Skip to main content
SpringerLink
Log in

The role of IAP antagonist proteins in the core apoptosis pathway of the mosquito disease vector Aedes aegypti

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

While apoptosis regulation has been studied extensively in Drosophila melanogaster, similar studies in other insects, including disease vectors, lag far behind. In D. melanogaster, the inhibitor of apoptosis (IAP) protein DIAP1 is the major negative regulator of caspases, while IAP antagonists induce apoptosis, in part, by binding to DIAP1 and inhibiting its ability to regulate caspases. In this study, we characterized the roles of two IAP antagonists, Michelob_x (Mx) and IMP, in apoptosis in the yellow fever mosquito Aedes aegypti. Overexpression of Mx or IMP caused apoptosis in A. aegypti Aag2 cells, while silencing expression of mx or imp attenuated apoptosis. Addition of recombinant Mx or IMP, but not cytochrome c, to Aag2 cytosolic extract caused caspase activation. Consistent with this finding, AeIAP1 bound and inhibited both initiator and effector caspases from A. aegypti, and Mx and IMP competed with caspases for binding to AeIAP1. However, a difference was observed in the BIR domains responsible for Dronc binding by AeIAP1 versus DIAP1. These findings demonstrate that the mechanisms by which IAP antagonists regulate apoptosis are largely conserved between A. aegypti and D. melanogaster, although subtle differences exist.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Hay BA, Guo M (2006) Caspase-dependent cell death in Drosophila. Annu Rev Cell Dev Biol 22:623–650

    Article  PubMed  CAS  Google Scholar 

  2. Yan N, Shi Y (2005) Mechanisms of apoptosis through structural biology. Annu Rev Cell Dev Biol 21:35–56

    Article  PubMed  CAS  Google Scholar 

  3. Grimaldi D, Engel MS (2005) Evolution of the insects. Cambridge University Press, Cambridge

    Google Scholar 

  4. Bryant B, Ungerer MC, Liu Q, Waterhouse RM, Clem RJ (2010) A caspase-like decoy molecule enhances the activity of a paralogous caspase in the yellow fever mosquito, Aedes aegypti. Insect Biochem Mol Biol 40:516–523

    Article  PubMed  CAS  Google Scholar 

  5. Muro I, Berry DL, Huh JR, Chen CH, Huang H, Yoo SJ, Guo M, Baehrecke EH, Hay BA (2006) The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process. Development 133:3305–3315

    Article  PubMed  CAS  Google Scholar 

  6. Xu D, Wang Y, Willecke R, Chen Z, Ding T, Bergmann A (2006) The effector caspases drICE and dcp-1 have partially overlapping functions in the apoptotic pathway in Drosophila. Cell Death Differ 13:1697–1706

    Article  PubMed  CAS  Google Scholar 

  7. Dorstyn L, Kumar S (2008) A biochemical analysis of the activation of the Drosophila caspase DRONC. Cell Death Differ 15:461–470

    Article  PubMed  CAS  Google Scholar 

  8. Muro I, Monser K, Clem RJ (2004) Mechanism of Dronc activation in Drosophila cells. J Cell Sci 117:5035–5041

    Article  PubMed  CAS  Google Scholar 

  9. Shi Y (2008) Apoptosome assembly. Methods Enzymol 442:141–156

    Article  PubMed  CAS  Google Scholar 

  10. Snipas SJ, Drag M, Stennicke HR, Salvesen GS (2008) Activation mechanism and substrate specificity of the Drosophila initiator caspase DRONC. Cell Death Differ 15:938–945

    Article  PubMed  CAS  Google Scholar 

  11. Wang SL, Hawkins CJ, Yoo SJ, Muller H-AJ, Hay BA (1999) The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 98:453–463

    Article  PubMed  CAS  Google Scholar 

  12. Muro I, Hay BA, Clem RJ (2002) The Drosophila DIAP1 protein is required to prevent accumulation of a continuously generated, processed form of the apical caspase DRONC. J Biol Chem 277:49644–49650

    Article  PubMed  CAS  Google Scholar 

  13. Dorstyn L, Mills K, Lazebnik Y, Kumar S (2004) The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells. J Cell Biol 167:405–410

