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

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.

1. Introduction

Programmed cell death, or apoptosis, regulates the critical balance between cellular life and death, and deregulated apoptosis can lead to a variety of human diseases [1]. Apoptosis can occur through two pathways: the TNF/Fas death receptor dependent extrinsic pathway and the BCL-2 protein family dependent intrinsic pathway [1]. The intrinsic pathway is characterized by permeabilization of the outer mitochondrial membrane and release of soluble factors from the mitochondria that are important in caspase activation [2]. The BCL-2 family of proteins, which regulate the intrinsic apoptotic pathway, is divided into antiapoptotic, proapoptotic, and BH3-only proteins, and their complex interactions can prevent or promote mitochondrial dysfunction [3]. By sequestering the BH3 helices of both BH3-only and multidomain propapoptotic proteins, antiapoptotic BCL-2 proteins promote cell survival [3]. The BH3-only proteins, on the other hand, function as stress sensors of cellular damage and transmit prodeath signals from various signaling pathways to the core apoptotic machinery [3]. A subset of BH3-only proteins, such as BIM and BID, can directly interact and activate BAX and BAK [4]. Upon activation, proapoptotics BAX and BAK oligomerize and promote mitochondrial outer membrane permeabilization (MOMP) leading to the release of apoptogenic factors (e.g., cytochrome c and Smac/DIABLO) that irreversibly initiate the caspase cascade and ultimately execute the death program [4].

Binding of the BCL-2 members occurs primarily at the mitochondrial outer membrane (MOM) [2]. Using artificial liposomes, a biochemical assay was developed to study the integrity of liposomal membranes’ upon BCL-2 family members’ interaction [5, 6]. Using the liposome permeabilization assay, as well as other supportive techniques, it was demonstrated for instance that anti-apopoptotics such as BCL-2 and BCL-X_L inhibit membrane permeabilization by directly sequestering tBID and/or BAX [5]. Additionally, the liposome permeabilization assay has been adapted as a screening tool to identify and characterize peptides, small molecules or antibodies that specifically bind to BCL-2 family members and modulate their function [7–9]. For instance, a small molecule that directly induces the activation of the BAX trigger site was characterized using this permeabilization assay [10]. Additionally, the inhibitory potential of MCL-1 small molecules binders was determined with this biochemical technique [11]. Synthetic antibodies that bind to the N-terminal activation site of BAX were shown to directly inhibit BAX-mediated liposomal permeabilization [9]. Hence, the liposome permeabilization assay constitutes a simple and well-established cell-free system that explores the function of recombinant BCL-2 family members in the absence of other mitochondrial proteins [5, 12, 13].

In general, liposomes are composed of defined lipids identified in lipid composition studies from solvent extracted Xenopus mitochondria that mimic the composition of the MOM [14]. The liposome permeabilization assay relies on the ability of proapoptotic proteins, such as BAX and BAK, to promote membrane permeabilization upon activation. To quantify membrane permeabilization, a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and a cationic quencher (DPX: p-xylene-bis-pyridinium bromide) are incorporated within the liposomes [6]. As the liposome is permeabilized by activated BAX [10] or BAK [15], ANTS and DPX diffuse apart, and an increase in fluorescence is detected using a fluorescence plate reader. Here we will discuss in more detail the experimental procedures to measure tBID-induced BAX-mediated permeabilization of liposomes.

2. Materials

2.1. Lipid Composition of Liposomes

See Table 1 (see Note 1)

Table 1.

Composition of lipids for preparation of liposomes (see Note 1))

Lipid	Mole %	Chloroform stock (mg/mL)	Volume f mg lipid or 1 film (μL)
POPC	48	25 mg/mL	18.3
POPE	28	25 mg/mL	10.1
PI	10	10 mg/mL	11.7
DOPS	10	10 mg/mL	10.2
TOCL	4	10 mg/mL	7.5

2.2. Reagents

1. Liposome Buffer (see Note 2): 10 mM HEPES pH 7.2, 200 mM KCl, 0.2 mM EDTA, 5 mM MgCl₂.

2. ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid) (see Note 3).

3. DPX ( p-xylene-bis-pyridinium bromide) (see Note 3).

4. Sepharose CL2B.

5. Recombinant human BID Caspase-8 cleaved (tBID).

6. Recombinant human BAX.

7. Triton X-100.

8. Liquid nitrogen.

9. Gas nitrogen.

2.3. Equipment

1. Ultrasonic water bath.

2. Gravity-flow chromatography column (14 × 1.5 cm).

3. Mini-extruder.

4. 96-well back plates.

5. 10 mm filter support.

6. Gas-tight syringe (1000 μL).

7. Nuclepore Track-Etch polycarbonate membranes (diameter 19 mm, pore size 0.1 μm).

8. 5 mL glass tubes.

9. Fluorescence plate reader (Excitation wavelength: 355 nm; Emission wavelength: 520 nm).

3. Methods

3.1. Making Lipid Film

1. Under a fume hood, use a pipette to add appropriate amounts of chloroform-solubilized lipids into a glass tube (see Note 4) to a total of 1 mg lipid (see Table 1, see Note 1).

