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• W2005026342 abstract "Insulin-degrading enzyme (IDE) is central to the turnover of insulin and degrades amyloid β (Aβ) 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 Aβ 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. Aβ sequence 17–27, known to be crucial in amyloid assembly, was sufficient to form a stable complex with IDE. Monomeric as opposed to aggregated Aβ was competent to associate irreversibly with IDE following a very slow kinetics (t½ ∼ 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 Aβ takes place with at least part of the substrate binding site of the protease. Limited proteolysis showed that Aβ remained bound to a ∼25-kDa N-terminal fragment of IDE in an SDS-resistant manner. Mass spectrometry after in gel digestion of the IDE ·Aβ complex showed that peptides derived from the region that includes the catalytic site of IDE were recovered with Aβ. Taken together, these results are suggestive of an unprecedented mechanism of conformation-dependent substrate binding that may perturb Aβ clearance, insulin turnover, and promote AD pathogenesis. Insulin-degrading enzyme (IDE) is central to the turnover of insulin and degrades amyloid β (Aβ) 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 Aβ 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. Aβ sequence 17–27, known to be crucial in amyloid assembly, was sufficient to form a stable complex with IDE. Monomeric as opposed to aggregated Aβ was competent to associate irreversibly with IDE following a very slow kinetics (t½ ∼ 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 Aβ takes place with at least part of the substrate binding site of the protease. Limited proteolysis showed that Aβ remained bound to a ∼25-kDa N-terminal fragment of IDE in an SDS-resistant manner. Mass spectrometry after in gel digestion of the IDE ·Aβ complex showed that peptides derived from the region that includes the catalytic site of IDE were recovered with Aβ. Taken together, these results are suggestive of an unprecedented mechanism of conformation-dependent substrate binding that may perturb Aβ clearance, insulin turnover, and promote AD pathogenesis. Several neurodegenerative disorders are associated with the progressive accumulation of amyloid β (Aβ) 3The abbreviations used are: Aβ, amyloid β; ACN, acetonitrile; AD, Alzheimer disease; APP, amyloid precursor protein; endo-LysC, endoprotease lysine C; CysC, cystatin C; ELISA, enzyme-linked immunoadsorbent assay; FAβ, fluorescein-labeled Aβ; GRF, growth hormone releasing factor; HCHWA-D, hereditary cerebral hemorrhage with amyloidosis, Dutch type; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; HPLC, high performance liquid chromatography; IDE, insulin-degrading enzyme; IDE ·AβSCx, IDE ·Aβ stable complex; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; PVDF, polyvinylidene difluoride; rIDE, recombinant IDE; TBS, Tris-buffered saline; SB, sample buffer; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 3The abbreviations used are: Aβ, amyloid β; ACN, acetonitrile; AD, Alzheimer disease; APP, amyloid precursor protein; endo-LysC, endoprotease lysine C; CysC, cystatin C; ELISA, enzyme-linked immunoadsorbent assay; FAβ, fluorescein-labeled Aβ; GRF, growth hormone releasing factor; HCHWA-D, hereditary cerebral hemorrhage with amyloidosis, Dutch type; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; HPLC, high performance liquid chromatography; IDE, insulin-degrading enzyme; IDE ·AβSCx, IDE ·Aβ stable complex; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; PVDF, polyvinylidene difluoride; rIDE, recombinant IDE; TBS, Tris-buffered saline; SB, sample buffer; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. in the brain, including Alzheimer disease (AD) (reviewed in Ref. 1Morelli L. Llovera R. Ibendhal S. Castanño E.M. Neurochem. Res. 2002; 27: 1387-1399Crossref PubMed Scopus (26) Google Scholar). Aβ accumulation has been attributed to several convergent mechanisms. These include a high rate of Aβ production, a fast kinetic of aggregation, and a defective clearance in the brain as the result of impaired transport, protein-protein interactions, or specific protease deficiencies (reviewed in Ref. 1Morelli L. Llovera R. Ibendhal S. Castanño E.M. Neurochem. Res. 2002; 27: 1387-1399Crossref PubMed Scopus (26) Google Scholar). The first two mechanisms may account for the accelerated Aβ deposition in rare hereditary disorders caused by mutations in the amyloid β precursor protein (APP) or presenilin genes, whereas the latter may be relevant in the complex pathogenesis of the more frequent sporadic AD. It is now accepted that self-assembly of Aβ follows a nucleation kinetic (2Harper J.D. Lansbury Jr., P.T. Annu. Rev. Biochem. 