To determine the class of intramembrane proteases that is responsible for the release of the TfR1-Cp, we tested different inhibitors targeting I-CLiPs. After treatment of HEK293 cells with the indicated inhibitors, the TfR1-Cp generated from endogenous I-CLiPs was isolated from conditioned media using anti-V5 IgG. The metalloprotease inhibitor 1,10-phenanthroline, which inhibits site-2 proteases , did not change the amount of TfR1-Cp released to the supernatant compared to control cells (Fig. 6A). Different studies have shown that (Z-LL)2-ketone inhibits SPP and SPPL2a/b but not γ-secretase [20, 22-24], whereas L-685,458 targets γ-secretase, SPP and SPPL2a/b [21, 23, 24, 32]. N-[N-(3,5-difluorophenylacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) only inhibits γ-secretase activity but fails to block SPP and SPPL2b activity [21, 23, 32]. In our experiments, (Z-LL)2-ketone and L685,458 strongly reduced TfR1-Cp secretion from HEK293 cells, whereas treatment with DAPT had no detectable effect (Fig. 6A). Moreover, the inhibition of TfR1-Cp secretion by (Z-LL)2-ketone was concentration-dependent (Fig. 6B). These results suggest that GxGD intramembrane aspartyl proteases of the SPP/SPPL-family are involved in the intramembrane cleavage of the TfR1-NTF. To further support this hypothesis, HEK293 cells, stably expressing the indicated members of the SPP/SPPL-family or their catalytically inactive mutants (D/A) were transiently transfected with Flag-TfR1-NTF-V5 and conditioned media were analyzed for TfR1-Cp secretion (Fig. 6C). The exposure time of the western blotting was optimized to the TfR1-Cp signal obtained after the overexpression of SPPLs. Therefore, the bands of TfR1-Cp released by endogenous proteases are only very faint (Fig. 6C, lane 2) and identical in intensity to cells expressing SPPL3 and SPP, demonstrating that these proteases have no impact on TfR1-NTF cleavage. Only the expression of catalytically active SPPL2a and SPPL2b resulted in increased release of TfR1-Cp, strongly indicating that these two I-CLiPs are involved in processing of the TfR1-NTF, with SPPL2b being the most prominent enzyme (Fig. 6C). Expression of the inactive mutant SPPL2a D/A resulted in TfR1-Cp release comparable to the endogenous level, whereas SPPL2b D/A led to the complete abolishment of this fragment, suggesting that this mutant is able to suppress endogenous SPPL2a by a feedback mechanism. To detect the corresponding intracellular cleavage product of TfR1-NTF (TfR1-ICD), we analyzed membrane lysates, cytosol and nuclei of the cells but were unable to find them during a first attempt. A sophisticated methanol/chloroform precipitation of the membrane lysates was finally required to detect the TfR1-ICD as a low-molecular weight fragment in cells co-expressing SPPL2b and TfR1-NTF (Fig. 6C, lane 5), which is in line with the existence of two palmitoylation sites within TfR1-ICD. By contrast to SPPL2b, the expression of SPPL2a did not result in a detectable generation of TfR1-ICD, indicating that TfR1-NTF is mainly processed by SPPL2b in HEK293 cells. This is further corroborated by confocal immunofluorescence microscopy experiments, demonstrating a clear colocalization between SPPL2b and TfR1-NTF, whereas colocalization with SPPL2a can only be detected sporadically (Fig. S2). Taken together, these results demonstrate that TfR1-NTF is mainly cleaved by SPPL2b and, to a lesser extent, by SPPL2a to release a 16 amino acid long Cp and a corresponding ICD.
Figure 6. Intramembrane proteolysis of TfR1-NTF. (A, B) The conditioned media of HEK293 cells expressing Flag-TfR1-NTF-V5 were subjected to immunoprecipitation with anti-V5 IgG and TfR1-V5-Cp was detected by western blotting. (A) The cells were incubated overnight with the indicated inhibitors. The solvent dimethysulfoxide served as a control. (B) Concentration-dependent inhibition of the endogenous protease by the inhibitor (Z-LL)2-ketone. (C) HEK293 cells, stably expressing the indicated SPPL variants, were transfected with Flag-TfR1-NTF-V5; D/A indicates dominant negative mutants (D421A for SPPL2a and SPPL2b, D272A for SPPL3, D265A for SPP). The conditioned media were used for immunoprecipitation with anti-V5 IgG to detect TfR1-Cp, whereas a membrane preparation of the cells was applied to identify the intracellular domain of the TfR1 (TfR1-ICD). The band designated as Flag-TfR1-ICD was identified as a result of the N-terminal Flag tag and a molecular mass of 10 kDa. An unspecific signal can be excluded because the control lanes do not show this signal; however, we cannot exclude the possibility that this Flag-TfR1-ICD is C-terminally shortened for some amino acids.
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