    Article  PubMed  CAS  Google Scholar 

  14. Means JC, Muro I, Clem RJ (2006) Lack of involvement of mitochondrial factors in caspase activation in a Drosophila cell-free system. Cell Death Diffeer 13:1222–1234

    Article  CAS  Google Scholar 

  15. Mendes CS, Arama E, Brown S, Scherr H, Srivastava M, Bergmann A, Steller H, Mollereau B (2006) Cytochrome c-d regulates developmental apoptosis in the Drosophila retina. EMBO Rep 7:933–939

    Article  PubMed  CAS  Google Scholar 

  16. Arama E, Agapite J, Steller H (2003) Caspase activity and a specific cytochrome C are required for sperm differentiation in Drosophila. Dev. Cell 4:687–697

    Article  PubMed  CAS  Google Scholar 

  17. Abdelwahid E, Yokokura T, Krieser RJ, Balasundaram S, Fowle WH, White K (2007) Mitochondrial disruption in Drosophila apoptosis. Dev Cell 12:793–806

    Article  PubMed  CAS  Google Scholar 

  18. Goyal G, Fell B, Sarin A, Youle RJ, Sriram V (2007) Role of mitochondrial remodeling in programmed cell death in Drosophila melanogaster. Dev Cell 12:807–816

    Article  PubMed  CAS  Google Scholar 

  19. Kumarswamy R, Seth RK, Dwarakanath BS, Chadna S (2009) Mitochondrial regulation of insect cell apoptosis: Evidence for permeability transition pore-independent cytochrome-c release in the Lepidopteran Sf9 cells. Int J Biochem Cell Biol 41:1430–1440

    Article  PubMed  CAS  Google Scholar 

  20. Wilson R, Goyal L, Ditzel M, Zachariou A, Baker DA, Agapite J, Steller H, Meier P (2002) The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis. Nat Cell Biol 4:445–450

    Article  PubMed  CAS  Google Scholar 

  21. Ditzel M, Broemer M, Tenev T, Bolduc C, Lee TV, Rigbolt KT, Elliott R, Zvelebil M, Blagoev B, Bergmann A, Meier P (2008) Inactivation of effector caspases through nondegradative polyubiquitylation. Mol Cell 32:540–553

    Article  PubMed  CAS  Google Scholar 

  22. Igaki T, Yamamoto-Goto Y, Tokushige N, Kanda H, Miura M (2002) Downregulation of DIAP1 triggers a novel Drosophila cell death pathway mediated by Dark and Dronc. J Biol Chem. 277:23103–23106

    Article  PubMed  CAS  Google Scholar 

  23. Zimmermann KC, Ricci J-E, Droin NM, Green DR (2002) The role of ARK in stress-induced apoptosis in Drosophila cells. J Cell Biol 156:1077–1087

    Article  PubMed  CAS  Google Scholar 

  24. Chai J, Yan N, Huh JR, Wu J-W, Li W, Hay BA, Shi Y (2003) Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination. Nat Struct Biol 10:892–898

    Article  PubMed  CAS  Google Scholar 

  25. Tenev T, Zachariou A, Wilson R, Ditzel M, Meier P (2005) IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. Nat Cell Biol 7:70–77

    Article  PubMed  CAS  Google Scholar 

  26. Yan SJ, Wu JW, Chai J, Li W, Shi Y (2004) Molecular mechanisms of DrICE inhibition by DIAP1 and removal of inhibition by Reaper, Hid and Grim. Nat Struct Mol Biol 11:420–428

    Article  PubMed  CAS  Google Scholar 

  27. Ditzel M, Wilson R, Tenev T, Zachariou A, Paul A, Deas E, Meier P (2003) Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis. Nat Cell Biol 5:373–376

    Article  Google Scholar 

  28. Zachariou A, Tenev T, Goyal L, Agapite J, Steller H, Meier P (2003) IAP-antagonists exhibit non-redundant modes of action through differential DIAP1 binding. EMBO J 22:6642–6652

    Article  PubMed  CAS  Google Scholar 

  29. Ryoo HD, Bergmann A, Gonen H, Ciechanover A, Steller H (2002) Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1. Nat Cell Biol 4:432–438