1997; 66: 385-407Crossref PubMed Scopus (1403) Google Scholar). In this process, nucleation takes place above the critical concentration of the peptide, and, therefore, the transient and steady levels of monomeric Aβ in the brain and the mechanisms that regulate them acquire a major importance. Although the input of Aβ depends largely on the rate of proteolytic release from APP, its clearance depends on diffusion, transport, and degradation. Neprilysin, insulin-degrading enzyme (IDE), and endothelin-converting enzyme are thought to be Aβ proteases of major physiological relevance, as shown by gene knock-out and overexpression animal models (3Iwata N. Tsubuki S. Takaki Y. Shirotani K. Lu B. Gerard N.P. Gerard C. Hama E. Lee H.J. Saido T.C. Science. 2001; 292: 1550-1552Crossref PubMed Scopus (828) Google Scholar, 4Farris W. Mansourian S. Chang Y. Lindsley L. Eckman E.A. Frosch M.P. Eckman C.B. Tanzi R.E. Selkoe D.J. Guenette S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4162-4167Crossref PubMed Scopus (1185) Google Scholar, 5Miller B.C. Eckman E.A. Sambamurti K. Dobbs N. Chow K.M. Eckman C.B. Hersh L.B. Thiele D.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6221-6226Crossref PubMed Scopus (261) Google Scholar, 6Eckman E.A. Watson M. Marlow L. Sambamurti K. Eckman C.B. J. Biol. Chem. 2003; 278: 2081-2084Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 7Leissring M.A. Farris W. Chang A.Y. Walsh D.M. Wu X. Sun X. Frosch M.P. Selkoe D.J. Neuron. 2003; 40: 1087-1093Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar). IDE is a highly conserved and ubiquitous Zn2+ metalloendopeptidase that belongs to the M16 family defined by an “inverted” canonical sequence in the active site (HXXEH instead of HEXXH), as compared with other members of the clan (8Roth R Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Elsevier Academic Press, New York2004: 868-876Crossref Scopus (3) Google Scholar). Although the precise physiological role of IDE is unknown, its deficiency after gene targeting in mice leads to a biochemical phenotype that includes hyperinsulinemia, glucose intolerance, elevated levels of soluble Aβ in the brain, and a substantial increase in the ∼50-residue C-terminal intracellular domain of APP (4Farris W. Mansourian S. Chang Y. Lindsley L. Eckman E.A. Frosch M.P. Eckman C.B. Tanzi R.E. Selkoe D.J. Guenette S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4162-4167Crossref PubMed Scopus (1185) Google Scholar, 5Miller B.C. Eckman E.A. Sambamurti K. Dobbs N. Chow K.M. Eckman C.B. Hersh L.B. Thiele D.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6221-6226Crossref PubMed Scopus (261) Google Scholar). These findings, together with preceding work on cell cultures, support that IDE participates in the degradation of these peptides in vivo, regulating their physiological levels (9Qiu W.Q. Walsh D.M. Ye Z. Vekrellis K. Zhang J. Podlisny M.B. Rosner M.R. Safavi A. Hersh L.B. Selkoe D.J. J. Biol. Chem. 1998; 273: 32730-32738Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 10Sudoh S. Frosch M.P. Wolf B.A. Biochemistry. 2002; 41: 1091-1099Crossref PubMed Scopus (71) Google Scholar). In addition, IDE has been shown to degrade a number of peptides of diverse sequence and functions, including several with the potential to form amyloid in vivo or in vitro. Apart from insulin, IDE can degrade glucagon, atrial natriuretic peptide, calcitonin, amylin, Aβ, including all its genetic variants, amyloid Bri, and amyloid Dan (reviewed in Refs. 11Kurochkin I.V. Trends Biochem. Sci. 2001; 26: 421-425Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 12Morelli L. Llovera R. Gonzalez S.A. Affranchino J.L. Prelli F. Frangione B. Ghiso J. Castanño E.M. J. Biol. Chem. 2003; 278: 23221-23226Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 13Morelli L. Llovera R.E. Alonso L.G. Frangione B. de Prat Gay G. Ghiso J. Castanño E.M. Biochem. Biophys. Res. Commun. 