FIGURE 2.
The N-terminal region of F1L is sufficient for C9 interaction and inhibition. A, F1LΔTM or different N-terminal truncation mutants (2 μm) lacking the first 23, 34, or 47 residues were preincubated with C9 (200 nm) that was activated using constitutively active Apaf-1ΔWD-40 (1 μm) for 10 min. C9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± σ; n = 3). The first column shows C9 activity in the absence of F1LΔTM. B, shown are co-IP assays of F1L and truncation mutants with C9. HEK29T cells were co-transfected with plasmids expressing FLAG-C9 and either GFP, GFP-F1L, GFP-F1LΔ34, or GFP-F1LΔ44 as described under “Experimental Procedures.” IPs and cell lysates were also immunoblotted (WB) with anti-Bak antibodies as indicated. The Bak immunoblot contains a nonspecific band above the band indicated for Bak. C9 FL and C9 LS represent full-length (proC9) and the large subunit of processed C9, respectively. C, shown is N-terminal peptide containing an authentic N terminus inhibit C9. A series of F1L N-terminal peptides was mixed at different concentrations with pro-C9 (200 nm) in HEPES buffer and apoptosome components (cytochrome c, dATP, and recombinant full-length Apaf-1 (1 μm)) and incubated at 37 °C for 10 min. C9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). F1L-(1–47) peptide was purified from bacterial cultures; other peptides were chemically synthesized. D, the point mutation, C7A, ablates the inhibitory activity of N-terminal peptides. Inhibition of C9 activity by F1L-(1–36) and F1L (1–36)-C7A was quantified under the same condition as in C. The value of Ki,app for F1L-(1–36), in the low μm range, was very similar that determined for F1LΔTM (see Table 1), whereas Ki,app for the mutant was estimated at >600 μm. E and F, GFP-F1L-(1–15) is sufficient to protect cells from apoptosis induced either by C9 transfection or staurosporine. E, HEK293T cells were co-transfected with FLAG-C9 plasmid (0.5 μg) and increasing amounts (0–2 μg) of GFP-F1L-(1–15) or GFP-F1L-(1–15)-C7A plasmids as indicated and incubated for 20 h. Total transfected DNA was kept constant at 3 μg by the addition of GFP plasmid. F, procedures were as in E, except that cells were treated with 0.2 μm staurosporine for 10 h rather than being transfected with FLAG-C9. In both cases, floating and adherent cells were collected, fixed, and stained with 0.1 μg/ml DAPI. The percentage of apoptotic cells was determined by counting GFP-positive cells showing nuclear fragmentation and/or chromatin condensation (mean ± σ; n = 3). Statistical pairwise comparisons were performed using the two-tailed Student's t test, where p ≤ 0.05 is considered significant, shown on E and F schematically by asterisks. For C9-induced apoptosis, p values are 0.008, 0.015, and 0.006 (wt) and 0.53, 0.1, and 0.02 (C7A). For staurosporine-induced apoptosis, p values are 0.04, 0.002, and 0.0004 (wild type) and 0.12, 0.12, and 0.03 (C7A). G and H, aliquots of the cell lysates prepared in E or F were normalized for protein content (10 μg) and incubated with the caspase-3/7 substrate Ac-DEVD-AFC. Enzyme activity was determined as in C (mean ± σ; n = 3).