Examining BCL-2 Family Function with Large Unilamellar Vesicles

Summary

Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.

Abstract

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis¹. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)¹. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues².

In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)^3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation^7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members^7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.

Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization¹⁰. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)¹⁰. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)¹¹. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Protocol

1. Combine Lipids and Create a Lipid Film

1. Working in a fume hood with minimal lighting, chloroform-solubilized lipids are combined into a 1.5 ml amber glass vial using a chloroform-rinsed Hamilton gas-tight glass syringe. This combination will yield approximately 4 mg of lipid (Figure 1A).
   • Cardiolipin 10 mg/ml (CL) - 63 μl
   • Phosphatidylcholine 10 mg/ml (PC) - 84 μl
   • Phosphatidylethanoloamine 10 mg/ml (PE) - 125 μl
   • Phosphatidylinositol 10 mg/ml (PI) - 59.5 μl
   • Phosphatidylserine 10 mg/ml (PS) - 65 μl
2. To remove the chloroform, apply a gentle stream of an ....

Discussion

The described method for generating LUVs enables a rapid and efficient means to test the function of various BCL-2 family proteins, peptides, and related reagents in a biochemically-defined membrane environment similar to the OMM. If using end point values to determine LUV permeabilization, multiple plates can be set-up to analyze hundreds of conditions within a single day. We find that the limiting reagents in these assays tend to be the quality and quantity of recombinant proteins, so dedicating sufficient time and res.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,2-Dioleoyl-sn-Glycero-3-Phosph–thanolamine "PE"	Avanti	850725C
L-α-Phosphatidylcholine (Egg, Chicken) "PC"	Avanti	840051C
L-α-Phosphatidylinositol (Liver, Bovine) "PI"	Avanti	840042C
L-α-Phosphatidylserine (Brain, Porcine) "PS"	Avanti	8400320
Cardiolipin (Heart, Bovine - Sodium Salt) "CL"	Avanti	840012C
Mini-extruder set	Avanti	610023
PC membrane 0.2 mM	Avanti	610006
Costar black 96 well plate	Fisher Scientific	07-200-590
Caspase-8 cleaved human BID	R&D Systems	882-B8-050
Human BCL-xL (minus C-terminus)	R&D Systems	894-BX-050
BID/PUMA BH3 domain peptides	Anaspec	61711/62404
Synergy H1 hybrid multi-mode microplate reader	BioTek	None