2005; 332: 808-816Crossref PubMed Scopus (25) Google Scholar). It has been long proposed that IDE specificity is dictated in part by substrate backbone conformation, a prediction that has been confirmed by recent crystallographic data (11Kurochkin I.V. Trends Biochem. Sci. 2001; 26: 421-425Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 14Shen Y. Joachimiak A. Rosner M.R. Tang W.J. Nature. 2006; 443: 870-887Crossref PubMed Scopus (272) Google Scholar). A growing number of studies suggest a possible genetic association of IDE polymorphisms with sporadic late-onset AD (reviewed in Ref. 15Vepsalainen S. Parkinson M. Helisalmi S. Mannermaa A. Soininen H. Tanzi R. Bertram L. Hiltunen M. J. Med. Genet. 2007; 44: 606-608Crossref PubMed Scopus (38) Google Scholar). In addition, recent work supports the association between diabetes mellitus type 2, hyperinsulinemia, and the risk for developing AD, placing IDE as a protein that may link several biochemical features of these diseases (reviewed in Refs. 16Qiu W.Q. Folstein M.F. Neurobiol. Aging. 2006; 27: 190-198Crossref PubMed Scopus (419) Google Scholar and 17Luchsinger J.A. Tang M.X. Shea S. Mayeux R. Neurology. 2004; 63: 1187-1192Crossref PubMed Scopus (559) Google Scholar). The expression and activity of IDE have been reported to be reduced in the hippocampus and cortex in sporadic AD and in cortical microvessels affected with amyloid angiopathy (18Pérez A. Morelli L. Cresto J.C. Castanño E.M. Neurochem. Res. 2000; 25: 247-255Crossref PubMed Scopus (209) Google Scholar, 19Cook D.G. Leverenz J.B. McMillan P.J. Kulstad J.J. Ericksen S. Roth R.A. Schellenberg G.D. Jin L.W. Kovacina K.S. Craft S. Am. J. Pathol. 2003; 162: 313-319Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 20Morelli L. Llovera R.E. Mathov I. Lue L. Frangione B. Ghiso J. Castanño E.M. J. Biol. Chem. 2004; 279: 56004-56013Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). It has been proposed that part of the loss of IDE activity may be accounted for by lower IDE mRNA levels or post-translational modifications such as oxidative damage (21Shinall H. Song E.S. Hersh L.B. Biochemistry. 2005; 44: 15345-15350Crossref PubMed Scopus (95) Google Scholar, 22Caccamo A. Oddo S. Sugarman M.C. Akbari Y. LaFerla F.M. Neurobiol. Aging. 2005; 26: 645-654Crossref PubMed Scopus (275) Google Scholar). In this study we report the unexpected finding that Aβ forms a highly stable complex that comprises part of the active site of IDE, suggesting a novel interaction between IDE and Aβ with potential implications in AD pathogenesis. Peptides and Antibodies—Aβ1–28, bovine insulin, bovine ubiquitin, and human growth hormone releasing factor 1–40 (GRF) were from Sigma; Aβ1–40 and Aβ1–42 were from Bachem; Aβ17–40, AβCys16–28, and AβCys17–27 were synthesized at the W. M. Keck facility at Yale University. [125I]Insulin (specific activity, 270 μCi/μg) was a gift of Edgardo Poskus, University of Buenos Aires. Synthetic Aβ1–40E22Q was kindly provided by Dr. Jorge Ghiso (New York University). Human cystatin C (CysC) was from Merck Biosciences. Antibodies against Aβ 6E10 (positions 4–13) and 4G8 (positions 17–24) were from Signet Labs, Dedham, MA. Rabbit polyclonal anti-ubiquitin was from Sigma. The rabbit antisera S40 and S42, specific for Aβ40 and Aβ42, respectively, were gifts of Dr. Mikio Shoji (Okayama University, Japan). Rabbit antiserum BC2 was generated against a glutathione S-transferase fusion protein comprising residues 97–273 of rat IDE, as previously described (12Morelli L. Llovera R. Gonzalez S.A. Affranchino J.L. Prelli F. Frangione B. Ghiso J. Castanño E.M. J. Biol. Chem. 2003; 278: 23221-23226Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Monoclonal antibody 3A2 against rat IDE was produced and purified as reported previously (20Morelli L. Llovera R.E. Mathov I. Lue L. Frangione B. Ghiso