Structure of substrate-free human insulin-degrading enzyme (IDE) and biophysical analysis of ATP-induced conformational switch of IDE

Insulin-degrading enzyme (IDE) is a zinc metalloprotease that hydrolyzes amyloid-beta (Abeta) and insulin, which are peptides associated with Alzheimer disease (AD) and diabetes, respectively. Our previous structural analysis of substrate-bound human 113-kDa IDE reveals that the N- and C-terminal domains of IDE, IDE-N and IDE-C, make substantial contact to form an enclosed catalytic chamber to entrap its substrates. Furthermore, IDE undergoes a switch between the closed and open conformations for catalysis. Here we report a substrate-free IDE structure in its closed conformation, revealing the molecular details of the active conformation of the catalytic site of IDE and new insights as to how the closed conformation of IDE may be kept in its resting, inactive conformation. We also show that Abeta is degraded more efficiently by IDE carrying destabilizing mutations at the interface of IDE-N and IDE-C (D426C and K899C), resulting in an increase in Vmax with only minimal changes to Km. Because ATP is known to activate the ability of IDE to degrade short peptides, we investigated the interaction between ATP and activating mutations. We found that these mutations rendered IDE less sensitive to ATP activation, suggesting that ATP might facilitate the transition from the closed state to the open conformation. Consistent with this notion, we found that ATP induced an increase in hydrodynamic radius, a shift in electrophoretic mobility, and changes in secondary structure. Together, our results highlight the importance of the closed conformation for regulating the activity of IDE and provide new molecular details that will facilitate the development of activators and inhibitors of IDE.     

The catalytic domain of insulin-degrading enzyme forms a denaturant-resistant complex with amyloid beta peptide: implications for Alzheimer disease pathogenesis

Insulin-degrading enzyme (IDE) is central to the turnover of insulin and degrades amyloid beta (Abeta) in the mammalian brain. Biochemical and genetic data support the notion that IDE may play a role in late onset Alzheimer disease (AD), and recent studies suggest an association between AD and diabetes mellitus type 2. Here we show that a natively folded recombinant IDE was capable of forming a stable complex with Abeta that resisted dissociation after treatment with strong denaturants. This interaction was also observed with rat brain IDE and detected in an SDS-soluble fraction from AD cortical tissue. Abeta sequence 17-27, known to be crucial in amyloid assembly, was sufficient to form a stable complex with IDE. Monomeric as opposed to aggregated Abeta was competent to associate irreversibly with IDE following a very slow kinetics (t(1/2) approximately 45 min). Partial denaturation of IDE as well as preincubation with a 10-fold molar excess of insulin prevented complex formation, suggesting that the irreversible interaction of Abeta takes place with at least part of the substrate binding site of the protease. Limited proteolysis showed that Abeta remained bound to a approximately 25-kDa N-terminal fragment of IDE in an SDS-resistant manner. Mass spectrometry after in gel digestion of the IDE .Abeta complex showed that peptides derived from the region that includes the catalytic site of IDE were recovered with Abeta. Taken together, these results are suggestive of an unprecedented mechanism of conformation-dependent substrate binding that may perturb Abeta clearance, insulin turnover, and promote AD pathogenesis.     

Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme

Insulin is a hormone vital for glucose homeostasis, and insulin-degrading enzyme (IDE) plays a key role in its clearance. IDE exhibits a remarkable specificity to degrade insulin without breaking the disulfide bonds that hold the insulin A and B chains together. Using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain high mass accuracy, and electron capture dissociation (ECD) to selectively break the disulfide bonds in gas phase fragmentation, we determined the cleavage sites and composition of human insulin fragments generated by human IDE. Our time-dependent analysis of IDE-digested insulin fragments reveals that IDE is highly processive in its initial cleavage at the middle of both the insulin A and B chains. This ensures that IDE effectively splits insulin into inactive N- and C-terminal halves without breaking the disulfide bonds. To understand the molecular basis of the recognition and unfolding of insulin by IDE, we determined a 2.6-Å resolution insulin-bound IDE structure. Our structure reveals that IDE forms an enclosed catalytic chamber that completely engulfs and intimately interacts with a partially unfolded insulin molecule. This structure also highlights how the unique size, shape, charge distribution, and exosite of the IDE catalytic chamber contribute to its high affinity (∼100 nm) for insulin. In addition, this structure shows how IDE utilizes the interaction of its exosite with the N terminus of the insulin A chain as well as other properties of the catalytic chamber to guide the unfolding of insulin and allowing for the processive cleavages.IDE³ is an ∼110-kDa zinc metalloprotease that is evolutionarily conserved from bacteria to humans (1, 2). It was first discovered based on its high affinity to bind insulin (∼100 nm) and degrade it into pieces (3, 4). Insulin is a 5.8-kDa hormone that plays a central role in glucose homeostasis and the development of diabetes in humans. Consistent with the in vitro activity of IDE for insulin degradation, loss-of-function mutations of IDE in rodents result in elevated insulin levels and glucose intolerance (5). In addition, a nucleotide polymorphism of the human IDE gene is linked to type 2 diabetes (6). Later studies showed that IDE can also degrade amyloid-β (Aβ), a peptide vital to the progression of Alzheimer disease (7, 8). Accumulating evidence from rodent models and human genetic analyses also indicate the physiological role of IDE in the clearance of Aβ (5, 9–12).Despite nearly 60 years of studies on IDE, the molecular basis by which IDE binds, unfolds, and degrades insulin has only begun to be elucidated. Different from ATP-dependent proteases, IDE does not require the additional energy source such as ATP to unfold, bind, and cleave its substrates (4, 13). Insulin consists of the A and B chains that are held together by two inter- and one intra-chain disulfide bonds. Remarkably, IDE does not require disulfide bond isomerase activity to unfold and cleave insulin (4). Thus, IDE needs to overcome the stability created by the disulfide bonds of insulin. Structural analysis reveals that human IDE contains a catalytic chamber formed by the internal cavity of two roughly equally sized ∼55-kDa N- and C-terminal halves (IDE-N and IDE-C, respectively) (2). Within this chamber, only one catalytic center exists. However, IDE cleaves insulin at multiple sites on both the insulin A and B chains to completely inactivate this hormone. It remains unclear whether the cleavages of insulin by IDE proceed in a sequential or stochastic manner.IDE represents an emerging protease family that utilizes an enclosed catalytic chamber to selectively recognize and unfold the substrates for their degradation (1). The volume of the enclosed chamber of IDE (∼16,000 ų) allows the preferential exclusion of peptides that are greater than ∼75 amino acids long. This chamber also has unique electrostatic properties; the internal cavity of IDE-N is predominantly negative, whereas that of IDE-C is positive. Inside the catalytic chamber, IDE has an exosite that is an evolutionarily conserved substrate-binding site ∼30 Å away from the catalytic groove. This exosite is used to anchor the N-terminal end of IDE substrates. The unique size, electrostatic potential, and exosite of Ides'' catalytic chamber are postulated as key factors for the selective binding and unfolding of IDE substrates (1, 2, 14). In addition, one common feature among the known IDE substrates is their higher propensity to form amyloid fibers (8). Amyloidogenic peptides tend to unfold by themselves, which could facilitate their unfolding and subsequent cleavage by IDE. However, the molecular basis of how the catalytic chamber of IDE binds, unfolds, and cleaves insulin into pieces and how the flexibility of this substrate contributes to its cleavage by IDE remain elusive.IDE is known to cut insulin at multiple sites, and the resulting cleavage products are quite complex (4, 15–18). Here we took advantage of the high mass accuracy of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and the selective targeting of disulfide bonds by electron capture dissociation (ECD) in our mass spectrometry (MS) analysis to unambiguously identify IDE-degraded fragments of human insulin, as well as the time-dependent production of these fragments. We also present a 2.6- Å insulin-bound IDE structure, revealing extensive shape and charge complementarity of the partially unfolded insulin with the enclosed catalytic chamber and a potential path for the unfolding of insulin. Together, our data elucidate the molecular basis by which IDE engulfs, unfolds, and effectively cleaves insulin into pieces.     

