Determination of the Proteolytic Cleavage Sites of the Amyloid Precursor-Like Protein 2 by the Proteases ADAM10, BACE1 and γ-Secretase (original) (raw)
Figure 2
Transient and stable knock-down of ADAM10 suppresses APLP2 shedding.
(A) SH-SY5Y cells were transfected with siRNA pools against the proteases ADAM10 (A10KD) or ADAM17 (A17KD) or with control siRNA (Con). Both proteases were detected in membrane preparations. The mature active form is indicated with ##, the immature form with #. Actin and full-length APLP2 levels (cell. APLP2) were detected in the cell lysate. Conditioned media were analyzed for total secreted APLP2 (sAPLP2). All three species of sAPLP2 (* CS-GAG modified, ** 115 kDa, *** 100 kDa) were strongly decreased upon knock-down of ADAM10, but not of ADAM17. (B) Quantification of experiments in A (mean +/− SEM). ADAM10 knock-down significantly reduced sAPLP2 (p<0.001 for all three species, n = 6), while ADAM17 knock-down did not lead to any significant changes in sAPLP2 levels. (C) Knock-down of ADAM10 and ADAM17 in HEK293 cells, carried out as in A. (D) Quantification of experiments in C (mean +/− SEM). ADAM10 knock-down significantly reduced sAPLP2 (p<0.001 for CS-GAG modified and 100 kDa APLP2, p = 0.001 for 115 kDa APLP2, n = 6). (E) SH-SY5Y cells with stable shRNA-mediated knock-down of ADAM10 were used. shRNAs sh7 and sh9 are targeting two different regions of ADAM10. As control, a stable SH-SY5Y cell line expressing a non-targeting shRNA was used (Con). sAPLP2 levels were clearly reduced upon ADAM10 knock-down. (F) Quantification of experiments in E (mean +/− SEM). Both shRNAs significantly reduced sAPLP2 (p<0.001 for all three species, n = 6). (G) SH-SY5Y cells were transiently transfected with a siRNA pool against ADAM10 (A10KD) or control siRNA (Con). Cells were treated with DAPT (1 µM). Additionally C3 (1 µM) or DMSO (as a solvent control) was applied. sAPLP2 levels were clearly reduced upon ADAM10 knock-down while cellular APLP2 levels (cell. APLP2) remained unchanged. Two forms of APLP2 C-terminal fragments (CTFs) were detected at around 10 kDa in the cell lysate (-,--). ADAM10 knock-down led to the elimination of the lower molecular weight APLP2 CTF (--) while C3-treatment abolished the higher molecular weight species (-).