2. After adding each lipid into glass tube, evaporate off chloroform with a “low” stream of nitrogen or argon gas.

3. Protect lipid film from light exposure and place under vacuum for a minimum of 3 h at room temperature to remove any remaining chloroform (see Note 5).

4. Dried lipid film can be used immediately for experimentation or for storage cover film with nitrogen gas, seal with parafilm, protect from light and place in −20 °C (see Note 6).

3.2. Preparing Liposomes

1. Add 12.5 mM ANTS and 45 mM DPX to a 1 mg dry lipid film (see Note 7).

2. Hydrate dry lipid film with 1 mL of liposome buffer (10 mM HEPES pH 7.2, 200 mM KCl, 0.2 mM EDTA, and 5 mM MgCl₂). Keep solution on ice and protect from light. Additionally vortex thoroughly for 10 min with intervals of 1 min until ANTS and DPX are completely dissolved (see Note 8).

3. To generate unilamellar liposomes, sonicate the lipid film using an ultrasonic water bath sonicator for 10 min (see Notes 9 and 10).

4. In order to generate liposomes of a uniform size, the hydrated lipid film is extruded through a filter with 0.1 μm pore size. Assemble the extruder according to the manufacturer’s guidelines (see Note 11). Once the extruder is properly assembled, extrude lipid solution 11 times (see Note 12).

5. Next, set up a 10 mL bed volume Sepharose CL2B size-exclusion column. This step allows you to separate excess ANTS and DPX in solution from ANTS/DPX encapsulated liposomes. Wash with three column volumes of liposome buffer for three times until all the buffer has run out and the flow has stopped.

6. Carefully add the extruded ANTS/DPX liposome solution to the column and collect flow through as your first fraction (approximately 1 mL); cap the column once the flow has stopped (see Note 13).

7. Apply 6 mL of liposome buffer to the column and collect fractions (1 mL each) in glass tubes (see Note 13). Liposomes will elute in fractions 4 and 5 (see Note 14). Combine the two ANTS/DPX liposome-containing fractions (~0.5 mg/mL lipid). Protect from light and store at 4 °C until use.

3.3. Assessing the Stability of ANTS/ DPX Liposome-Containing Fractions

1. Confirm the stability of ANTS/DPX liposomes before running an experiment. Liposome stability may be compromised if any of the steps provided above were not followed accurately (see Note 15).

2. Test ANTS/DPX liposomes stability by comparing the fluorescence intensity of ANTS/DPX exposed to liposome buffer alone or liposome buffer supplemented with 0.2% Triton. It is important to assess if ANTS/DPX liposome are stable over time.

3. For control experiment, on a black 96-well plate, add 90 μL of liposome buffer and 10 μL of ANTS/DPX liposome per well (triplicates). Also, on separate wells, add 90 μL of liposome buffer, 10 μL of ANTS/DPX liposome and 2 μL of 10% Triton X-100 (final concentration 0.2%) (see Note 16).

4. Next, read the plate at 30 °C for 1 h using a fluorescence plate reader set to excite at 355 nm (5 nm bandwidth) and emission at 520 nm (12 nm bandwidth) (see Note 17).

5. If ANTS/DPX liposomes are stable, there is a four- to fivefold increase in fluorescence between control (no Triton) and burst (Triton) samples over time (see Note 18).

3.4. tBID Induced BAX-Mediated Permeabilization in Liposomes

1. To study the permeabilization induced by BAX upon tBID activation ANTS/DPX liposomes are assayed in a 96-well format using a black plate. The total volume per reaction (well) is 100 μL where 10 μL corresponds to ANTS/DPX liposomes (see Notes 19 and 20).

2. Next, add 10 μL of a 10× solution of your desired BAX concentration and 10 μL of a 10× solution of your desired tBID concentration. For recombinant full length BAX a concentration ranging from 100 nM to 500 nM is sufficient to detect liposome permeabilization induced after tBID activation. 20 nM tBID and higher concentrations are sufficient to induce BAX activation (see Notes 21–23).

3. Read fluorescence emission of ANTS using a fluorescence plate reader set to excite at 355 nm (5 nm bandwidth) and emission at 520 nm (12 nm bandwidth). Fluorescence emission is recorded every minute for 2.5 h at room temperature (see Notes 17 and 24).

4. After 2.5 h, remove plate from plate reader and add 2 μL of 10% Triton and read plate again for 10 min every minute. Triton is added to record the maximal liposomal release per well.

5. To normalize the raw data, the percentage release of ANTS/ DPX at every minute is calculated as percentage of fluorescence emission = ((F − F₀)/(F₁₀₀ − F₀)) × 100, where F₀ corresponds to the fluorescence intensity at 1 min and F₁₀₀ to maximal fluorescence from Triton treatment and F to the fluorescence intensity at a given time. The normalized data is either plotted as kinetic study (Fig. 1a) or as a single time point (Fig. 1b).

Fig. 1.

Kinetic (a) or single-time point (b) study of the permeabilization of ANTS/ DPX liposomes upon tBID-induced BAX activation.