Differential degradation of amyloid beta genetic variants associated with hereditary dementia or stroke by insulin-degrading enzyme

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid beta (Abeta) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Abeta wild type, yet whether IDE is capable of degrading Abeta bearing pathogenic substitutions is not known. We studied the degradation of all of the published Abeta genetic variants by recombinant rat IDE (rIDE). Monomeric Abeta wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Abeta Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Abeta wild type and the rest of the mutant peptides. In the case of Abeta Dutch, inefficient proteolysis was related to a high content of beta structure as assessed by circular dichroism. All of the Abeta variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13-15 and 18-21. SDS-stable Abeta dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Abeta. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Abeta in hereditary forms of Abeta diseases.     

Insulin-degrading enzyme in brain microvessels: proteolysis of amyloid {beta} vasculotropic variants and reduced activity in cerebral amyloid angiopathy

The accumulation of amyloid beta (Abeta) in the walls of small vessels in the cerebral cortex is associated with diseases characterized by dementia or stroke. These include Alzheimer's disease, Down syndrome, and sporadic and hereditary cerebral amyloid angiopathies (CAAs) related to mutations within the Abeta sequence. A higher tendency of Abeta to aggregate, a defective clearance to the systemic circulation, and insufficient proteolytic removal have been proposed as mechanisms that lead to Abeta accumulation in the brain. By using immunoprecipitation and mass spectrometry, we show that insulin-degrading enzyme (IDE) from isolated human brain microvessels was capable of degrading (125)I-insulin and cleaved Abeta-(1-40) wild type and the genetic variants Abeta A21G (Flemish), Abeta E22Q (Dutch), and Abeta E22K (Italian) at the predicted sites. In microvessels from Alzheimer's disease cases with CAA, IDE protein levels showed a 44% increase as determined by sandwich enzyme-linked immunosorbent assay and Western blot. However, the activity of IDE upon radiolabeled insulin was significantly reduced in CAA as compared with age-matched controls. These results support the notion that a defect in Abeta proteolysis by IDE contributes to the accumulation of this peptide in the cortical microvasculature. Moreover they raise the possibility that IDE inhibition or inactivation is a pathogenic mechanism that may open novel strategies for the treatment of cerebrovascular Abeta amyloidoses.     

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.     

Deletion of the Fission Yeast Homologue of Human Insulinase Reveals a TORC1-Dependent Pathway Mediating Resistance to Proteotoxic Stress

Insulin Degrading Enzyme (IDE) is a protease conserved through evolution with a role in diabetes and Alzheimer''s disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1) modulates cellular sensitivity to endoplasmic reticulum (ER) stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1). Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in iph1 null cells when the TORC1 complex was inhibited by rapamycin or by heat inactivation of the Tor2 kinase. Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of iph1⁺ promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway.     

A Monomeric Variant of Insulin Degrading Enzyme (IDE) Loses Its Regulatory Properties

Background

Insulin degrading enzyme (IDE) is a key enzyme in the metabolism of both insulin and amyloid beta peptides. IDE is unique in that it is subject to allosteric activation which is hypothesized to occur through an oligomeric structuture.

Methodology/Principal Findings

IDE is known to exist as an equilibrium mixture of monomers, dimers, and higher oligomers, with the dimer being the predominant form. Based on the crystal structure of IDE we deleted the putative dimer interface in the C-terminal region, which resulted in a monomeric variant. Monomeric IDE retained enzymatic activity, however instead of the allosteric behavior seen with wild type enzyme it displayed Michaelis-Menten kinetic behavior. With the substrate Abz-GGFLRKHGQ-EDDnp, monomeric IDE retained ∼25% of the wild type activity. In contrast with the larger peptide substrates β-endorphin and amyloid β peptide 1–40, monomeric IDE retained only 1 to 0.25% of wild type activity. Unlike wild type IDE neither bradykinin nor dynorphin B-9 activated the monomeric variant of the enzyme. Similarly, monomeric IDE was not activated by polyphosphates under conditions in which the activity of wild type enzyme was increased more than 50 fold.

Conclusions/Significance

These findings serve to establish the dimer interface in IDE and demonstrate the requirement for an oligomeric form of the enzyme for its regulatory properties. The data support a mechanism where the binding of activators to oligomeric IDE induces a conformational change that cannot occur in the monomeric variant. Since a conformational change from a closed to a more open structure is likely the rate-determining step in the IDE reaction, the subunit induced conformational change likely shifts the structure of the oligomeric enzyme to a more open conformation.     