J. Castanño E.M. J. Biol. Chem. 2004; 279: 56004-56013Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Rabbit anti-CysC was raised with purified human CysC from urine following standard protocols and characterized by ELISA and Western blot. Human endogenous Aβ1–40 with the E22Q substitution was purified and characterized from the leptomeninges of a case of hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D), as previously reported (23Castanño E.M. Prelli F. Soto C. Beavis R. Matsubara E. Shoji M. Frangione B. J. Biol. Chem. 1996; 271: 32185-32191Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Fluorescein Labeling of Aβ—AβCys16–28 and AβCys17–27 (1.5 mg/ml) were incubated with a 10-fold molar excess of Tris(2-carboxyethyl)phosphine HCl in 50 mm phosphate, pH 7, for 1 h at room temperature followed by the addition of 60 μl of 2.5 mg of fluoresceinmaleimide (Molecular Probes) in DMSO. The reaction was incubated overnight at 4 °C in the dark and stopped by the addition of β-mercaptoethanol to a final concentration of 50 mm. Peptides were purified by reversed-phase chromatography with a C18 column (Beckman Ultrasphere ODS), and the eluate was monitored at 215 nm. HPLC peaks were analyzed by mass spectrometry as described below to assess the incorporation of fluorescein. The peaks containing single peptides with m/z of 1983.3 and 1726.5, corresponding to fluorescein-labeled AβCys16–28 and AβCys17–27 (FAβs) at a 1:1 stoichiometry, respectively, were identified. The concentrations of these derivatives were determined by absorbance at 492 nm in 0.1 m Tris HCl, pH 9 (extinction coefficient 83,000 cm–1 m–1) with a Beckman-Coulter spectrophotometer and stored in the HPLC elution solvent at –80 °C for binding experiments. Recombinant IDE Purification—Recombinant rat IDE 42–1019 (rIDE) was subcloned from pECE-IDE into pET-30a(+) (Novagen, Darmstadt, Germany), expressed in Escherichia coli BL21 and purified using a Hi-Trap Ni2+ chelating column as described (20Morelli L. Llovera R.E. Mathov I. Lue L. Frangione B. Ghiso J. Castanño E.M. J. Biol. Chem. 2004; 279: 56004-56013Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Further purification was obtained by size exclusion chromatography on a Superdex 200 column (Amersham Biosciences). On-line laser light scattering data were collected with a PD2010 detector. The relation between a 90° static light scattering signal and UV absorbance or refractive index signal was used to calculate molecular mass and hydrodynamic diameter with the Discovery 32 software (Precision Detectors, Bellingham, MA). The purity, folding parameters, and activity of rIDE are shown in supplemental Fig. S1. Protein concentration was determined by absorbance at 280 nm (extinction coefficient, 115,810 cm–1 m–1) and by a bicinchoninic acid assay (Pierce) and expressed as monomeric rIDE. Spectroscopic Studies—CD measurements were carried out on a Jasco J-810 spectropolarimeter. Far-UV spectra were collected using a Peltier temperature-controlled sample holder at 25 °C in a 0.2-cm path length cell, with rIDE concentration of 0.8 μm in 50 mm sodium phosphate, pH 7.2. Fluorescence emission spectra were recorded on an Aminco-Bowman spectrofluorometer with an excitation wavelength of 295 nm in the same buffer and rIDE concentration as above. Denaturation of rIDE induced by urea was assessed by the loss of molar ellipticity at 222 nm and changes in the center of spectral mass of Trp fluorescence at the indicated urea concentrations in the same buffer as above. rIDE ·Aβ Complex Formation and Treatments—Peptides and rIDE were incubated at the indicated concentrations and molar ratios in 0.1 m phosphate, pH 7.2, for 3 h at 37°C with constant agitation at 350 rpm in a Thermomixer Comfort block (Eppendorf). The reaction was stopped by the addition of an equal volume of 0.25 m Tris-HCl, pH 6.8, 40% glycerol, 4% SDS containing 0.1 m dithiothreitol (sample buffer, SB). To assess the effect of urea and formic acid, the reaction volume was dried under vacuum followed by the addition of 8 m urea in 0.1 m phosphate, pH 7.2, or 70% formic acid, respectively and incubated for 2 h at 37°C. Acid-treated samples were dried under a