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. 2005 Aug;6(8):769-74.
doi: 10.1038/sj.embor.7400473.

Michelob_x is the missing inhibitor of apoptosis protein antagonist in mosquito genomes

Lei Zhou  1 , Guohua Jiang, Gina Chan, Carl P Santos, David W Severson, Lei Xiao
Affiliations

Michelob_x is the missing inhibitor of apoptosis protein antagonist in mosquito genomes

Lei Zhou et al. EMBO Rep. 2005 Aug.

Abstract

Apoptosis is implicated in the life cycle of the malaria parasite in mosquitoes. The genome project for the primary malaria vector Anopheles gambiae showed a significant expansion of the inhibitor of apoptosis protein (IAP) and caspase gene families in comparison with Drosophila. However, because of extensive sequence divergence, no orthologue was identified for the reaper/grim-like IAP antagonist genes that have a pivotal role in cell death regulation in Drosophila. Using a customized searching strategy, we identified michelob_x(mx), a gene not predicted by the genome project, as the missing IAP antagonist in the An. gambiae and other mosquito genomes. Mx has a highly conserved amino-terminal IAP-binding motif. Expression of Mx induces rapid cell death in insect cell lines and is a potent tissue ablator in vivo. Its proapoptotic activity is totally dependent on the IAP-binding motif. Like reaper in Drosophila, mx is transcriptionally induced by ultraviolet irradiation to mediate cell death.

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Figures

Figure 1
Figure 1
Comparison of Mx sequences with Reaper and Grim. (A) Alignment of Mx sequences from Anopheles gambiae (An.gamb), Aedes albopictus (Ae.albo) and Aedes aegypti (Ae.aegy), and Grim (NP_524137) and Reaper (NP_524138) sequences from the fruitfly. The relative position of the single intron (indicated with solid triangle) in mx genes is conserved between Anopheles and Aedes. There are no introns in grim or reaper. The colour scheme reflects percentage identity among the five aligned sequences (red, 100%; blue, >75%; and green, >50%). (B) Protein distance tree showing that Mx is slightly closer to Reaper and Grim than to Hid and Sickle. The scale bar reflects the rate of nucleotide change per position. (C) Comparison of the inhibitor of apoptosis protein (IAP)-binding motifs in fly (Reaper, Grim, Hid (NP_524136), Sickle (NP_524139) and Jafrac2 (Q9V3Q4)), mosquito (Michlob_x) and human (Smac/IDIA (NP_063940), OmiI/HtrA (O43464)) IAP antagonists. Numbers indicate the position of the first Ala in the host proteins.
Figure 2
Figure 2
The IAP (inhibitor of apoptosis protein)-binding motif (IBM) is required for the proapoptotic activity of Mx. (A) Expression of Mx in S2 cells induces cell apoptosis similar to Reaper, or Reaper(107), which has two helix-disturbing substitutions in its GH3 (Grim helix 3) domain (Leu35Pro and Ala36Pro). Although Reaper (−IBM) retained partial proapoptotic activity, removal of the IBM (amino acids 2–8) from Mx totally abolished its proapoptotic activity. Error bars represent the standard error of 4–5 measurements. mx.ag, mx cloned from Anopheles gambiae; mx.ae, mx cloned from Aedes albopictus; mx from Aedes aegypti behaved the same as the two presented here (data not shown). (B) Expression of mx induced cell death in mosquito C6/36 cells, which is dependent on the IBM domain. Mx from Ae. albopictus was used. (C) Western blot analysis of cell lysates from transfected H1299 cells. At 48 h after transfection, there is significant accumulation of mutant protein Mx(−IBM) in cells transfected with pRK5-Mx(−IBM):HA (haemagglutinin) or pRK5-Mx(−IBM):Flag. In contrast, there is little accumulation of the wild-type protein in cells transfected with pRK5-Mx:HA or pRK5-Mx:Flag. Equal amounts of whole-cell lysates were loaded on all lanes. (D–G) Expression of mx in transgenic fly strain induces cell/tissue ablation. P52Gal4 is an enhancer trap insertion in the GP150 gene (Melnattur et al, 2002). It guides the expression of UAS-lacZ in ventral musculature (arrowhead in (D), ventral view, stage 15) as well as in the central nervous system midline glia and neurons (arrow in (F), sagittal view, stage 15). Expression of mx (E,G) caused the elimination of muscle cells; the arrowhead in (E) (ventral view, stage 15) points to the empty space that should be occupied by the β-galactosidase (β-Gal)-expressing muscle cells (compare with D). Corresponding with the disappearance of β-Gal-positive muscle cells is the emergence of round β-Gal-positive apoptotic inclusion bodies (blue triangles in E) inside migrating microphages. Similarly, expression of mx also eliminated midline neurons (arrow in G, compare with F).
Figure 3
Figure 3
Mx-induced cell death is blocked by Diap1 and P35. (A) Unlike Reaper- or Grim-induced cell death, which cannot be totally blocked by Diap1, cell death induced by Mx is readily blocked by Diap1, the principal death-inhibiting IAP (inhibitor of apoptosis protein) from the fruitfly, and by p35, a viral caspase inhibitor. In this regard, it is similar to Reaper(107), which has two helix-disrupting substitutions in the GH3 domain. The colour of the bars reflects the ratio of the two testing constructs in a total of 0.9 μg per sample. Error bars represent the standard error of repeated measurements (n=3). (B) Direct interaction between Diap1 and Mx is detected by co-immunoprecipitation (IP). This interaction is abolished when the IBM (amino acids 2–8) is removed from Mx. NCI-H1299 cells were transfected with 1 μg of pRK5-HA (haemagglutinin):Diap along with 3 μg of pRK5-Mx:Flag, or pRK5-Mx(IBM):Flag, or empty vector. At 48 h after transfection, cells were lysed. Equal amounts of whole-cell lysates were immunoprecipitated with an anti-HA antibody (12CA5). The immunoprecipitate was resolved by SDS–polyacrylamide gel electrophoresis and transferred to the membrane, followed by immunoblotting (IB) with anti-Flag.
Figure 4
Figure 4
mx is transcriptionally upregulated by ultraviolet light to mediate cell death. (A) mx RNA is rapidly induced after ultraviolet (UV) treatment of C6/36 mosquito cells. Units are relative to the messenger RNA level of the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Error bars represent standard errors (n=3). (B) Transfection of double-stranded mx RNA (ds_mx) suppresses UV-induced cell death in C6/36 cells. Double-stranded green fluorescent protein (GFP) RNA (ds_gfp) was used as a control. Transfection of dsRNA and DNA was followed by 2 h recovery before UV treatment. Error bars represent standard errors (n=4 for 1 mJ and n=3 for 6 mJ).
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