Fig 1: PepE target the TrxR1 through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 µM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 µM) or NADPH reduced TrxR1 nSec498 (0.9 µM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 µM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 µM) to measure the binding affinity of PepE to the proteins (Kon) and subsequently moved to wells containing buffer to measure dissociation rates (Kdis). The affinity constant (KD) was calculated as the ratio of the Kdis to the Kon. (E) Kinetic analysis of the interaction between TrxR1 (0.9 µM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 µM) by BLI. (F) The interaction of the PepE with the Sec residue in the C-terminal active center of TrxR1. HRP-conjugated streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 µM), DMSO(negative control), and DNCB (positive control, 5 µM), **P < 0.01, vs. the control group, data is presented as means ± SD in triplicate experiments. (G) The NADPH oxidase activity of TrxR1 (0.2 µM) induced by PepE (5 µM), DMSO (control), and DNCB (positive control, 5 µM). *P < 0.05 and **P < 0.01 vs. the control group, all data is presented as means ± SD, n = 6.
Fig 2: PepE and DMAPE targeting the TrxR in CD34+AML cells. (A) Imaging the TrxR activity in live KG-1a CD34+ cells by TR-Green. After treatment of cells with 2 and 6 µM of PepE, DMAPE or 6 µM of PTL (positive control) for 24 h, the TrxR activity was stained by the TrxR probe TR-Green. The bright field pictures (top panel) and the fluorescence pictures (bottom panel) were captured by inverted fluorescence microscope. Scale bar: 20 µm. (B) 10 cells were randomly selected and relative fluorescence intensity (RFI) was quantified in individual cells Metamorph Software (**P < 0.01 vs. the control group; ##P < 0.01 vs. the 2.0 µM group, ??P < 0.01 vs. the PTL group). Data are expressed as means ± SD of three experiments. (C) No obvious changes of TrxR1 expression levels in KG-1a CD34+ cells after PepE and DMAPE treatment. Cells were treated with the indicated concentration of DMAPE and PepE for 24 h, and the cell extracts were assessed by Western blots. (D) Quantification of TrxR1 expressions in control and KG-1a TrxR1 knockdown cells. TrxR1 expressions in different cells were analyzed by western blot using ß-actin as the internal standard and qRT-PCR using GAPDH as the internal standard (means ± SD, n = 3, **P < 0.01 vs. the control cells). (E) Quantification of TrxR1 expressions in control and KG-1a TrxR1 activation cells. TrxR1 expressions in different cells were analyzed by western blot using ß-actin as the internal standard and qRT-PCR using GAPDH as the internal standard. (means ± SD, n = 3, **P < 0.01 vs. the control activation cells). (F) and (G) Growth inhibition of PepE and DMAPE to TrxR1 knockdown cells (KD cells), control knockdown cells (CTRL KD cells), TrxR1 activation cells (ACTV cells) and control activation cells (CTRL ACTV cells). These cells were treated with PepE or DMAPE for 48 h, and the viability was determined by the CCK-8 method. Data were expressed as mean ± SD of three experiments. *P < 0.05 and **P < 0.01 vs. the CTRL ACTV cells, ##P < 0.01 vs. the CTRL KD cells.
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