stream of N2 before the addition of electrophoresis SB. For guanidine-HCl treatment, to a 20-μl reaction of rIDE and Aβ1–28, 180 μl of 7.5 m guanidine in HCl (10 mm) or 180 μl of milli-Q water were added, and the samples were incubated for 1 additional hour at 37 °C. After bringing the final volume to 1.8 ml with water, proteins were precipitated with trichloroacetic acid, centrifuged at 15,000 g at 4 °C for 30 min, and carefully washed with ice-chilled acetone to remove the excess of guanidine. This last step was repeated three times. The pellet was dried under vacuum, and then resuspended with SB for SDS-PAGE and Western blot. Aβ1–40E22Q was incubated at 250 μm in PBS for 3 weeks or applied freshly after disaggregation (see below) for incubation with rIDE. For the time-course experiments, Aβ1–28 was pretreated with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) as described (24Wood S.J. Maleeff B. Hart T. Wetzel R. J. Mol. Biol. 1996; 256: 870-877Crossref PubMed Scopus (339) Google Scholar) and incubated at 100:1 molar ratio (Aβ:IDE) in 20 μl of 0.1 m sodium phosphate, pH 7.2, in the presence or absence of 5 mm EDTA for the indicated times at 37 °C. The reaction was stopped by boiling in 20 μl of SB, and samples were stored at –80 °C until the electrophoresis was performed. Data from two experiments in duplicate were fit to a single exponential equation and analyzed with GraphPad Prism 4 software. Competition experiments were carried out by incubating increasing concentrations of insulin or GRF with 2.5 μm FAβ16–28 and 50 nm rIDE for the indicated periods of time at 37 °C in 0.1 m phosphate, pH 7.2, containing 5 mm EDTA. Dynamic Light Scattering and Aggregation Assay—Aβ1–28 was pre-treated with HFIP to disaggregate oligomers, dried under nitrogen, dissolved to a final concentration of 100 μm in 0.1 m phosphate, pH 7.2, and filtered through a 0.22-μm membrane. This sample was incubated al 37 °C for up to 72 h, and particle size was assessed at the indicated times by dynamic light scattering using the excitation laser at 633 nm and a detector angle: of 173° with a Zetasizer Nano-S (Malvern Instruments). After the indicated incubation times, data were expressed as particle relative abundance versus particle hydrodynamic diameter. To study the aggregation kinetic of FAβCys16–28, the peptide stock was lyophilized, dissolved at the indicated concentrations in 0.1 m phosphate, pH 7.2, and layered upon an equal volume of 20% sucrose in the same buffer. After incubation for the indicated times at 37 °C with continuous agitation at 350 rpm, samples were centrifuged at 10,000 × g for 30 min. Aliquots from the supernatant were carefully removed and dissolved in 50 mm Tris-HCl, pH 9, and absorbance was measured at 492 nm. Data from two independent experiments in duplicate were expressed as percentage of soluble peptide, taking the dead time of the experiment as 100%. IDE ·AβSCx Formation with Endogenous IDE—Brains were obtained from adult Sprague-Dawley rats following protocols approved by the Fundación Instituto Leloir Ethical Committee. Tissue was homogenized in Tris-HCl-buffered saline, pH 8 (TBS), containing a mixture of protease inhibitors (Sigma) and centrifuged at 100,000 × g for 1 h at 4°C. One milligram of proteins from the supernatant was immunoprecipitated with monoclonal antibody 3A2 coupled to CNBr-activated Sepharose overnight at 4 °C. After washing with 0.4 m NaCl, 0.1 m Tris-HCl, pH 8, the pellets were incubated with 2 μg of Aβ1–40 in 40 μl of 0.1 m phosphate, pH 8, for 3 h at 37°Cand centrifuged at 3000 rpm for 3 min, and the beads were boiled in 30 μl of SB. Proteins were separated on 7.5% SDS-PAGE and IDE ·AβSCx detected by Western blot with antibody S40 as described below. Negative controls included the immunoprecipitation with an unrelated mouse IgG, 3A2-Sepharose beads with buffer alone, and rat brain proteins with protein G alone. IDE ·AβSCx Detection in Rat Brain—The pellet obtained from the rat brain homogenate as described above was homogenized in 10 volumes of TBS containing protease inhibitors, 1% Triton X-100, and 0.1% SDS and centrifuged at 100,000 × g for 1 h at 4 °C. One milligram of proteins from