umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Impact of oxidized phospholipids on the structural and dynamic organization of phospholipid membranes: a combined DSC and solid state NMR study
Umeå University, Faculty of Science and Technology, Department of Chemistry.
J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Show others and affiliations
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. Vol. 161, 499-513 p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:umu:diva-58036DOI: 10.1039/C2FD20089AOAI: oai:DiVA.org:umu-58036DiVA: diva2:546626
Note

First published online 27 Jun 2012

Available from: 2012-08-24 Created: 2012-08-24 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Insight into the mitochondrial apoptotic pathway: The interplay of the pro-apoptotic Bax protein with oxidized phospholipids and its counterplayer, the pro-survival Bcl-2 protein
Open this publication in new window or tab >>Insight into the mitochondrial apoptotic pathway: The interplay of the pro-apoptotic Bax protein with oxidized phospholipids and its counterplayer, the pro-survival Bcl-2 protein
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis plays a crucial role in multicellular organisms by preserving tissue homeostasis and removing harmful cells. The anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and the pro-apoptotic Bcl-2-associated X protein (Bax) act as major regulators of the mitochondrial apoptotic pathway. Activation of Bax via stress signals causes its translocation to the mitochondrial outer membrane (MOM). There, Bax forms homo-oligomeric pores, leading to the release of apoptogenic factors, caspase activation and ultimately cell death. However, the underlying mechanism for the recruitment and pore forming activity of Bax is still not elucidated. Nevertheless, the mitochondrial membrane system seems to play an active and crucial role, presumably being directly involved in the onset of the mitochondrial apoptosis. Since the formation of reactive oxygen species (ROS) is a common stress signal and one of the hallmarks of the mitochondrial apoptosis, direct damage can occur to these membranes by the generation of oxidized phospholipids (OxPls), whose presence can crucially influence the pro-apoptotic action of Bax there. To better understand the impact of OxPls on membranes as well as their potential role in the mitochondrial apoptotic process, defined OxPl species were incorporated into phospholipid vesicles and studied with various biophysical techniques. Differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy were used to gain insight into changes in membrane properties in the presence of OxPls. In addition to circular dichroism (CD) spectroscopy, DSC and solid state NMR were furthermore performed to elucidate the impact of OxPls on Bax-membrane interactions. The occurrence of OxPls gave rise to dramatic changes in membrane organization and dynamics, manifested as lateral phase separation into OxPl-rich and -poor domains and modified hydration at the membrane interface. The presence of OxPls also had a great impact on the interaction between Bax and mitochondria-mimicking vesicles, strongly promoting the association of the protein with the membrane.

At the MOM, Bax is believed to be inhibited by Bcl-2. How this inhibition occurs is still a mystery due to the lack of biophysical information on Bcl-2, in particular on the full-length protein variant. Since Bcl-2 is also one of the main culprits in the progression of various forms of cancer, knowledge of the structural and mechanistic properties of the full-length protein is essential for a fundamental understanding of its function at a molecular level. To this end, a method for the production of full-length Bcl-2 was developed. By performing cell-free protein synthesis, preparative amounts of the protein were obtained, which enabled a biophysical characterization of the putative interaction between Bax and Bcl-2 using CD and fluorescence spectroscopy. A protocol for the reconstitution of Bcl-2 into proteoliposomes was also developed, promising for future studies of the full-length protein in its native membrane environment; a prerequisite to fully understand its pro-survival functions as well as providing crucial information for the design of novel anti-cancer drugs.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2012. 62 p.
Keyword
apoptosis, Bax, Bcl-2, CD, cell-free protein synthesis, DSC, membrane, mitochondria, oxidized phospholipids, reconstitution, solid state NMR
National Category
Biophysics
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-61290 (URN)978-91-7459-490-4 (ISBN)
Public defence
2012-11-30, KBC-huset, KB3B1, Umeå universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2012-11-09 Created: 2012-11-07 Last updated: 2012-11-08Bibliographically approved
2. 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
Open this publication in new window or tab >>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
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
Bax, Bc-2, apoptosis, mitochondria, membranes, oxidized lipids, NMR, calorimetry, circular dichroism
National Category
Chemical Sciences
Research subject
Biochemistry; Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-110701 (URN)978-91-7601-375-5 (ISBN)
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

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Wallgren, MarcusPham, Quoc DatKhanh, LinhLidman, MartinGröbner, Gerhard
By organisation
Department of Chemistry
In the same journal
Faraday discussions (Online)
Chemical Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 461 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf