Functional Human Insulin-Degrading Enzyme Can Be Expressed in Bacteria

Chesneau, Valérie; Rosner, Marsha Rich

doi:10.1006/prep.2000.1217

Cited by 41 publications

(39 citation statements)

References 38 publications

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“…1B). These results are fully consistent with the reported characterization of human recombinant IDE expressed in E. coli (34) and rat recombinant IDE produced in a baculovirus-insect cell system (32).…”

Section: Expression and Characterization Of Recombinant Rat Ide-supporting

confidence: 92%

“…The PCR DNA fragment was digested with BamHI and XhoI and cloned into pET-30a(ϩ) (Novagen) previously digested with the same restriction enzymes to generate the pET-IDE construct. Recombinant IDE 42-1019 (rIDE) was expressed in Escherichia coli BL21 and purified to homogeneity using a Hi Trap Ni-chelating columm (Amersham Biosciences) following the method described by Chesneau and Rosner (34). Purity, as assessed by SDS-PAGE, was Ͼ95%.…”

Section: Methodsmentioning

confidence: 99%

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Differential Degradation of Amyloid β Genetic Variants Associated with Hereditary Dementia or Stroke by Insulin-degrading Enzyme

Morelli

Llovera

González

et al. 2003

Journal of Biological Chemistry

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Inherited amino acid substitutions at position 21, 22, or 23 of amyloid ␤ (A␤) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze A␤ wild type, yet whether IDE is capable of degrading A␤ bearing pathogenic substitutions is not known. We studied the degradation of all of the published A␤ genetic variants by recombinant rat IDE (rIDE). Monomeric A␤ wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of A␤ Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with A␤ wild type and the rest of the mutant peptides. In the case of A␤ Dutch, inefficient proteolysis was related to a high content of ␤ structure as assessed by circular dichroism. All of the A␤ variants were cleaved at Glu 3 -Phe 4 and Phe 4 -Arg 5 in addition to the previously described major sites within positions 13-15 and 18 -21. SDS-stable A␤ 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 A␤. These results raise the possibility that upregulation of IDE may promote the clearance of soluble A␤ in hereditary forms of A␤ diseases.The accumulation of amyloid ␤ peptide (A␤) 1 in the brain is a central process in a number of human neurodegenerative disorders that may be grouped as "amyloid ␤ diseases" (1). In Alzheimer's disease, A␤ is mainly found within senile plaques in the neurophil and vascular lesions, whereas in sporadic and hereditary amyloid angiopathies, A␤ deposits are mainly associated with cortical and leptomeningeal vessels leading to stroke or multi-infarct dementia. To a lesser extent, cerebral A␤ deposits are also present in normal aging (2). Autosomal dominant mutations in the amyloid ␤ precursor protein (A␤ PP) gene result in amino acid substitutions at position 21, 22, or 23 of A␤ sequence. Although these A␤ variants present with a primarily vascular deposition, they translate into different clinical phenotypes. In this regard, A␤ Arctic (E22G) and A␤ Iowa (D23N) are characterized by presenile dementia and A␤ Flemish (A21G) is associated with early onset dementia and cerebral hemorrhage, whereas A␤ Dutch (E22Q) and A␤ Italian (E22K) variants have a predominant vascular phenotype characterized by massive strokes (3-7). The underlying mechanism of aggregation and deposition in vivo may be strongly influenced by the type of amino acid substitution as well as the location of mutations in the A␤ peptide. In vitro studies have shown that A␤ E22Q and A␤ D23N form typical amyloid fibrils at a higher rate than wild-type A␤ and that A␤ E22G assembles into unique protofibrils that may be toxic to neurons (3, 8 -10). In the case of A␤ A21G, overproduction of the peptide may contribute as a pathogenic mechanism (11, 12). Regarding A␤ E22K, deposition seems not to be related with fibril formation rate, and yet this variant may be toxic to human cerebrovascular smooth muscle cells in cult...

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Section: Expression and Characterization Of Recombinant Rat Ide-supporting

confidence: 92%

Section: Methodsmentioning

confidence: 99%

Differential Degradation of Amyloid β Genetic Variants Associated with Hereditary Dementia or Stroke by Insulin-degrading Enzyme

Morelli

Llovera

González

et al. 2003

Journal of Biological Chemistry

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“…Using 80 M insulin, which is significantly higher than the K m value (85 nM), and the time-dependent production of a major cleavage product, 2,355 Da, the estimated catalytic rate is ϳ2 s Ϫ1 . This is in reasonable agreement with the known rate (0.5 s Ϫ1 ) determined by using 125 I-labeled insulin (33).…”

Section: Maldi-tof Analysis Of the Degradation Of Human Insulinsupporting

confidence: 90%

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

Manolopoulou

Guo

Malito

et al. 2009

Journal of Biological Chemistry

174

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Insulin is a hormone vital for glucose homeostasis, and insulindegrading 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 timedependent 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 (ϳ100 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 ϳ110-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 (ϳ100 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-␤ (A␤), 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 A␤ (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 d...

