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The role of the mitochondrial membrane system in apoptosis: the influence of oxidative stress on membranes and their interactions with apoptosis-regulating Bcl-2 proteins
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Gerhard Gröbner)ORCID iD: 0000-0003-2432-8118
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis is a crucial process in multicellular organisms in sculpting them, especially during embryogenesis. In addition, apoptosis is responsible for the clearance of harmful or damaged cells which can otherwise be detrimental to the organism. The Bcl-2 family proteins are key players in the regulation of the intrinsic pathway of the apoptotic machinery. This family consists of three subfamilies with B-cell CLL/lymphoma 2 (Bcl-2) protein itself representing anti-apoptotic members, the Bcl-2-associated X protein (Bax), and pro-apoptotic BH3-only signaling proteins. The interplay between pro- and anti-apoptotic proteins on the mitochondrial membranes is central to the balance between the life and death decision of whether the membrane should be permeabilized or not. The cytosolic Bax protein can upon cellular stress translocate to the mitochondrial membrane where it can either carry out its action of forming homo-oligomers that cause outer membrane permeabilization or be inhibited there by the anti-apoptotic membrane protein Bcl-2. Upon mitochondrial outer membrane permeabilization (MOMP) apoptogenic factors leak out from the intermembrane space (IMS) of the mitochondria, leading to caspase activation and ultimately cell death. A common stress signal initiating apoptosis is an increased formation of reactive oxygen species (ROS in the mitochondria, who can cause oxidative damage to lipid membranes. This membrane damage presumably influences the lipid landscape and the membrane features and hence the interactions of the Bcl-2 family proteins with each other and the mitochondrial outer membrane (MOM). To investigate the significance of membrane oxidation on the behavior of the Bcl-2 family proteins, especially Bax, synthetically produced oxidized phospholipids (OxPls) were incorporated in MOM-mimicking vesicles. Differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) spectroscopy revealed a major perturbation in membrane organization in the presence of OxPls. These changes in membrane properties increase the affinity of Bax to its target membrane and enable its partial penetration and formation of pores, as fluorescence leakage assays confirmed. However, in the absence of BH3-only proteins these pores are not sufficiently large for the release of apopototic factors such as cytochrome C (CytC). To understand the inhibition of Bax by the full-length Bcl-2 protein, suitable detergent solubilizing conditions were carefully chosen to enable the measurement of their direct binding to each other outside the membrane, by an antimycin A2 fluorescence assay. The observed protein-protein interaction was confirmed by surface plasmon resonance (SPR). An established protocol for the reconstitution of Bcl-2 into stable proteoliposomes now paves the way for structural studies of this key protein, in its membrane environment near physiological conditions; information essential for understanding its function, on a molecular level, and its potential as a cancer drug target.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2015. , 68 p.
Keyword [en]
Bax, Bc-2, apoptosis, mitochondria, membranes, oxidized lipids, NMR, calorimetry, circular dichroism
National Category
Chemical Sciences
Research subject
Biochemistry; Physical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-110701ISBN: 978-91-7601-375-5 (print)OAI: oai:DiVA.org:umu-110701DiVA: diva2:864265
Public defence
2015-11-20, Stora Hörsalen, KBC-huset (KB3B1), Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-10-30 Created: 2015-10-26 Last updated: 2015-11-13Bibliographically approved
List of papers
1. Impact of oxidized phospholipids on the structural and dynamic organization of phospholipid membranes: a combined DSC and solid state NMR study
Open this publication in new window or tab >>Impact of oxidized phospholipids on the structural and dynamic organization of phospholipid membranes: a combined DSC and solid state NMR study
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2013 (English)In: Faraday discussions (Online), ISSN 1359-6640, E-ISSN 1364-5498, Vol. 161, 499-513 p.Article in journal (Refereed) Published
Abstract [en]

Membranes undergo severe changes under oxidative stress conditions due to the creation of oxidized phospholipid (OxPls) species which possess molecular properties quite different from their parental lipid components. These OxPls play crucial roles in various pathological disorders and their occurrence is involved in the onset of intrinsic apoptosis, a fundamental pathway in programmed mammalian cell death. However, the molecular mechanisms by which these lipids can exert their apoptotic action via their host membranes (e.g. altering membrane protein function) are poorly understood. Therefore, we studied the impact of OxPls on the organization and biophysical properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) based lipid membranes by differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy. Incorporation of defined OxPls with either a carboxyl group (1-Palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC)) or aldehyde (1-Palmitoyl-(9´oxononanoyl)-sn-glycero-3-phosphocholine (PoxnoPC)) at their truncated sn-2-chain ends enabled us to reveal OxPls species dependent differences. The calorimetric studies revealed significant effects of OxPls on the thermotropic phase behavior of DMPC bilayers, especially at elevated levels where PazePC induced more pronounced effects than PoxnoPC. Temperature dependent changes in the solid state 31P NMR spectra which provided information of the of lipid headgroup region in these mixed membrane system, reflected this complex phase behavior. In the temperature region between 293 K (onset of L-phase) and 298 K two overlapping NMR spectra were visible which reflect the co-existence of two liquid-crystalline lamellar phases with presumably one reflecting OxPls-poor domains and the other OxPls-rich domains. Deconvolution of the DSC profiles also revealed these two partially overlapping thermal events. In addition, also a third thermal, non NMR-visible, event occurred at low temperatures, which mostly likely can be associated with a solid-phase mixing/demixing process of the OxPl-containing membranes. The observed phase transitions were moved to higher temperatures in the presence of heavy water due its condensing effect, where additional wideline 2H NMR studies revealed a complex hydration pattern in the presence of OxPls.