Identification of the allosteric regulatory site of insulysin

Background

Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the Aβ peptide associated with Alzheimer''s disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP.

Principal Findings

The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric kinetics and activation by a small peptide, as well as greatly reducing activation by ATP, demonstrating that this site plays a key role in allostery. Comparison of the peptide bound IDE structure (using a low activity E111F IDE mutant) with unliganded wild type IDE shows a change in the interface between two halves of the clamshell-like molecule, which may enhance enzyme activity by altering the equilibrium between closed and open conformations. In addition, changes in the dimer interface suggest a basis for communication between subunits.

Conclusions/Significance

Our findings indicate that a region remote from the active site mediates allosteric activation of insulysin by peptides. Activation may involve a small conformational change that weakens the interface between two halves of the enzyme.     

Mutation of active site residues of insulin-degrading enzyme alters allosteric interactions

The active site glutamate (Glu(111)) and the active site histidine (His(112)) of insulin-degrading enzyme (IDE) were mutated. These mutant enzymes exhibit, in addition to a large decrease in catalytic activity, a change in the substrate-velocity response from a sigmoidal one seen with the native enzyme (Hill coefficient > 2), to a hyperbolic response. With 2-aminobenzoyl-GGFLRKHGQ-N-(2,4-dinitrophenyl)ethylenediamine as substrate, ATP and triphosphate increase the reaction rate of the wild type enzyme some 50-80-fold. This effect is dampened with glutamate mutants to no effect or less than a 3-fold increase in activity and changed to inhibition with the histidine mutants. Sedimentation equilibrium shows the IDE mutants exhibit a similar oligomeric distribution as the wild type enzyme, being predominantly monomeric, with triphosphate having little if any effect on the oligomeric state. Triphosphate did induce aggregation of many of the IDE mutants. Thus, the oligomeric state of IDE does not correlate with kinetic properties. The His(112) mutants were shown to bind zinc, but with a lower affinity than the wild type enzyme. The glutamate mutants displayed an altered cleavage profile for the peptide beta-endorphin. Wild type IDE cleaved beta-endorphin at Leu(17)-Phe(18) and Phe(18)-Lys(19), whereas the glutamate mutants cleaved at these sites, but in addition at Lys(19)-Asn(20) and at Met(5)-Thr(6). Thus, active site mutations of IDE are suggested to not only reduce catalytic activity but also cause local conformational changes that affect the allosteric properties of the enzyme.     

染色质免疫沉淀技术分析HePG2细胞TCF7L2蛋白与IDE基因转录启动子的结合

TCF7L2是一种重要的转录因子,通过Wnt信号途径,调节葡萄糖代谢.胰岛素降解酶(IDE)是细胞水平催化胰岛素降解的最关键的酶,与2型糖尿病(T2DM)高血糖、胰岛素抵抗、高胰岛素血症密切相关.为了检测HePG2细胞内转录因子TCF7L2与IDE基因启动子区的结合情况,采用染色质免疫沉淀技术结合PCR技术检测IDE基因启动子序列.结果表明,在特异性TCF7L2抗体免疫沉淀的DNA片段中扩增出IDE基因启动子序列,因此证实在HePG2细胞内,TCF7L2蛋白可与IDE基因转录启动子的特异区域结合,进而可能参与IDE基因的表达调控.     

IL-4-induced selective clearance of oligomeric beta-amyloid peptide(1-42) by rat primary type 2 microglia