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“…Rabbit anti-IDE antibody IDE-1 was directed against amino acids 56 to 68 of human IDE exon 2 (24). Rabbit anti-IDE antibody UCG43 was a gift from Marsha Rosner (University of Chicago) (9). Anti-hemagglutinin (anti-HA) monoclonal antibody HA.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids encoding Histagged wild-type human IDE and IDE mutants IDE-E111Q, IDE-N, and IDE-C were expressed and purified as described previously (9,23). Briefly, E. coli Rosetta cells (Novagene/EMD Biosciences, San Diego, CA) were transformed with the plasmids, and the cells were cultured at 30°C until the optical density at 600 nm reached 0.6.…”

mentioning

confidence: 99%

The Amino Terminus of Varicella-Zoster Virus (VZV) Glycoprotein E Is Required for Binding to Insulin-Degrading Enzyme, a VZV Receptor

Krogmann

Ali

et al. 2007

J Virol

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Varicella-zoster virus (VZV) glycoprotein E (gE) is required for VZV infection.Although gE is well conserved among alphaherpesviruses, the amino terminus of VZV gE is unique. Previously, we showed that gE interacts with insulin-degrading enzyme (IDE) and facilitates VZV infection and cell-to-cell spread of the virus. Here we define the region of VZV gE required to bind IDE. Deletion of amino acids 32 to 71 of gE, located immediately after the predicted signal peptide, resulted in loss of the ability of gE to bind IDE. A synthetic peptide corresponding to amino acids 24 to 50 of gE blocked its interaction with IDE in a concentration-dependent manner. However, a chimeric gE in which amino acids 1 to 71 of VZV gE were fused to amino acids 30 to 545 of herpes simplex virus type 2 gE did not show an increased level of binding to IDE compared with that of full-length HSV gE. Thus, amino acids 24 to 71 of gE are required for IDE binding, and the secondary structure of gE is critical for the interaction. VZV gE also forms a heterodimer with glycoprotein gI. Deletion of amino acids 163 to 208 of gE severely reduced its ability to form a complex with gI. The amino portion of IDE, as well an IDE mutant in the catalytic domain of the protein, bound to gE. Therefore, distinct motifs of VZV gE are important for binding to IDE or to gI.Varicella-zoster virus (VZV) is a member of the alphaherpesvirus subfamily. VZV causes chickenpox during primary infection, in which it disseminates throughout the body. The virus establishes latency in dorsal root and cranial nerve ganglia and can reactivate to cause shingles. During acute infections, VZV is probably transmitted to other persons as a cellfree virus; during viremia, the virus circulates in T cells (1). In cell cultures, VZV is highly cell associated, and cell-to-cell spread is the exclusive route of infection.The early events of VZV infection are similar to those observed with the other human alphaherpesvirus, herpes simplex virus (HSV). Both viruses attach to cell surface heparan sulfate and engage cellular receptor(s) (44, 49). Mannose 6-phosphate inhibits the infectivity of cell-free VZV, which implicates the cation-independent mannose 6-phosphate receptor (MPR ci ) in facilitating entry of cell-free viruses by interacting with viral glycoproteins that contain phosphorylated N-linked complex oligosaccharides (17,49). A soluble form of mannose 6-phosphate receptor blocks cell-to-cell spread of HSV, and HSV gD colocalizes with the mannose 6-phosphate receptor in endosomes (5, 6). The involvement of MPR ci at an early step of VZV infection is further supported by the finding that stable MPR ci -deficient cell lines are refractory to infection by cellfree VZV (8).Previously, we showed that insulin-degrading enzyme (IDE) binds VZV glycoprotein E (gE) and serves as a cellular receptor for the virus (24). Down-regulation of IDE by small interfering RNAs or blocking of IDE by antibodies or by bacitracin inhibited VZV infection and impaired the cell-to-cell spread of the virus. Human ...

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Functional Human Insulin-Degrading Enzyme Can Be Expressed in Bacteria

Cited by 41 publications

References 38 publications

Differential Degradation of Amyloid β Genetic Variants Associated with Hereditary Dementia or Stroke by Insulin-degrading Enzyme

Differential Degradation of Amyloid β Genetic Variants Associated with Hereditary Dementia or Stroke by Insulin-degrading Enzyme

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

The Amino Terminus of Varicella-Zoster Virus (VZV) Glycoprotein E Is Required for Binding to Insulin-Degrading Enzyme, a VZV Receptor

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