Place, publisher, year, edition, pages
RSC Publishing, 2013
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-58036 (URN)10.1039/C2FD20089A (DOI)
Note

First published online 27 Jun 2012

Available from: 2012-08-24 Created: 2012-08-24 Last updated: 2017-12-07Bibliographically approved
2. The oxidized phospholipid PazePC modulates interactions between Bax and mitochondrial membranes
Open this publication in new window or tab >>The oxidized phospholipid PazePC modulates interactions between Bax and mitochondrial membranes
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2012 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1818, no 11, 2718-2724 p.Article in journal (Refereed) Published
Abstract [en]

Activation of the pro-apoptotic protein Bax under intracellular oxidative stress is closely related to its association with the mitochondrial outer membrane (MOM) system, ultimately resulting in cell death. The precise mechanism by which this activation and the subsequent structural changes in the protein occur is currently unknown. In addition to triggering the onset of apoptosis, oxidative stress generates oxidized lipids whose impact on mitochondrial membrane integrity and the activity of membrane-associated Bax is unclear. We therefore devised a model system that mimics oxidative stress conditions by incorporating oxidized phospholipids (OxPls) into mitochondria-like liposomes, and studied the OxPls' impact on Bax-membrane interactions. Differential scanning calorimetry (DSC) was used to study membrane organization and protein stability, while conformational changes in the protein upon contact with lipid vesicles were monitored using far-UV circular dichroism (CD) spectroscopy. The thermograms for liposomes containing the OxPl 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) differed dramatically from those for unmodified liposomes. Moreover, Bax exhibited enhanced thermal stability in the presence of the modified liposomes, indicating that it interacted strongly with PazePC-containing membranes. The presence of PazePC also increased the α-helical character of Bax compared to the protein alone or with PazePC-free vesicles, at 10°C, 20°C, and 37°C. Presumably, the presence of PazePC-like OxPls a) increases the population of membrane-associated Bax and b) facilitates the protein's insertion into the membrane by distorting the bilayer's organization, as seen by solid-state high-resolution (1)H and (31)P magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy.

Place, publisher, year, edition, pages
Elsevier, 2012
Keyword
Apoptosis, Bax, CD, DSC, MAS NMR, PazePC
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-56762 (URN)10.1016/j.bbamem.2012.06.005 (DOI)22705638 (PubMedID)
Available from: 2012-06-26 Created: 2012-06-26 Last updated: 2017-12-07Bibliographically approved
3. Reconstitution of the anti-apoptotic bcl-2 protein into lipid membranes and biophysical evidence for its detergent-driven association with the pro-apoptotic bax protein
Open this publication in new window or tab >>Reconstitution of the anti-apoptotic bcl-2 protein into lipid membranes and biophysical evidence for its detergent-driven association with the pro-apoptotic bax protein
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 4, e61452- p.Article in journal (Refereed) Published
Abstract [en]

The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2) protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax), are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM) and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD) spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23)-lauryl-ether (Brij-35) detergent at a level below its critical micelle concentration (CMC). Additional surface plasmon resonance (SPR) measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A2 (a known inhibitory ligand of Bcl-2) to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC). Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.

Place, publisher, year, edition, pages
PLoS ONE, 2013
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-70192 (URN)10.1371/journal.pone.0061452 (DOI)23626686 (PubMedID)
Available from: 2013-05-07 Created: 2013-05-07 Last updated: 2017-12-06Bibliographically approved
4. Membranes and their lipids: a molecular insight into their organization and function
Open this publication in new window or tab >>Membranes and their lipids: a molecular insight into their organization and function
2014 (English)In: Advances in Biological Solid-State NMR: proteins and Membrane-Active Peptides / [ed] Frances Separovic and Akira Naito, Cambridge, GBR: Royal Society of Chemistry, 2014, 1, 113-132 p.Chapter in book (Refereed)
Place, publisher, year, edition, pages
Cambridge, GBR: Royal Society of Chemistry, 2014 Edition: 1
Series
New Developments in NMR, ISSN 2044-253X ; 3
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-110698 (URN)978-1-84973-910-8 (pISBN) (ISBN)9781782627449 (ISBN)
Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2015-10-28Bibliographically approved
5. The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax
Open this publication in new window or tab >>The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax
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2016 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 6, 1288-1297 p.Article in journal (Refereed) Published
Abstract [en]

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 15N-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.

Keyword
Apoptosis, Bax-protein, Calorimetry, Membranes, NMR, Oxidized phospholipids, Leakage
National Category
Chemical Sciences
Research subject
Biochemistry; Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-110699 (URN)10.1016/j.bbamem.2016.03.003 (DOI)000375356900023 ()
Note

Originally published in manuscript form with the title The oxidized phospholipid PazePC promotes the formation of Bax pores in mitochondrial membranes

Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2017-12-01Bibliographically approved

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