Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , HEK293-CD13-myc ISG15 stable cells were infected with HCoV-229E at an MOI of 0.01 for 72 h prior to collection of cell lysates (lysates) and supernatant (S/N) prior to western blot with anti-myc Ab (as above) to detect transfected ISG15. The level of ISG15 in the supernatant was normalized to cell lysates and loading control (GAPDH), and was expressed relative to the most abundant protein in the supernatant ( Mops condylurus ). Only ISG15 of Mops condylurus was significantly secreted into the supernatant and secretion further increased during HCoV-229E infection. Mean and standard error of the mean are displayed, including individual points (black) for n = 3 independent experiments. Significance was tested with a two-tailed t-test comparing infection to ISG15 alone. Raw data are provided in Supplementary Table . b , An example western blot image (as per panel a) showing high and low exposure of ISG15 supernatant (S/N), HCoV-229E N protein, GAPDH and ISG15 bands in the HEK293-subclone-ΔISG15 cell lysate with protein ladders. Lanes are as indicated. c , Example western blot image with low and high contrast showing ISG15 conjugation (α-myc) after transfection with ISG15’s and HCoV-229E infection (−/+). Arrows indicate bands not seen in the empty vector control (conjugated proteins). d , Western blot of E1 ligase (UBE1L), E2 ligase (UBE2L6) and E3 ligase (HERC5) expression, together with GAPDH in the same samples shown in panel c, indicating sufficient expression of ISGylation machinery in the HEK293-subclone-ΔISG15 cell line. e , HEK293-subclone-ΔISG15 cells were transfected with ISG15 constructs as indicated and with/without NSP3C/L (SARS-CoV-2 PLpro). As per panel c, arrows indicate ISGylation bands, or ISG15 dimer/monomer. The amount of ISG15 present was calculated, normalized to GAPDH and quantified from three western blots. One of the three western blots is shown as an example. f , Representative western blot for the amount of ISG15 present in the supernatant (matched to e). g , ISGylation machinery expression, as per panel d, for NSP3C samples used in panel e and f and Fig. . Western blots in panels b-g are matched to the quantification in Fig. .

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Infection, Western Blot, Transfection, Control, Two Tailed Test, Conjugation Assay, Plasmid Preparation, Expressing, Construct

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: Interaction of peptide G4 with the catalytic site of CD13. A Competitive inhibition of rCD13 enzymatic activity by peptide G4. Steady-state kinetic analysis of rCD13 in the presence of different concentrations of l -alanine p-nitroanilide hydrochloride substrate and G4, as indicated, in 60 mM potassium phosphate buffer, pH 7.4, at 37 °C, is shown. The initial velocities ( Vo ) were calculated from the slopes of the first 5 min of reaction. Two independent experiments, each in duplicate, were performed. The results of one representative experiment are shown in the left panel (mean ± SEM). The indicated enzyme inhibitory constant ( K i = 21 ± 7 nM , mean ± SEM of two independent experiments) was calculated using the competitive enzyme inhibition model of GraphPad Prism Software. The Lineweaver–Burk plot of the data (right panel) shows that G4 changed the apparent Km of the enzyme but not the Vmax, which is consistent with a competitive mechanism of inhibition. B Representative docking poses of G4 in complex with the closed (left) and intermediate open forms of human CD13 (center). For comparison, the crystallographic structure of CNGRCG in complex with the open form of porcine CD13 (pdb code: 4OU3) is shown on the right. CD13 and peptides are represented in cartoon and CPK, respectively. This model suggests that G4 fits into the catalytic pocket of the human CD13. C The best docking pose, in terms of ΔG MMGBSA , of G4 in complex with the closed and intermediate open form of human CD13 is shown on the left and on the right panels, respectively. CD13 residues involved in electrostatic interactions engaged by Val-1 are highlighted and labeled on the upper panels. The corresponding ligand interaction diagrams are shown in the lower panels. In the diagrams, protein pockets within 4 Å from G4 are displayed as colored spheres, where red, green, purple, and blue indicate acidic, hydrophobic, basic, and polar residues, respectively. H-bonds and salt bridges are shown as lines and arrows. G4 atoms exposed to solvent are highlighted with gray spheres

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: Inhibition, Activity Assay, Enzyme Inhibition Assay, Software, Comparison, Labeling, Solvent

