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| 2. |
- Dingeldein, Artur Peter Günther, et al.
(författare)
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Apoptotic Bax at Oxidatively Stressed Mitochondrial Membranes : Lipid Dynamics and Permeabilization
- 2017
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 112:10, s. 2147-2158
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using P-31 solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing effects on Bax pore formation.
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| 3. |
- Dingeldein, Artur P G, et al.
(författare)
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BCL-2 Family Proteins Effect on Mitochondrial-Mimicking Membrane Structure by Solid State NMR
- 2015
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Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 108:2, s. 251A-252A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondria are not only the cells' powerhouse, but also involved in their suicide via apoptosis. Key regulators of this pathway are members of the Bcl-2 protein family which interact with the outer mitochondrial membrane to modulate permeability and enable the release of apoptotic stimuli like cytochrome c. For a long time the mitochondrial membrane forming lipids have been seen as merely structural building units with proteins doing the actual work. This view changed in recent years, since lipids were shown to be also directly involved in apoptotic events e.g. under intracellular oxidative stress. Oxidized phospholipids (OxPls) generated under these stress conditions might trigger mitochondria-mediated apoptosis. Their presence in mitochondrial membranes can severely alter the properties of these membranes with yet unknown consequences regarding the formation of pores through membrane-mediated interplay with apoptotic Bax protein. We therefore devised a model system that embodies oxidative stress conditions by incorporating OxPls into mitochondria mimicking model membranes composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) to study the impact of OxPls on apoptotic Bax-membrane interactions. To obtain molecular insight into hydrophobic fatty acid regions of membranes and their hydrophilic interface which is responsible for first protein-membrane contacts, we used differential scanning calorimetry (DSC) and solid state NMR spectroscopy. Upon incorporating OxPls with carboxyl (PoxnoPC) or aldehyde (PazePC) groups at their truncated sn-2-chains into our mitochondria model membranes, calorimetric and NMR measurements showed dramatic changes. 31P NMR experiments revealed major perturbation effects in these membranes; an effect which presumably elevates the membrane binding of apoptotic Bax to the charged membranes and its partial penetration, being a prerequisite for its final formation of pores which enable cytochrome c release from the mitochondrial interior. Currently structural studies of various Bax-lipid assemblies are ongoing.
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| 4. |
- Humpolickova, Jana, et al.
(författare)
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Dynamics and size of crosslinking-induced lipid nanodomains in model membranes
- 2012
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 102:3, s. 294a-
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Tidskriftsartikel (refereegranskat)abstract
- Changes of membrane organization upon crosslinking of its components trigger cellsignaling response to various exogenous factors. Crosslinking of raft gangliosides GM1with cholera toxin (CTxB) was demonstrated to cause microscopic phase separation inmodel membranes and the CTxB-GM1 complexes forming a minimal lipid raft unit aresubject of ongoing cell membrane research. Yet, those subdiffraction sized rafts havenever been described in terms of size and dynamics. By means of two-color z-scanfluorescence correlation spectroscopy, we show that the nano-sized domains are formedin model membranes at lower sphingomyelin content than needed for the large scalephase separation and that the CTxB-GM1 complexes are confined in the domains poorlystabilized with sphingomyelin. Fluorescence resonance energy transfer together withMonte Carlo modeling of the donor decay response reveal the domain radius ofapproximately 8 nm, which increases at higher sphingomyelin content. We observed twotypes of differently behaving domains, which suggests a dual role of the crosslinker: first,local transient condensation of the GM1 molecules compensating lack of sphingomyelinand second, coalescence of existing nanodomains ending in large scale phase separation.
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| 5. |
- Lidman, Martin, 1985-, et al.
(författare)
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The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax
- 2016
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1858:6, s. 1288-1297
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Tidskriftsartikel (refereegranskat)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 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.
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| 8. |
- Sachl, Radek, 1982-, et al.
(författare)
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Limitations of electronic energy transfer in the determination of lipid nanodomain sizes
- 2011
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Ingår i: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 101:11, s. L60-L62
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Tidskriftsartikel (refereegranskat)abstract
- Even though superresolution microscopy indicates that size of plasma membrane rafts is <20 nm, those structures have never been observed. Forster resonance energy transfer (FRET) is therefore still the most powerful optical method for characterization of such domains. In this letter we investigate relation between nanodomain affinity of a donor-acceptor (D/A) pair and the detectable nanodomain size/area. We show that probes with high affinity to the liquid-ordered (L(o)) phase are required for detecting domain sizes of a few nanometers, and/or domains that occupy a few percent of the bilayer area. A combination of donors and acceptors that prefer different phases is the more favorable approach. For instance, a D/A pair with the distribution constant of donors K(D) = 5 and acceptors K(A) = 0.01 can resolve a broad spectrum of nanodomain sizes. On the other hand, currently available donors and acceptors that prefer the same phase, either the liquid-disordered (L(d)) or L(o) phase, are not so convenient for determining domain sizes <20 nm. Here the detection limits of FRET experiments employing several commonly used D/A pairs have been investigated.
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| 9. |
- Stefl, Martin, et al.
(författare)
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Dynamics and size of cross-linking-induced lipid Nanodomains in model Membranes
- 2012
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 102:9, s. 2104-2113
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Tidskriftsartikel (refereegranskat)abstract
- Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin ( CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics. By means of two-color z-scan fluorescence correlation spectroscopy, we show that the nanosized domains are formed in model membranes at lower sphingomyelin (Sph) content than needed for the large-scale phase separation and that the CTxB-GM1 complexes are confined in the domains poorly stabilized with Sph. Forster resonance energy transfer together with Monte Carlo modeling of the donor decay response reveal the domain radius of similar to 8 nm, which increases at higher Sph content. We observed two types of domains behaving differently, which suggests a dual role of the cross-linker: first, local transient condensation of the GM1 molecules compensating for a lack of Sph and second, coalescence of existing nanodomains ending in large-scale phase separation.
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| 10. |
- Wallgren, Marcus, 1978-, et al.
(författare)
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Impact of oxidized phospholipids on the structural and dynamic organization of phospholipid membranes : a combined DSC and solid state NMR study
- 2013
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Ingår i: Faraday discussions. - : RSC Publishing. - 1359-6640 .- 1364-5498. ; 161, s. 499-513
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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