title: The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax.
abstract: Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface.
authors.affiliation: Department of Chemistry, University of Umeå, SE-901 87 Umeå, Sweden.
authors.forename: Martin
authors.initials: M
authors.lastname: Lidman
authors.affiliation: J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v. v. i., Dolejškova 3, 182 23 Prague 8, Czech Republic; Department of Biochemistry, Charles University, Hlavova 8, 128 40 Prague 2, Czech Republic.
authors.forename: Šárka
authors.initials: Š
authors.lastname: Pokorná
authors.affiliation: Department of Chemistry, University of Umeå, SE-901 87 Umeå, Sweden.
authors.forename: Artur P G
authors.initials: AP
authors.lastname: Dingeldein
authors.affiliation: Department of Chemistry, University of Umeå, SE-901 87 Umeå, Sweden.
authors.forename: Tobias
authors.initials: T
authors.lastname: Sparrman
authors.affiliation: Department of Medical Chemistry and Biophysics, University of Umeå, SE-901 87 Umeå, Sweden.
authors.forename: Marcus
authors.initials: M
authors.lastname: Wallgren
authors.affiliation: J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v. v. i., Dolejškova 3, 182 23 Prague 8, Czech Republic.
authors.forename: Radek
authors.initials: R
authors.lastname: Šachl
authors.affiliation: J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v. v. i., Dolejškova 3, 182 23 Prague 8, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Hof
authors.affiliation: Department of Chemistry, University of Umeå, SE-901 87 Umeå, Sweden. Electronic address: gerhard.grobner@chem.umu.se.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Gröbner
mesh_terms: D002152:Calorimetry, Differential Scanning; D066241:Carbon-13 Magnetic Resonance Spectroscopy; D006801:Humans; D051336:Mitochondrial Membranes / Q000378:metabolism*; D010084:Oxidation-Reduction; D010539:Permeability; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000378:metabolism; D066244:Proton Magnetic Resonance Spectroscopy; D053835:Unilamellar Liposomes; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
chemical_list: C528536:1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; C494826:BAX protein, human; D053835:Unilamellar Liposomes; D051028:bcl-2-Associated X Protein; D010767:Phosphorylcholine
keywords: Apoptosis; Bax-protein; Calorimetry; Leakage; Membranes; NMR; Oxidized phospholipids