A hallmark of immunopathology associated with Alzheimer's disease is the presence of activated microglia (MG) surrounding senile plaque deposition of beta-amyloid (Abeta) peptides. Abeta peptides are believed to be potent activators of MG, which leads to Alzheimer's disease pathology, but the role of MG subtypes in Abeta clearance still remains unclear. In this study, we found that IL-4 treatment of rat primary-type 2 MG enhanced uptake and degradation of oligomeric Abeta(1-42) (o-Abeta(1-42)). IL-4 treatment induced significant expression of the scavenger receptor CD36 and the Abeta-degrading enzymes neprilysin (NEP) and insulin-degrading enzyme (IDE) but reduced expression of certain other scavenger receptors. Of cytokines and stimulants tested, the anti-inflammatory cytokines IL-4 and IL-13 effectively enhanced CD36, NEP, and IDE. We demonstrated the CD36 contribution to IL-4-induced Abeta clearance: Chinese hamster ovary cells overexpressing CD36 exhibited marked, dose-dependent degradation of (125)I-labeled o-Abeta(1-42) compared with controls, the degradation being blocked by anti-CD36 Ab. Also, we found IL-4-induced clearance of o-Abeta(1-42) in type 2 MG from CD36-expressing WKY/NCrj rats but not in cells from SHR/NCrj rats with dysfunctional CD36 expression. NEP and IDE also contributed to IL-4-induced degradation of Abeta(1-42), because their inhibitors, thiorphan and insulin, respectively, significantly suppressed this activity. IL-4-stimulated uptake and degradation of o-Abeta(1-42) were selectively enhanced in type 2, but not type 1 MG that express CD40, which suggests that the two MG types may play different neuroimmunomodulating roles in the Abeta-overproducing brain. Thus, selective o-Abeta(1-42) clearance, which is induced by IL-4, may provide an additional focus for developing strategies to prevent and treat Alzheimer's disease.     

Structure based discovery of small molecules to regulate the activity of human insulin degrading enzyme

Background

Insulin-degrading enzyme (IDE) is an allosteric Zn⁺² metalloprotease involved in the degradation of many peptides including amyloid-β, and insulin that play key roles in Alzheimer''s disease (AD) and type 2 diabetes mellitus (T2DM), respectively. Therefore, the use of therapeutic agents that regulate the activity of IDE would be a viable approach towards generating pharmaceutical treatments for these diseases. Crystal structure of IDE revealed that N-terminal has an exosite which is ∼30 Å away from the catalytic region and serves as a regulation site by orientation of the substrates of IDE to the catalytic site. It is possible to find small molecules that bind to the exosite of IDE and enhance its proteolytic activity towards different substrates.

Methodology/Principal Findings

In this study, we applied structure based drug design method combined with experimental methods to discover four novel molecules that enhance the activity of human IDE. The novel compounds, designated as D3, D4, D6, and D10 enhanced IDE mediated proteolysis of substrate V, insulin and amyloid-β, while enhanced degradation profiles were obtained towards substrate V and insulin in the presence of D10 only.

Conclusion/Significance

This paper describes the first examples of a computer-aided discovery of IDE regulators, showing that in vitro and in vivo activation of this important enzyme with small molecules is possible.     

Decreased catalytic activity of the insulin-degrading enzyme in chromosome 10-linked Alzheimer disease families

Insulin-degrading enzyme (IDE) is a zinc metalloprotease that degrades the amyloid beta-peptide, the key component of Alzheimer disease (AD)-associated senile plaques. We have previously reported evidence for genetic linkage and association of AD on chromosome 10q23-24 in the region harboring the IDE gene. Here we have presented the first functional assessment of IDE in AD families showing the strongest evidence of the genetic linkage. We have examined the catalytic activity and expression of IDE in lymphoblast samples from 12 affected and unaffected members of three chromosome 10-linked AD pedigrees in the National Institute of Mental Health AD Genetics Initiative family sample. We have shown that the catalytic activity of cytosolic IDE to degrade insulin is reduced in affected versus unaffected subjects of these families. Further, we have shown the decrease in activity is not due to reduced IDE expression, suggesting the possible defects in IDE function in these AD families. In attempts to find potential mutations in the IDE gene in these families, we have found no coding region substitutions or alterations in splicing of the canonical exons and exon 15b of IDE. We have also found that total IDE mRNA levels are not significantly different in sporadic AD versus age-matched control brains. Collectively, our data suggest that the genetic linkage of AD in this set of chromosome 10-linked AD families may be the result of systemic defects in IDE activity in the absence of altered IDE expression, further supporting a role for IDE in AD pathogenesis.     

Degradation of Alzheimer’s Amyloid-β by a Catalytically Inactive Insulin-Degrading Enzyme

It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer’s amyloid-β (Aβ). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aβ degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aβ(1–40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aβ(1–40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aβ fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aβ(1–40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aβ(1–40) inactivates cf-E111Q-IDE’s catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aβ degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE’s function.     

