recombinant human insulysin ide  (R&D Systems)

Journal: Neuronal Signaling

Article Title: Inhibition of insulin-degrading enzyme in human neurons promotes amyloid-β deposition

doi: 10.1042/NS20230016

Figure Lengend Snippet: ( A ) Schematic diagram of the FAM-Aβ-biotin substrate used in the fluorescence polarisation assay. Incubation of the Aβ substrate (500 nM) with ( B ) 25 ng recombinant NEP (rNEP) and ( C ) 25 ng recombinant IDE (rIDE) in the absence or presence of the general metalloprotease inhibitor 1,10-phenanthroline (1 mM), the NEP inhibitor phosphoramidon (100 μM) or the IDE inhibitors 6bK (10 μM), ML345 (10 μM) and insulin (100 μM). NEP and IDE were pre-incubated with the inhibitors for 30 min at 37°C before addition of the FAM-Aβ-biotin substrate and a further incubation for 4 h at 37°C. The cleaved substrate was separated and the fluorescence measured as described in the Experimental section. Data shown as mean ± SEM, n = 3 experimental repeats. ( D ) OX1-19 iPSCs were differentiated to cortical neurons and neuronal identity was confirmed at day 80 by identification of neuronal markers using immunofluorescence microscopy. Representative images demonstrate staining for the neuronal markers MAP2 and β-III tubulin (β-III). Nuclei were stained with DAPI, scale bar represents 200 µm. ( E ) Membrane potential was measured in neurons using the FLIPR® Membrane Potential Assay Kit. Representative traces shown for neurons at day 60 and day 80 of differentiation. Experiments were repeated in three independent cell preparations. ( F ) Incubation of the Aβ substrate with human OX1-19 iPSC-derived neurons (hiPSC-Ns) in the absence or presence of the indicated inhibitors. n = 3 neuronal inductions (three technical repeats were performed per neuronal induction). ** P <0.005, **** P <0.0001 using one-way ANOVA with Dunnett’s multiple comparisons test to compared with control.

Article Snippet: For experiments with recombinant IDE and NEP the following amounts were used: 25 ng NEP (BioTechne #1182-ZNC-010 (lot:RXD0217041)) or 25 ng IDE (Bio-Techne #2496-ZN-010 (lot:NSA1016031)).

Techniques: Fluorescence, Incubation, Recombinant, Immunofluorescence, Microscopy, Staining, Membrane, Derivative Assay

Journal: Neuronal Signaling

Article Title: Inhibition of insulin-degrading enzyme in human neurons promotes amyloid-β deposition

doi: 10.1042/NS20230016

Figure Lengend Snippet: ( A ) OX1-19 iPSC-derived neurons seeded into 3D matrigel cultures were imaged for neuronal markers MAP2 and β-III tubulin, scale bar = 200 μm. ( B ) Neuronal membrane potential was measured in neurons using the FLIPR® Membrane Potential Assay Kit. Representative trace of membrane potential from neurons in 3D matrigel cultures. Day 60 OX1-19 neurons were cultured in a 3D Matrigel matrix for 21 days in the absence or presence of the IDE inhibitor, ML345 (10 μM) or the NEP inhibitor, phosphoramidon (10 μM). ( C ) Representative images of OX1-19 neurons following treatment without or with ML345 or phosphoramidon using immunofluorescence microscopy to determine the number of Aβ deposits; composite images of βIII-tubulin and Aβ are shown and Aβ staining (with antibody 4G8) is highlighted by the arrowheads, scale bar = 200 µm. ( D ) Representative images of OX1-19 neurons following ML345 treatment showing only the Aβ deposits stained with antibody 4G8 with (i) being the same field of view as shown in ( C ), scale bar = 200 µm and (ii) a larger magnification image from a separate field of view, scale bar = 100 µm. ( E ) The number of Aβ deposits in each image was quantified and the spread of data from images for each condition are shown by the violin plot. Data are shown with median (solid line) and quartiles (dashed line) for 36 data points for control and ML345 and 25 data points for phosphoramidon. ( F ) The number of Aβ deposits was then averaged for each neuronal induction for each condition and statistical analysis performed. Data shown as mean ± SEM, n = 3 neuronal inductions (three technical repeats were performed per neuronal induction). **** P <0.0001 using an ordinary one-way ANOVA test with Dunnett’s multiple comparisons test to compared with control. ( G ) Representative images taken for the analysis of Aβ deposition in day 60 SBAD neurons cultured in a 3D Matrigel matrix for 21 days in the absence or presence of the IDE inhibitor, ML345 (10 μM) using immunofluorescence microscopy, scale bar = 200 µm.