    Article  PubMed  CAS  Google Scholar 

  30. Yoo SJ, Huh JR, Muro I, Yu H, Wang L, Wang SL, Feldman RMR, Clem RJ, Müller H-AJ, Hay BA (2002) Hid, Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms. Nat Cell Biol 4:416–424

    Article  PubMed  CAS  Google Scholar 

  31. Colon-Ramos DA, Shenvi CL, Weitzel DH, Gan EC, Matts R, Cate J, Kornbluth S (2006) Direct ribosomal binding by a cellular inhibitor of translation. Nat Struct Mol Biol 13:103–111

    Article  PubMed  CAS  Google Scholar 

  32. Claveria C, Caminero E, Martinez AC, Campuzano S, Torres M (2002) GH3, a novel proapoptotic domain in Drosophila Grim, promotes a mitochondrial death pathway. EMBO J 21:3327–3336

    Article  PubMed  CAS  Google Scholar 

  33. Waterhouse RM, Kriventseva EV, Meister S, Xi Z, Alvarez KS, Bartholomay LC, Barillas-Mury C, Bian G, Blandin S, Christensen BM, Dong Y, Jiang H, Kanost MR, Koutsos AC, Levashina EA, Li J, Ligoxygakis P, Maccallum RM, Mayhew GF, Mendes A, Michel K, Osta MA, Paskewitz S, Shin SW, Vlachou D, Wang L, Wei W, Zheng L, Zou Z, Severson DW, Raikhel AS, Kafatos FC, Dimopoulos G, Zdobnov EM, Christophides GK (2007) Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes. Science 316:1738–1743

    Article  PubMed  CAS  Google Scholar 

  34. Bryant B, Blair CD, Olson KE, Clem RJ (2008) Annotation and expression profiling of apoptosis-related genes in the yellow fever mosquito, Aedes aegypti. Insect Biochem Mol Biol 38:331–345

    PubMed  CAS  Google Scholar 

  35. Bartholomay LC, Waterhouse RM, Mayhew GF, Campbell CL, Michel K, Zou Z, Ramirez JL, Das S, Alvarez K, Arensburger P, Bryant B, Chapman SB, Dong Y, Erickson SM, Karunaratne SH, Kokoza V, Kodira CD, Pignatelli P, Shin SW, Vanlandingham DL, Atkinson PW, Birren B, Christophides GK, Clem RJ, Hemingway J, Higgs S, Megy K, Ranson H, Zdobnov EM, Raikhel AS, Christensen BM, Dimopoulos G, Muskavitch MA (2010) Pathogenomics of Culex quinquefasciatus and meta-analysis of infection responses to diverse pathogens. Science 330:88–90

    Article  PubMed  CAS  Google Scholar 

  36. Li Q, Li H, Blitvich BJ, Zhang J (2007) The Aedes albopictus inhibitor of apoptosis 1 gene protects vertebrate cells from bluetongue virus-induced apoptosis. Insect Mol Biol 16:93–105

    Article  PubMed  CAS  Google Scholar 

  37. Pridgeon JW, Zhao L, Becnel JJ, Clark GG, Linthicum KJ (2008) Developmental and environmental regulation of AaeIAP1 transcript in Aedes aegypti. J Med Entomol 45:1071–1079

    Article  PubMed  CAS  Google Scholar 

  38. Liu Q, Clem RJ (2010) Defining the core apoptosis pathway in the mosquito disease vector Aedes aegypti: the roles of iap1, ark, dronc, and effector caspases. Apoptosis. doi:10.1007/s10495-010-0558-9

  39. Zhou L, Jiang G, Chan G, Santos CP, Severson DW, Xiao L (2005) Michelob_x is the missing inhibitor of apoptosis protein antagonist in mosquito genomes. EMBO Rep 6:769–774

    Article  PubMed  CAS  Google Scholar 

  40. Bryant B, Zhang Y, Zhang C, Santos CP, Clem RJ, Zhou L (2010) A lepidopteran orthologue of reaper reveals functional conservation and evolution of IAP antagonists. Insect Mol Biol 18:341–351

    Article  Google Scholar 

  41. Wang H, Blair CD, Olson KE, Clem RJ (2008) Effects of inducing or inhibiting apoptosis on Sindbis virus replication in mosquito cells. J Gen Virol 89:2651–2661

