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Isolation of Synthetic Antibodies Against BCL-2-Associated X Protein (BAX)

  • Zhou Dai
  • Jonathan R. Lai
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)

Abstract

The BCL-2 protein family plays central roles in the mitochondrial pathway of cell apoptosis. The BCL-2-Associated X protein (BAX), along with other proapoptotic proteins, induces cell death in response to a variety of stress stimuli. Upon receipt of killing signals, cytosolic BAX is activated and translocates to mitochondria where it causes mitochondrial outer membrane permeabilization (MOMP) and initials a series of cellular events that eventually lead to cell destruction. Despite recent progress toward understanding the structure, function, and activation mechanism of BAX, detailed information about how cytosolic BAX can be inhibited is still limited. Here we describe a method of selecting synthetic antibody fragments (Fabs) against BAX using phage display. Synthetic antibodies discovered from the selection have been used as structural probes to gain novel mechanistic details on BAX inhibition. This synthetic antibody selection method could be potentially applied to other BCL-2 proteins.

Key words

BAX Apoptosis Synthetic antibodies Fab Phage display Antibody selection 

Notes

Acknowledgment

J.R.L. gratefully acknowledges funding from the Irma T. Hirschl Foundation, and the NIH (R01 AI125462).

References

  1. 1.
    Danial NN, Korsmeyer SJ (2004) Cell death. Cell 116(2):205–219CrossRefPubMedGoogle Scholar
  2. 2.
    Fuchs Y, Steller H (2011) Programmed cell death in animal development and disease. Cell 147(7):1640CrossRefGoogle Scholar
  3. 3.
    Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR (2010) The BCL-2 family reunion. Mol Cell 37(3):299–310CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Youle RJ, Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9(1):47–59CrossRefPubMedGoogle Scholar
  5. 5.
    Westphal D, Kluck RM, Dewson G (2014) Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis. Cell Death Differ 21(2):196–205CrossRefPubMedGoogle Scholar
  6. 6.
    Walensky LD, Gavathiotis E (2011) BAX unleashed: the biochemical transformation of an inactive cytosolic monomer into a toxic mitochondrial pore. Trends Biochem Sci 36(12):642–652CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Gavathiotis E, Suzuki M, Davis ML, Pitter K, Bird GH, Katz SG, Tu HC, Kim H, Cheng EH, Tjandra N, Walensky LD (2008) BAX activation is initiated at a novel interaction site. Nature 455(7216):1076–1081CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Gavathiotis E, Reyna DE, Davis ML, Bird GH, Walensky LD (2010) BH3-triggered structural reorganization drives the activation of proapoptotic BAX. Mol Cell 40(3):481–492CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Garner TP, Reyna DE, Priyadarshi A, Chen HC, Li S, Wu Y, Ganesan YT, Malashkevich VN, Almo SS, Cheng EH, Gavathiotis E (2016) An autoinhibited dimeric form of BAX regulates the BAX activation pathway. Mol Cell 63(3):485–497CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Czabotar PE, Westphal D, Dewson G, Ma S, Hockings C, Fairlie WD, Lee EF, Yao S, Robin AY, Smith BJ, Huang DC, Kluck RM, Adams JM, Colman PM (2013) Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis. Cell 152(3):519–531CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhang Z, Subramaniam S, Kale J, Liao C, Huang B, Brahmbhatt H, Condon SG, Lapolla SM, Hays FA, Ding J, He F, Zhang XC, Li J, Senes A, Andrews DW, Lin J (2016) BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes. EMBO J 35(2):208–236CrossRefPubMedGoogle Scholar
  12. 12.
    Hsu Y-T, Wolter KG, Youle RJ (1997) Cytosol-to-membrane redistribution of Bax and Bcl-XL during apoptosis. Proc Natl Acad Sci U S A 94(8):3668–3672CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Wolter KG, Hsu Y-T, Smith CL, Nechushtan A, Xi X-G, Youle RJ (1997) Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 139(5):1281–1292CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Pancera M, Zhou T, Druz A, Georgiev IS, Soto C, Gorman J et al (2014) Structure and immune recognition of trimeric pre-fusion HIV-1 Env. Nature 514(7523):455–461CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Koellhoffer JF, Chen G, Sandesara RG, Bale S, Ollmann Saphire E, Chandran K et al (2012) Two synthetic antibodies that recognize and neutralize distinct proteolytic forms of the Ebola virus envelope glycoprotein. Chembiochem 13(17):2549–2557CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Gao J, Sidhu SS, Wells JA (2009) Two-state selection of conformation-specific antibodies. Proc Natl Acad Sci U S A 106(9):3071–3076CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Koerber JT, Thomsen ND, Hannigan BT, Degrado WF, Wells JA (2013) Nature-inspired design of motif-specific antibody scaffolds. Nat Biotechnol 31(10):916–921CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Fellouse FA, Esaki K, Birtalan S, Raptis D, Cancasci VJ, Koide A et al (2007) High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J Mol Biol 373(4):924–940CrossRefPubMedGoogle Scholar
  19. 19.
    Uchime O, Dai Z, Biris N, Lee D, Sidhu SS, Li S et al (2016) Synthetic antibodies inhibit Bcl-2-associated X protein (BAX) through blockade of the N-terminal activation site. J Biol Chem 291(1):89–102CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiochemistryAlbert Einstein College of MedicineBronxUSA

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