Dissection of the BCL-2 family signaling network with stabilized alpha-helices of BCL-2 domains

Methods Enzymol. 2008:446:387-408. doi: 10.1016/S0076-6879(08)01623-6.

Abstract

The BCL-2 family of apoptotic proteins regulates the critical balance between cellular life and death and, thus, has become the focus of intensive basic science inquiry and a fundamental target for therapeutic development in oncology and other diseases. Classified based on the presence of conserved alpha-helical motifs and pro- and anti-apoptotic functionalities, BCL-2 proteins participate in a complex interaction network that determines cellular fate. The identification of BCL-2 homology domain 3 (BH3) as a critical death helix that engages and regulates BCL-2 family proteins has inspired the development of molecular tools to decode and drug the interaction network. Stabilized Alpha-Helices of BCL-2 domains (SAHBs) are structurally reinforced, protease-resistant, and cell-permeable compounds that retain the specificity of native BH3 death ligands and, therefore, serve as ideal reagents to dissect BCL-2 family interactions in vitro and in vivo. Here, we describe the in vitro and cell-based methods that exploit SAHB compounds to determine the functional consequences of BH3 interactions in regulating apoptosis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Annexin A5 / pharmacology
  • Apoptosis / drug effects*
  • BH3 Interacting Domain Death Agonist Protein / metabolism*
  • Cytochromes c / metabolism
  • Dextrans / metabolism
  • Fluorescein-5-isothiocyanate / analogs & derivatives
  • Fluorescein-5-isothiocyanate / metabolism
  • Fluorescence Polarization
  • Humans
  • Liposomes / metabolism
  • Mice
  • Mitochondria / metabolism
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins c-bcl-2 / physiology
  • Signal Transduction
  • bcl-2-Associated X Protein / physiology

Substances

  • Annexin A5
  • BH3 Interacting Domain Death Agonist Protein
  • Dextrans
  • Liposomes
  • Proto-Oncogene Proteins c-bcl-2
  • bcl-2-Associated X Protein
  • fluorescein isothiocyanate dextran
  • Cytochromes c
  • Fluorescein-5-isothiocyanate