PMC

Ibm1[ΔGH3], when transfected alone, induced cell death in S2 (A) and SF9 (B) cells similar to that induced by Ibm1. However, Ibm1[ΔGH3] was much more sensitive to the inhibitory effect of Diap1 in co-transfections.

The level of Ibm1 mRNA relative to that of gapdh (A) or actin (B) was determined by quantitative RT-PCR (represent as percent to the control). The day larvae hatched or cocoons appeared was counted as day 1 of larval and pupal stages, respectively. The shortening of larvae was observed on day 27 or 28 of the larva stage. Data are represented as Mean±SE of 3 – 4 QPCR measurements.

(A) Expression of FLAG-tagged Ibm1 and Ibm1[ΔIBM] and HA-tagged BmIAP1 was assessed (either singly or in combination) by western blotting. Note that BmIAP1 is heavily ubiquitinated in the presence of Ibm1 (as indicated by the bracket). (B) Interaction between Ibm1 and BmIAP1 was detected by immunoprecipitation with α-FLAG followed by western blotting with α-HA. A non-specific background band is designated by an asterisk. An arrow indicates band of interest and an asterisk designates a non-specific background band. The upper blot contains immunoprecipitated material, while the lower blot contains the unbound material that remained in the supernatant. Note that BmIAP1 levels are increased when co-expressed with Ibm1.

A) SF21 cell viability following transfection of Ibm1 or Ibm1[ΔIBM]. The cells were heat shocked prior to measuring viability, which sensitizes them to apoptosis. B) Caspase activity in SF21 cells transfected with Ibm1 or Ibm1[ΔIBM] was determined by incubation of cell lysate with DEVDafc (Human caspase 3 substrate). Transfections were done in the presence or absence of the pan-caspase inhibitor zVADfmk.

Plasmids expressing Ibm1 or Ibm1[Δ2–4] (−IBM) were transfected into Lepidopteran SF9 (A), mosquito C6/36 (B), and Drosophila S2 (C) cell lines. Ibm1 was also cotransfected with increasing ratios of Diap1 or P35-expressing plasmids. Cell survival of vector (pie3)-transfected samples was set as 100% and the relative cell survival rate was calculated for each gene or combination of genes. The results are the average of 3–4 experiments and the error bars reflect standard deviation. pie-reaper was included as a positive control.

(A) Global alignment of Grim (D. melanogaster), Reaper (D. melanogaster), Mx (Aedes aegypti), and Ibm1 (B. mori). The IAP-binding motif is indicated by the red bar. The GH3 domain previously identified in Grim and Reaper is indicated by the blue bar on top of the Grim sequence. The GH3-like motifs in Mx and Ibm1 sequences are not aligned with the GH3 region in Reaper and Grim. Note that while there is a very strong conservation of the IAP-binding motif in all of the IAP-antagonists, the conservation of other regions, including the GH3 region, is minimal. (B) A distance tree of seven IAP-antagonists from D. melanogaster (D.mel), D. virilis (D. viri), Aedes aegypti (Aedes), Anopheles gambiae (Anopheles), and Bombyx mori (Bombyx). The numbers at each branch are the boot-strapping values (% confirmation). The scale bar represents 0.1 substitution per position.

Seven IAP antagonist sequences were subjected to detection of shared sequence motifs using MEME. The program was run in the Zero or One Occurrence Per Sequence (zoops) mode. (A) All of the seven sequences were detected as having a shared motif at their N-terminal end, which corresponds to the IAP-binding motif. (B) Six of the seven sequences, but not Hid, were detected as having a second shared motif, which corresponds to the GH3 domain identified in Grim and Reaper. For both A & B, the number in front of each sequence indicates the starting amino acid position of the motif in the corresponding protein. The number after the sequence indicates the p value reported by the program as the possibility to identify such a match in random sequences. (C) A helix wheel drawing of the second shared motif detected in Ibm1 (aa 21–35) indicates that it has the potential to form an amphipathic helix, similar to the GH3 motifs in Grim and Reaper.