Figure 1
From: Harnessing the natural inhibitory domain to control TNFα Converting Enzyme (TACE) activity in vivo
The natively folded TPD inhibition in vitro and modulation of TNFα secretion in cell based assay and in vivo.
(a) Representative purification procedure of recombinant TPD: Molecular weight ladder (MW), whole bacteria lysate (1), lysate soluble fraction (2), Ni+ NTA elution (3) and anion exchange first peak elution (4). (b) Purified TPD eluted from gel filtration superdex 75 in a sharp single peak. (c) Circular dichroism measurement of purified TPD in room temperature. (d) Catalytic activity of TACE (
), MT1-MMP (
), MMP7 (
), MMP9 (
) and ADAM10 (
) on fluorogenic peptide in presents of TPD. Calculated IC50 value of TACE is 145 ± 1.9 nM and up to 2000 nM no significant inhibition was observed for other catalytic activity. (e) Secretion of TNFα in CHO stable transfected TNFα cells incubated with TPD treatment. *P < 0.05, n = 4 (Mean ± SD, Student’s t test) (f) Secretion of TNFα in primary activated macrophages. LPS is used to stimulate the macrophages prior to treatment. TAPI (small molecule inhibitor) in 20 μM concentration is used as positive control in both cell types assays. *P < 0.05, n = 3 (Mean ± SD, Student’s t test). (g) Serum level of TNFα in naive C57/BL mice and C57/BL mice injected with LPS and treated with control PBS or different concentrations of TPD (0.5, 1, 2 mg/Kg). *P < 0.05, n = 8 (Mean ± SEM, Student’s t test). (h) Shedding of Neuropilin-1 from HEK293 cells. Transfection of Neuropilin-1 only in the presence of TPD (lane 1–4) shows no effect on the shedding pattern by endogenous ADAM10. Overexpressing both Neuropilin-1 and TACE in the present of TPD (lane 5–9), generates a specific cleavage by TACE which is inhibited by TPD in a concentration-dependent manner. The black arrow indicates the cleavage product by ADAM10 and the grey arrow the cleavage product of TACE.