the supernatant was immunoprecipitated as indicated before with 3A2 or unrelated mouse IgG in the same buffer without SDS. After washing, proteins were separated on SDS-PAGE and subjected to Western blot with anti-Aβ S40 and S42. IDE ·AβSCx Detection in AD Brain—Brain samples of definite AD cases were obtained from participants enrolled in the Brain Donation Program of Sun Health Research Institute (Sun City, AZ). Approximately 300 mg of frontal cortex from each case was dissected on ice, pooled (n = 5), homogenized, and centrifuged as described above. The pellet was homogenized in TBS containing protease inhibitors and 2% SDS and centrifuged at 100,000 × g for 1 h at 4°C. One milligram of proteins from the water and SDS-soluble fractions were used for immunoprecipitation with anti-IDE 3A2 coupled to Sepharose in TBS-0.2% SDS. Proteins were resolved on SDS-PAGE and detected by Western blot using anti-Aβ S42, S40, and BC2 (see below). For urea treatment, the beads pellet was resuspended in 2 vol. of SB containing 9 m urea. Negative controls included immunoprecipitation of brain proteins with an unrelated mouse IgG coupled to Sepharose and 3A2 beads with buffer alone. High Performance Liquid Chromatography—The separation of rIDE ·Aβ complexes by HPLC was performed with a C4 Brownlee BU-300 (30 × 2.1 mm) column using a linear gradient from 0 to 100% acetonitrile (ACN) in 0.1% trifluoroacetic acid at 0.2 ml/min monitored at 214 nm. The peaks were analyzed by ELISA as described below. For the purification of FAβ peptides, a C18 Beckman Ultrasphere (250 × 4.6 mm) was used, with a linear gradient from 0 to 100% acetonitrile in 0.1% trifluoroacetic acid at 1 ml/min. Peaks were detected at 214 nm, collected, and concentrated with a SpeedVac, and peptides were quantitated as described above and analyzed by mass spectrometry (see below). After in gel endoproteinase LysC (endo-LysC) digestion of rIDE ·Aβ complex, peptides were separated using a linear gradient from 0 to 100% ACN in 0.1% trifluoroacetic acid in 75 min. Enzyme-linked Immunoadsorbent Assay—Aliquots of 20 μl from all the peaks obtained from HPLC were mixed with 80 μl of 0.1% trifluoroacetic acid, 50% ACN, placed on polystyrene microtiter plates (Nunc), and incubated at 37 °C until evaporation (typically overnight). After blocking with 3% bovine serum albumin in PBS for 2 h at 37°C, wells were incubated with antibodies 6E10, 4G8, S40, or BC2 overnight at 4 °C in 0.3% bovine serum albumin in PBS followed by horseradish peroxidase-conjugated anti-mouse or rabbit IgG (Amersham Biosciences) for 2 h at room temperature in the same buffer. Reactivity was developed with ortho-phenylenediamine and H2O2 for 10 min, stopped with 1 n SO4H2, and the optical density was measured at 490 nm with a microtiter plate reader 550 (Bio-Rad, Hercules, CA). After subtraction of background levels (wells without peptide), optical density values were plotted and analyzed with GraphPad Prism version 4. To better reflect the total immunoreactive proteins obtained from each HPLC peak, results were expressed as the product of optical density at 490 nm and peak volume, in arbitrary units. SDS-PAGE, Western Blot, and Fluorescence Detection—Proteins were run on 7.5 or 12.5% Tris-Tricine gels and transferred to polyvinylidene difluoride (PVDF) membranes. In some experiments, gels were cut horizontally at the level of the 67-kDa molecular mass marker. The lower half was stained with Coomassie Blue, and the upper half was transferred at 400 mA for 3 h to detect rIDE and rIDE ·Aβ complexes. Protein complexes formed between rIDE and unlabeled Aβ peptide were detected and quantified by Western blot. Membranes were blocked with 5% low fat milk in PBS for 2 h at 37°C and with the indicated primary antibodies overnight at 4 °C. Immunoreactivity was detected with anti-rabbit or anti-mouse horseradish peroxidase-labeled IgG and enhanced chemiluminescence using ECL Plus reagents (Amersham Biosciences). Western blots were scanned with a STORM 860 fluorometer and analyzed with ImageQuaNT 5.1 software (Molecular Dynamics). For fluorescence assessment, PVDF membranes were equilibrated in PBS for 45 min, air-dried, and