Insulin-degrading enzyme rapidly removes the beta-amyloid precursor protein intracellular domain (AICD).

The intramembranous gamma-secretase cleavage of the beta-amyloid precursor protein (APP) is dependent on biologically active presenilins (PS). Notch also undergoes a similar PS-dependent gamma-secretase-like cleavage, resulting in the liberation of the Notch intracellular domain (NICD), which is critically required for developmental signal transduction. gamma-Secretase processing of APP results in the production of a similar fragment called AICD (APP intracellular domain), which may function in nuclear signaling as well. AICD, like NICD, is rapidly removed. By using a battery of protease inhibitors we demonstrate that AICD, in contrast to NICD, is degraded by a cytoplasmic metalloprotease. In vitro degradation of AICD can be reconstituted with cytoplasmic fractions obtained from neuronal and non-neuronal cells. Taking into account the inhibition profile and the cytoplasmic localization, we identified three candidate enzymes (neurolysin, thimet oligopeptidase, and insulin-degrading enzyme (IDE), also known as insulysin), which all are involved in the degradation of bioactive peptides in the brain. When insulin, a well characterized substrate of IDE, was added to the in vitro degradation assay, removal of AICD was efficiently blocked. Moreover, overexpression of IDE resulted in enhanced degradation of AICD, whereas overexpression of the inactive IDE E111Q mutant did not affect AICD degradation. Finally, immunodepletion of IDE significantly reduced the AICD degrading activity. Therefore our data demonstrate that IDE, which is one of the proteases implicated in the removal of extracellular Abeta, also removes the cytoplasmic product of gamma-secretase cleaved APP.     

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 V_max 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.     

Natural regulatory mechanisms of insulin degradation by insulin degrading enzyme

Insulin-degrading enzyme (IDE) accounts for most of the insulin degrading activity in extracts of several tissues and plays an important role in the intracellular degradation of insulin. Using newly developed sandwich radioimmunoassay for rat IDE, this enzyme was detectable in all tissues we examined and liver had the highest level of IDE. The ratio of insulin degrading activity to IDE concentration was roughly the same in liver, brain and muscle, however, twice as high in kidney as compared with other tissues. On the contrary, its degrading activity in these tissue extracts, including kidney, was completely lost after immunoprecipitation of IDE. These results suggest that IDE degrades insulin in the initial step of cleavage and that there are some mechanisms to regulate insulin degrading activity by IDE in the tissues.     

Identification of residues in the insulin molecule important for binding to insulin-degrading enzyme

Insulin-degrading enzyme (IDE) hydrolyzes insulin at a limited number of sites. Although the positions of these cleavages are known, the residues of insulin important in its binding to IDE have not been defined. To this end, we have studied the binding of a variety of insulin analogues to the protease in a solid-phase binding assay using immunoimmobilized IDE. Since IDE binds insulin with 600-fold greater affinity than it does insulin-like growth factor I (25 nM and approximately 16,000 nM, respectively), the first set of analogues studied were hybrid molecules of insulin and IGF I. IGF I mutants [insB1-17,17-70]IGF I, [Tyr55,Gln56]IGF I, and [Phe23,Phe24,Tyr25]IGF I have been synthesized and share the property of having insulin-like amino acids at positions corresponding to primary sites of cleavage of insulin by IDE. Whereas the first two exhibit affinities for IDE similar to that of wild type IGF I, the [Phe23,Phe24,Tyr25]IGF I analogue has a 32-fold greater affinity for the immobilized enzyme. Replacement of Phe-23 by Ser eliminates this increase. Removal of the eight amino acid D-chain region of IGF I (which has been predicted to interfere with binding to the 23-25 region) results in a 25-fold increase in affinity for IDE, confirming the importance of residues 23-25 in the high-affinity recognition of IDE. A similar role for the corresponding (B24-26) residues of insulin is supported by the use of site-directed mutant and semisynthetic insulin analogues. Insulin mutants [B25-Asp]insulin and [B25-His]insulin display 16- and 20-fold decreases in IDE affinity versus wild-type insulin.(ABSTRACT TRUNCATED AT 250 WORDS)