Unsaturation of Fatty Acids Promotes Pore-Forming Activity of Bax and Bak in Apoptosis

Background

Apoptosis is the major form of programmed cell death, involved in fundamental biological processes such as embryonic development, tissue homeostasis, and immune system functions. Dysregulation of apoptosis can lead to neurodegenerative diseases and tumorigenesis, and most anti-cancer chemotherapies rely on inducing apoptosis in tumor cells. Mitochondrial outer membrane permeabilization (MOMP) is a key event in the mitochondrial apoptosis pathway, where the opening of the apoptotic pore allows the release of cytochrome c and other apoptogenic factors into the cytosol, activating caspases and eventually leading to cell death. However, the molecular structure of the apoptotic pore remains elusive, and the contribution of lipids to MOMP is particularly poorly understood.

The pro-apoptotic members Bax and Bak of the Bcl-2 protein family are critical effectors regulating MOMP. In healthy cells, Bax is predominantly cytosolic, while Bak is associated with the mitochondrial outer membrane (MOM). During apoptosis, Bax and Bak oligomerize and undergo conformational changes at the MOM, forming pores that release apoptogenic factors.

Although the role of lipids in modulating the function of Bcl-2 family proteins and mitochondrial permeability has been studied, our understanding of the specific lipid contributions at the apoptotic pore boundary remains limited. To address this question, the present study utilized nanodiscs to explore the lipid environment surrounding Bak during pore formation.

Source

This research was conducted by Shashank Dadsena, Rodrigo Cuevas Arenas, Gonçalo Vieira, Susanne Brodesser, Manuel N. Melo, and Ana J. García-Sáez from the CECAD Research Center, University of Cologne, Bijvoet Center for Biomolecular Research, Utrecht University, Instituto de Tecnologia Química e Biológica António Xavier, and Max Planck Institute for Biophysical Chemistry, respectively. The paper was published in Nature Communications in 2024.

Research Process

1. Extraction and Separation of Bak Protein with Nanodiscs: The researchers used SMA copolymers to extract and separate Bak protein and its surrounding membrane environment. GFP-tagged nanodiscs containing Bak were affinity-purified, and their size and homogeneity were assessed by Dynamic Light Scattering (DLS) and Negative Stain Transmission Electron Microscopy (TEM).

2. Lipidomic Analysis: Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) was employed to analyze the extracted lipids, with a focus on changes in major mitochondrial lipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The results indicated an enrichment of unsaturated lipid species in the Bak-containing nanodiscs under apoptotic conditions.

3. Effects on Membrane Permeability: The researchers investigated the impact of unsaturated lipids on Bax pore-forming activity using model membranes, giant unilamellar vesicles (GUVs), and cellular systems. The results demonstrated that unsaturated lipids significantly promoted Bax pore-forming activity in all systems. Furthermore, coarse-grained molecular dynamics (CG-MD) simulations supported these findings, suggesting that the enrichment of unsaturated lipids at the pore boundary facilitates pore formation.

4. Role of FADS2 Enzyme: The study found that the absence of FADS2, a key fatty acid desaturase enzyme, reduced cellular sensitivity to apoptosis and decreased the activation of the cGAS/STING pathway. Additionally, FADS2 levels correlated with the apoptotic sensitivity of different lung and kidney cancer cell lines upon co-treatment with unsaturated fatty acids.

Key Findings

1. Lipid Composition Analysis: LC-MS/MS analysis revealed a significant increase in unsaturated species of major phospholipids (PC and PE) in Bak-containing nanodiscs under apoptotic conditions, while saturated species decreased. Other phospholipids such as phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidic acid (PA) did not show significant changes.

2. Promotion of Membrane Permeability: Experiments with model membranes, GUVs, and cellular systems demonstrated that unsaturated lipids significantly enhanced Bax pore-forming activity, with higher levels of unsaturation leading to increased pore formation. CG-MD simulations supported these findings, showing a significant reduction in the energy barrier for pore formation with unsaturated PC compared to saturated PC, favoring pore formation.

3. Role of FADS2 in Apoptosis: Apoptotic sensitivity was correlated with FADS2 levels. FADS2 deficiency led to a decrease in unsaturated fatty acids in the membrane, reducing Bax/Bak pore-forming activity during MOMP and impacting the downstream cGAS/STING signaling pathway. Supplementation with unsaturated fatty acids restored this defect, indicating that FADS2 is an important regulator of apoptotic sensitivity.

Conclusions and Significance

This study revealed a significant enrichment of unsaturated lipids in the mitochondrial outer membrane during apoptosis, and this change in the lipid environment directly promoted the membrane permeabilizing activity of Bax and Bak, facilitating the formation of the apoptotic pore. This finding not only deepens our understanding of the MOMP process by unveiling the critical role of the lipid environment in apoptotic pore formation but also provides new insights for anti-cancer therapies, suggesting that modulating cellular lipid environments could enhance the efficacy of apoptosis-inducing agents.

The study highlights the importance of lipids in apoptosis regulation and provides a theoretical basis for lipid-modulating anti-cancer strategies. Future research could further explore the interplay between lipid environments and other apoptosis regulatory proteins, as well as their multifaceted roles in cell survival and death.