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: G4 recognizes soluble forms of CD13, with higher affinity and selectivity than bestatin, and the membrane forms expressed on the surface of HUVEC cells. A Inhibitory effects of G4 and bestatin on the enzymatic activities of different aminopeptidases (rCD13, APA, and APB). Various amounts of G4 or bestatin were incubated with rCD13 (0.2 µg/mL in 60 mM Tris–HCl pH 7.4), APA (0.2 µg/mL in 60 mM Tris–HCl pH 7.4, 50 mM calcium chloride, 200 mM sodium chloride) or APB (0.2 µg/mL in 60 mM Tris–HCl pH 7.4, 100 mM potassium chloride) and the corresponding substrates at 0.1 mM ( l -alanine-7-amido-4-methylcoumarin for rCD13, l -glutamic acid γ-(7-amido-4-methylcoumarin) for APA, and l -arginine-7-amido-4-methylcoumarin for APB). The mixtures were incubated for 30 min at 37 °C, and the cleavage of each substrate was monitored by measuring the fluorescence of methylcoumarin ( λ ex 341 nm; λ em 441 nm) using an Infinite 200 PRO plate reader (Tecan). Three independent experiments were performed for each enzyme. The results of one representative experiment for each enzyme are shown (mean ± SEM, duplicates). B Inhibitory effects of G4 and bestatin on soluble CD13 immunocaptured from human serum (sCD13) using microtiter plates coated with anti-CD13 mAb WM15. The microtiter plate was filled with a solution of mAb WM15 (5 μg/ml in DPBS) and incubated overnight at 4 °C. The plate was then washed with DPBS, blocked with 3% bovine serum albumin (BSA) in DPBS, and filled with human serum containing soluble CD13 (diluted 1:2 with DPBS containing 2% BSA). After washing with DPBS, mixtures of G4 or bestatin (0–100 µM in 60 mM Tris–HCl, pH 7.4) and l -alanine p-nitroanilide hydrochloride substrate (4.5 mM in 60 mM Tris–HCl, pH 7.4) were added and left to incubate at 37 °C for 2 h. Substrate cleavage by sCD13 dissociated from mAb WM15 was then monitored by measuring the absorbance at 405 nm in each well using a plate reader (Bio-Rad). The results of one experiment performed in triplicate are shown (mean ± SEM). C Effect of G4 on the binding of the anti-CD13 mAb WM15 to HUVEC. HUVEC were seeded at a density of 2 × 10 5 cells in 100 µL DPBS supplemented with 5% normal human serum (NHS) and incubated with a mixture of G4 peptide (0, 0.1, 1, and 10 µM) and mAb WM15 (0 or 0.13 nM) as indicated. As a negative control, we used the 11-mer peptide CgA 429–439 , which is a human chromogranin A fragment. Cells were incubated for 60 min at 4 °C, washed, and resuspended in 100 µL of DPBS containing 5% NHS and 1 µg/mL FITC-labeled goat anti-mouse IgG secondary antibody. After incubation for 60 min at 4 °C, the cells were washed and analyzed by flow cytometry. Data are expressed as the percentage of counts vs. fluorescence intensity units

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: Membrane, Incubation, Fluorescence, Binding Assay, Negative Control, Labeling, Flow Cytometry

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: Effect of G4-TNF on tumor growth and body weight of mice bearing WEHI-164 fibrosarcomas. A Mice bearing subcutaneous WEHI-164 tumors (5–6 mice/groups) were treated 5 days after tumor implantation with a single intraperitoneal injection of G4-TNF (0, 4, 20, or 100 pg). Tumor volume and body weight after treatment were monitored daily. B Effect of mAb R3-63 (anti-murine CD13 antibody, 25 µg) and CNGRC peptide (100 µg) on the antitumor activity of G4-TNF (100 pg) (5–6 mice/group). The arrows indicate the time of treatment. * P < 0.05; ** P < 0.01, by Mann–Whitney analysis of the area under the curve for each tumor, with the GraphPad Prism software (mean ± SEM)

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: Tumor Implantation, Injection, Activity Assay, MANN-WHITNEY, Software

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: The active site of membrane CD13 expressed by HUVEC, but not that of soluble CD13 or membrane CD13 expressed by HL60 cells, is accessible to G4-TNF. A Competitive effects of G4 and G4-TNF on the binding of mAb WM15 (anti-CD13) to sCD13 or rCD13 showing the lack of binding of G4-TNF to natural and recombinant soluble CD13 (sCD13 and rCD13). G4, scrambled G4 (GCRSNCYRVG), and TNF were used as positive (G4) and negative (scrambled G4, TNF) controls. The sCD13 competition assay was performed as follows: microplates were coated with mAb WM15 (5 μg/mL) in DPBS and incubated overnight at 4 °C. Plates were subsequently blocked with 3% BSA in DPBS and filled with a mixture of human serum containing sCD13 (diluted 1:2) and the indicated competitors at different concentrations in DPBS, 0.1% BSA, and 0.05% Tween-20. The mixtures were incubated for 2 h at room temperature and washed with DPBS. To detect bound sCD13, we added an anti-CD13 rabbit polyclonal antibody (1:1000, 1 h at room temperature), washed with DPBS, and added peroxidase-labeled goat anti-rabbit secondary antibody (1:2000, 1 h). Bound peroxidase was detected using o-phenylendiamine chromogenic substrate. The absorbance of o-phenylenediamine was measured at 490 nm using an ELISA plate reader (Bio-Rad). Cumulative results of two independent experiments, each performed in duplicate, are shown (mean ± SEM). The rCD13 competition assay was performed as described in Supplementary Fig. S2 (one experiment in duplicate, mean ± SEM). B Inhibitory effects of G4 or G4-TNF on the sCD13 or rCD13 enzymatic activity (one experiment performed in duplicates; mean ± SEM). C Competition of mAb WM15 binding to HUVEC or HL-60 cells with G4 and G4-TNF. Scrambled G4 and TNF were used as negative controls. Cumulative results of three (HUVEC cells) or two (HL-60 cells) independent experiments, each performed in duplicate or triplicate (mean ± SEM). D Competitive binding of mAb WM15 (anti-CD13) to HUVEC and HL-60 cells with G4 and G4-Avidin complex prepared by mixing a G4-biotin conjugate with avidin (see “Methods). Avidin was also used as control. The experiment was performed in duplicate (mean ± SEM). E Inhibitory effects of G4 and G4-Avidin on rCD13 enzymatic activity. The assay was performed as described in Supplementary Fig. S2. The results of one experiment, performed in duplicate, are shown (mean ± SEM)