Article Snippet: For experiments with recombinant IDE and NEP the following amounts were used: 25 ng NEP (BioTechne #1182-ZNC-010 (lot:RXD0217041)) or 25 ng IDE (Bio-Techne #2496-ZN-010 (lot:NSA1016031)).

Techniques: Derivative Assay, Membrane, Cell Culture, Immunofluorescence, Microscopy, Staining

Journal: The Journal of biological chemistry

Article Title: Antibody-guided proteases enable selective and catalytic degradation of challenging therapeutic targets.

doi: 10.1016/j.jbc.2023.104685

Figure Lengend Snippet: Figure 2. Protease and fusion format screening for targeted degradation of Aβ. A, in vitro protease screening assay for cleavage of Aβ(1–40) (blue circles) and Aβ(1–42) (red squares). B, expression yields of Fc and IgG protease fusion formats. Four different proteases were expressed in the context of the eight formats shown in the icons. IgG fusions contained variable regions of the anti-Aβ antibody crenezumab, and all heavy chain constant regions were human IgG1. The bar graph shows the expression yields from duplicate 30 ml HEK293 expressions of each construct. C, purification of crenezumab NEP protease fusion formats. Each NEP fusion format was expressed in HEK293 cells and initially purified using a protein A resin. Size exclusion chromatography (SEC) coupled with sample fractionation was used for further purification. SEC chromatograms revealed the presence of multiple species with each sample containing 2 to 3 peaks. Nonreducing SDS-PAGE analysis to identify the peaks of similar antibody–protease fusions is shown in Fig. S7. D, the central fraction associated with each peak in the chromatograms from (C) was tested for Aβ(1–40) cleavage, and fractions of active peaks were pooled to obtain samples free of unwanted species. Error bars in (A, B, and D) represent standard error values with n = 2. Aβ, amyloid-β; ECE, endothelin-converting enzyme; Fc, fragment crystallizable; IgG, immunoglobulin G; IDE, insulin-degrading enzyme; MMP, matrix metalloproteinase; MTSP1, matriptase; NEP, neprolysin.

Article Snippet: NEP (R&D, 1182-ZNC-010), NEP2 (R&D, 2340-ZN-010), ECE-1 (R&D, 1784-ZN-010), ECE-2 (R&D, 1645-ZN-010), ACE (R&D, 929-ZN-010), IDE (R&D, 2496-ZN-010), MMP2 (R&D, 902-MP-010), MMP9 (R&D, 911-MP-010), and MTSP1 (R&D, 3946-SEB-010) were purchased commercially for initial Aβ cleavage activity screening.

Techniques: In Vitro, Screening Assay, Expressing, Construct, Size-exclusion Chromatography, Fractionation, SDS Page

Journal: Diabetologia

Article Title: Presence of immunogenic alternatively spliced insulin gene product in human pancreatic delta cells

doi: 10.1007/s00125-023-05882-y

Figure Lengend Snippet: IDE is not expressed in delta cells and cleaves INS-splice. ( a ) Pancreatic IDE protein expression was determined by immunohistochemistry of human pancreas sections by staining for proinsulin (green), IDE (white) and somatostatin (red). IDE was expressed ubiquitously in the exocrine and endocrine pancreas except in delta cells. Scale bar, 10 μm. Nuclei were visualised by Hoechst staining (blue). ( b ) Co-localisation of IDE with insulin and IDE with somatostatin was quantified using MCC (QuPath). Thirty-six islets from six pancreas donors were analysed. Data are shown as mean ± SD and statistical analysis was performed using a paired two-tailed Student’s t test (*** p <0.001). ( c ) Coomassie staining of INS-splice after IDE cleavage assay. Absence or presence of IDE is indicated by − or +, respectively. Full-length recombinant INS-splice is 14 kDa. INS, insulin; M, protein marker; SST, somatostatin

Article Snippet: Recombinant alternatively spliced insulin product (INS-splice; 0.25 μg/μl) was incubated with recombinant human IDE (0.05 μg/μl, 2496-ZN; R&D Systems, USA) in cleavage buffer (50 mmol/l Tris, 1 mol/l NaCl, pH 7.5) at 37°C for 48 h, following the manufacturer’s recommendations.

Techniques: Expressing, Immunohistochemistry, Staining, Two Tailed Test, Cleavage Assay, Recombinant, Marker