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Liposomal Permeabilization Assay to Study the Functional Interactions of the BCL-2 Family

  • Denis E. Reyna
  • Evripidis Gavathiotis
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)

Abstract

Apoptosis, a form of programmed cell death that is important for development and homeostasis, is regulated by the BCL-2 family of proteins. Over twenty BCL-2 family members have been classified in three groups based on structural homology and function. The multidomain antiapoptotic proteins promote survival, whereas the multidomain and the BH3-only proapoptotic members induce cell death. Because the interaction among the BCL-2 family members occurs primarily at the mitochondrial outer membrane, biochemical assays using artificial liposomes have been developed to study the functional relationship between these proteins. The liposomal permeabilization assay is a cell-free system that relies on the ability of multidomain pro-apoptotic members to promote membrane permeabilization upon activation. By encapsulating a fluorophore and a quencher into liposomes, the degree of permeabilization can be quantified by the increase in fluorescence intensity as the fluorophore and quencher dissociate. The liposomal permeabilization assay has been used to delineate interactions among BCL-2 family members as well as to characterize peptides, small molecules, and lipids that modulate the function of BCL-2 family of proteins. Here, we describe in detail the permeabilization of liposomes induced by the interaction between BAX and BH3-only activator tBID.

Key words

BCL-2 family BAX BAK tBID BH3-domain Liposomal membrane Mitochondria MOMP Apoptosis 

Notes

Acknowledgments

We would like to thank current and past members of the Gavathiotis Laboratory for contributing to the optimization of this protocol and research. This work was supported by an NCI grant 1R01CA178394 and awards from the Sidney Kimmel Foundation for Cancer Research, the Gabrielle’s Angel Foundation for Cancer Research, the Alexandrine and Alexander L. Sinsheimer Foundation, the Pershing Square Sohn Cancer Research Alliance, the American Heart Association Collaborative Science Award (15CSA26240000), the Fondation Leducq Transatlantic Network of Excellence grant (RA15CVD04) and the Irma T. Hirschl Trust Career Award.

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Copyright information

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

Authors and Affiliations

  1. 1.Department of BiochemistryAlbert Einstein College of MedicineBronxUSA
  2. 2.Department of MedicineAlbert Einstein College of MedicineBronxUSA
  3. 3.Albert Einstein Cancer CenterAlbert Einstein College of MedicineBronxUSA
  4. 4.Wilf Family Cardiovascular Research InstituteAlbert Einstein College of MedicineBronxUSA

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