    Article  PubMed  CAS  Google Scholar 

  42. Brackney DE, Scott JC, Sagawa F, Woodward JE, Miller NA, Schilkey FD, Mudge J, Wilusz J, Olson KE, Blair CD, Ebel GD (2010) C6/36 Aedes albopictus cells have a dysfunctional antiviral RNA interference response. PLoS Negl Trop Dis 4:e856

    Article  PubMed  Google Scholar 

  43. Scott JC, Brackney DE, Campbell CL, Bondu-Hawkins V, Hjelle B, Ebel GD, Olson KE, Blair CD (2010) Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells. PLoS Negl Trop Dis 26:e848

    Article  Google Scholar 

  44. Crook NE, Clem RJ, Miller LK (1993) An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. J Virol 67:2168–2174

    PubMed  CAS  Google Scholar 

  45. Liu Z, Sun C, Olejniczak ET, Meadows RP, Betz SF, Oost T, Herrmann J, Wu JC, Fesik SW (2000) Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature 408:1004–1008

    Article  PubMed  CAS  Google Scholar 

  46. Wu G, Chai J, Suber TL, Wu J-W, Du C, Wang X, Shi Y (2000) Structural basis of IAP recognition by Smac/DIABLO. Nature 408:1008–1012

    Article  PubMed  CAS  Google Scholar 

  47. Wright CW, Clem RJ (2002) Sequence requirements for Hid binding and apoptosis regulation in the baculovirus inhibitor of apoptosis Op-IAP: Hid binds Op-IAP in a manner similar to Smac binding of XIAP. J Biol Chem 277:2454–2462

    Article  PubMed  CAS  Google Scholar 

  48. Hawkins CJ, Yoo SJ, Peterson EP, Wang SL, Vernooy SY, Hay BA (2000) The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM. J Biol Chem 275:27084–27093

    PubMed  CAS  Google Scholar 

  49. Meier P, Silke J, Leevers SJ, Evan GI (2000) The Drosophila caspase DRONC is regulated by DIAP1. EMBO J 19:598–611

    Article  PubMed  CAS  Google Scholar 

  50. Reibarkh M, Yamamoto Y, Singh CR, del Rio F, Fahmy A, Lee B, Luna RE, Ii M, Wagner G, Asano K (2008) Eukaryotic initiation factor (eIF) 1 carries two distinct eIF5-binding faces important for multifactor assembly and AUG selection. J Biol Chem 283:1094–1103

    Article  PubMed  CAS  Google Scholar 

  51. Varkey J, Chen P, Jemmerson R, Abrams JM (1999) Altered cytochrome c display precedes apoptotic cell death in Drosophila. J Cell Biol 144:701–710

    Article  PubMed  CAS  Google Scholar 

  52. Dorstyn L, Read S, Cakouros D, Huh JR, Hay BA, Kumar S (2002) The role of cytochrome c in caspase activation in Drosophila melanogaster cells. J Cell Biol 156:1089–1098

    Article  PubMed  CAS  Google Scholar 

  53. Clem RJ, Miller LK (1994) Control of programmed cell death by the baculovirus genes p35 and iap. Mol Cell Biol 14:5212–5222

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Katsura Asano (Kansas State University) for providing the GB expression plasmid and Taryn Penabaz for culturing cells. This work was supported by NIH grants R21 AI067642 (to R.J.C.) and P20 RR16475 from the BRIN program of the National Center for Research Resources, by the Terry C. Johnson Center for Basic Cancer Research, and by the Kansas Agricultural Experiment Station. This is contribution number 10-343-J from the Kansas Agricultural Experiment Station.

Author information

Author notes

  1. Hua Wang

    Present address: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

Authors and Affiliations

  1. Molecular, Cellular, and Developmental Biology Program, Arthropod Genomics Center, Division of Biology, Kansas State University, 268 Chalmers Hall, Manhattan, KS, 66506, USA

    Hua Wang & Rollie J. Clem

Authors
  1. Hua Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rollie J. Clem
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rollie J. Clem.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 449 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, H., Clem, R.J. The role of IAP antagonist proteins in the core apoptosis pathway of the mosquito disease vector Aedes aegypti . Apoptosis 16, 235–248 (2011). https://doi.org/10.1007/s10495-011-0575-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-011-0575-3

Keywords

Search

Navigation