scanned in blue fluorescence mode with the photomultiplier set at 975 V. The intensity of the fluorescent signal as a function of peptide mass was determined by dot-blot on PVDF membranes with FAβs. The amount of total rIDE was estimated by Coomassie Blue staining of the same membranes used for fluorescence detection. For N-terminal sequence, proteins were digested with trypsin at 1:200 for 15 min at room temperature, subjected to SDS-PAGE, and transferred to PVDF. After staining with 0.1% Coomassie Blue the band of interest was cut and subjected to Edman degradation with a 477A sequencer (Applied Biosystems). In-gel Digestion—After SDS-PAGE and Coomassie Blue staining, the band of 120 kDa containing the rIDE ·Aβ (FAβ17–27) complex was cut, and the gel slice was incubated in 100 mm Tris-HCl, pH 8.8 (digestion buffer), with 45 mm dithiothreitol for 30 min at 60 °C. The tube was cooled at room temperature, and 100 mm iodoacetamide was added, followed by incubation for 30 min in the dark at room temperature. The gel was then washed in 50% ACN with shaking for 1 h, cut in 1-mm pieces, and transferred to a small tube. The gel pieces were shrunk in ACN, dried in a rotator evaporator, and re-swollen with 10 μl of digestion buffer containing 0.1 mg/ml endo-LysC (Roche Applied Science). The sample was incubated overnight at 37 °C, and digestion products were extracted twice from the gel with 60% ACN/0.1% trifluoroacetic acid for 20 min. Combined extractions were loaded into a C18 HPLC column and separated as described above. Seventy-six 1-min fractions were collected, and volumes were reduced to ∼50 μl with a rotator evaporator and applied to a PVDF membrane in PBS for dot-blot fluorescence detection and 4G8 immunoreactivity as described before. Those fractions in which the presence of FAβ17–27 was detected were further analyzed by mass spectrometry. Negative controls included digestion of rIDE alone and a blank piece of gel. Mass Spectrometry Analysis—FAβ peptides and products of endo-LysC of rIDE in the presence or absence of Aβ were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). To produce the dried droplets, a saturated solution of α-cyano-4-hydroxycinnamic acid (Fluka) was prepared using 0.1% trifluoroacetic acid-30% ACN at room temperature. Equal volumes from this solution and the sample were mixed, and 1 μl was placed on the probe, allowed to dry, and analyzed on linear mode in an Omniflex spectrometer (Bruker Daltonics). Calibration was performed with the following peptides for which average m/z values are shown in parentheses: bradykinin 1–7 (757.86), angiotensin I (1297.49), renin substrate (1760.04), microperoxidase (1862.95), insulin B chain (3496.92), and insulin (5734.54). Peptides were identified with the Flex-Analysis 2.2 software (Bruker), and the Find Pep program available on the ExPASy Proteomic Server. Precision for analysis was set at <0.1%. All the peaks identified as fragments of rIDE were distributed in 100-residue segments of the recombinant enzyme, and the relative frequency for each segment was expressed as the percentage of the total peaks assigned to rIDE. IDE and Aβ Form a Complex That Resists Dissociation with Strong Denaturants—After incubating synthetic Aβ1–40 (25 μm) with rat rIDE (1 μm), a Western blot with antibody 6E10 specific for Aβ 4–13 showed, in addition to the digestion of the monomeric peptide, a 120-kDa component consistent with a complex between the protease (∼115 kDa) and Aβ (∼4 kDa). This high molecular weight component was not present when Aβ was incubated alone, ruling out an oli" @default.
• W2005026342 created "2016-06-24" @default.
• W2005026342 creator A5010512465 @default.
• W2005026342 creator A5011915671 @default.
• W2005026342 creator A5012408791 @default.
• W2005026342 creator A5026025869 @default.
• W2005026342 creator A5031895176 @default.
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• W2005026342 date "2008-06-01" @default.
• W2005026342 modified "2023-10-17" @default.
• W2005026342 title "The Catalytic Domain of Insulin-degrading Enzyme Forms a Denaturant-resistant Complex with Amyloid β Peptide" @default.
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