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: Membrane, Binding Assay, Recombinant, Competitive Binding Assay, Incubation, Labeling, Enzyme-linked Immunosorbent Assay, Activity Assay, Avidin-Biotin Assay

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: Quantification of CD13 expressed by HL-60 and HUVEC by western blotting and FACS analyses, and determination of their enzymatic activity. A Western blotting (WB) analysis of CD13 expression in HL-60 cells and HUVEC. For both cell lines, 2 µg of cell lysates were loaded in each well ( n = 4–5 replicates, as indicated). After blotting, the membrane was cut and analyzed for CD13 and actin expression as described in Material and methods. The results of one representative experiment, out of three independent experiments performed, are shown (bars, ratio of CD13/actin band intensity, mean ± SEM). Recombinant human CD13 (rCD13) (20 ng) was loaded as positive control. MW, molecular weight markers. B FACS analysis of CD13 expression in HUVEC and HL-60 cells. The cells were incubated with mAb WM15 (10 μg/mL) and stained with a FITC-labeled goat anti-mouse IgG (H + L) secondary antibody. Data were expressed as fluorescence intensity and normalized to the number of cells. The cumulative results of three independent experiments are shown (each performed in duplicate; mean ± SEM). C Enzyme activity assay of CD13 expressed in HUVEC and HL-60 cells. Cells were seeded into 96-well microtiter plates (3 × 10 5 cells/well) and incubated with l -alanine p-nitroanilide hydrochloride substrate (0.5 mM in DPBS) for 30 min at 37 °C. Absorbance at 405 nm was measured using a plate reader. The enzyme units in each sample were calculated using rCD13 as a standard and were normalized to the number of cells. Cumulative results of three independent experiments, each performed in duplicate, are shown (mean ± SEM). D Inhibition of the enzymatic activity of CD13 expressed in HUVEC and HL-60 cells by G4 and G4-TNF. Cells were seeded into 96-well microtiter plates (3 × 10 5 /well) and incubated with the l -alanine p-nitroanilide hydrochloride substrate and different amounts of G4-TNF, G4, TNF, or scrambled G4 peptide (30 min at 37 °C). The absorbance was measured at 405 nm using a plate reader. The results of one experiment performed in duplicate are shown (mean ± SEM)

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: Western Blot, Activity Assay, Expressing, Membrane, Recombinant, Positive Control, Molecular Weight, Incubation, Staining, Labeling, Fluorescence, Enzyme Activity Assay, Inhibition

Journal: Cellular and Molecular Life Sciences

Article Title: A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

doi: 10.1007/s00018-023-05102-1

Figure Lengend Snippet: Representative model of the interaction of G4 and G4-TNF with different human CD13 forms. Docking pose of G4 bound to CD13 in a closed and intermediate open form (left and center); model of G4-TNF in complex with the intermediate open form of CD13 (right). G4 and CD13 are represented by the pink and white surfaces, respectively. G4-TNF is shown in cyan

Article Snippet: Phenyl-Sepharose 6 Fast Flow (cat. 17097305), DEAE-Sepharose Fast Flow (cat. 17070901), and Sephacryl-S 300 HR (cat. 17059901) were obtained from Cytiva. l -alanine 7-amido-4-methylcoumarin (cat. 4028736), l -glutamic acid γ- (7-amido-4-methylcoumarin) (cat. 4002702), and l -arginine-7-amido-4-methylcoumarin (cat. 4002148) were obtained from Bachem; His-tagged recombinant human CD13 (rCD13) (cat. 10051-H08H) consisting of the CD13 extracellular domain expressed in HEK-293 cells, His-tagged recombinant human aminopeptidase A (APA) (cat. 10554-H07B) and rabbit anti-human CD13 polyclonal antibody (cat. 10051-RP02) were purchased from Sino Biological.

Techniques: