assembly, however, cross-seeding between different amyloid proteins has been described in vitro and in vivo.24,25 The resistance to denaturation and slow formation of IDE-AβSCx together with the structural data on IDE-substrate binding described above prompt us to postulate that IDE-AβSCx may be the result of an amyloid-like

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when the peptides are free in solution with a “template” within the catalytic chamber of IDE. In the case of Aβ1-40, it is noteworthy that the central region Lys16-Asp23 acquires an extended conformation in its IDE-bound form and displays extensive contacts between residues known to be crucial in Aβ self-assembly (Leu17-Val-PhePhe20) and extremely conserved amino acids in a β strand (β6) that contributes to the catalytic cleft of IDE. Additional important information derived indirectly from the crystal structure is that each IDE monomer may sway between an open and a closed conformer, based on the comparison with substrate-free E. coli pitrilysin (PDB 1Q2L). In pitrilysin, the C-terminal domain is rotated away from the N-terminal domain in such a way that the chamber is widely open to the substrate while in IDE, extensive contacts between the N and C-terminal domains keep this chamber closed. It has been proposed that, after binding, the peptide substrate remains entrapped in the IDE closed conformer during the catalytic cycle. Interestingly, a substrate-free human IDE has been recently crystallized in a closed state, suggesting that this conformer is stable and that reopening of the chamber may be a limiting step to the exit of proteolytic products.12

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Amyloid peptides bind irreversibly with insulin-degrading enzyme

Possible Physiological Significance of IDE-AβSCx

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The immunoprecipitation of IDE from rat and AD brains followed by Western blot with antibodies specific for Aβ carboxyl-terminal epitopes (ending at Val40 or Ala42, respectively) showed a component with the molecular mass of IDE-AβSCx, suggesting that this complex is formed in vivo.13 More recently, we have extended this study to a MALDI-TOF analysis of IDE immunoprecipitated from rat brain followed by in-gel digestion with endo-Lys C. This experiment showed a minor, though clearly detectable peptide with the mass of rodent Aβ 1–28, a predicted cleavage product of Aβ at Lys28-Gly29 by endo-Lys C Figure 1. A proposed model for the formation of IDE-AβSCx. Monomeric Aβ (Aβm) (Fig. 2). A definite biochemical proof awaits the MS/MS may follow a pathway of self-assembly to form oligomers (Aβo) or interact with the fragmentation analysis of this peptide after a large scale binding site (or at least a part of it) of fully active or inactive IDE conformers (IDEa and preparation yet, the available data support that IDE-AβSCx IDEi, respectively) to form a reversible IDE-Aβ encounter complex. IDEa and IDEi may is formed in the brain under physiological conditions. This be or not in equilibrium. IDE-AβSCx formation proceeds very slowly (represented by raises intriguing possibilities regarding IDE-AβSCx signifithe dashed arrows) as an irreversible process that involves a conformational change cance, of which we would like to discuss two examples, both in both molecules. In the case of IDEa-Aβ at neutral pH, the pathway is largely favored toward proteolysis of Aβ and free IDE (not depicted). Alternatively, it may lead to the from IDE and Aβ sides. slow formation of IDE-AβSCx. IDE-AβSCx may reflect a function of IDE alternative to enzymatic activity. The endocytic pathway has been proposed as a main subcellular site of Aβ generation from its precursor protein by β and γ-secretases while the compartment in which IDE-Aβ interaction takes place is at present unknown. IDE is a ubiquitous protein and has been detected in endosomes.26 However, IDE catalytic activity is maximal at pH 7, above the pH within the increasingly acidic environment along the endosomallysosomal pathway and our experiments in vitro indicate that IDE activity falls sharply below pH 6.5 (Llovera RE and Castaño EM, unpublished observations). Therefore, it is possible that within this compartment, an interaction between Aβ and IDE may be strongly displaced toward the formation of IDE-AβSCx, as expected from the combination of a high local Aβ concentration together with a pH-dependent impairment of IDE catalytic activity. Our experiments using an inactive mutant of IDE indicate that IDE-AβSCx is effectively formed in the pH range 5.5–7, supporting this possibility (de Tullio M., unpublished observations). In yeast, the facilitation of bona fide amyloid assembly of the prion [RNQ+] by the HSP40 chaperone Sis1 prevents the formation of soluble, non-amyloid toxic aggregates of the Rnq1 protein. Interestingly, Sis1 has been Figure 2. Endogenous IDE-AβSCx from rat brain. Rat brain IDE was immunoprecipitated with monoclonal anti-IDE 3A2, proteins resolved on SDS-PAGE shown to stably bind to Rnq1 in a 1:1 complex, suggesting that a and the 120 kDa specific band digested in-gel with endo-LysC. Products template-based mechanism is in play. These results indicate that were analyzed by MALDI-TOF as described 13 and 51 peaks were assigned eukaryotic cells are capable of sequestering intracellularly “benign” to IDE (not shown). The depicted spectrum shows a peak (peak A) with the amyloid-like species to prevent the accumulation of more toxic average mass of rodent Aβ1-28 (error