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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
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Gerhard Gröbner)
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 [en]
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: urn:nbn:se:umu:diva-61290ISBN: 978-91-7459-490-4 (print)OAI: oai:DiVA.org:umu-61290DiVA: diva2:565555
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
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. Expression and purification of full-length anti-apoptotic Bcl-2 using cell-free protein synthesis
Open this publication in new window or tab >>Expression and purification of full-length anti-apoptotic Bcl-2 using cell-free protein synthesis
2011 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 77, no 2, 220-223 p.Article in journal (Refereed) Published
Abstract [en]

The anti-apoptotic B cell CLL/lymphoma-2 (Bcl-2) protein is a key player in the regulation of programmed cell death and is linked to various types of cancer and their resistance to drug treatment. Biophysical and structural studies of the full-length intact Bcl-2 have been hampered due to difficulties in expression and severe solubility problems, precluding isolation of this hydrophobic membrane protein. Therefore, previous work has so far mainly been carried out using structurally modified Bcl-2 variants, lacking the transmembrane region. Thus, biophysical information regarding the full-length protein is still missing. Here, a protocol is presented for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)). A batch-based cell-free expression system, using extract isolated from Escherichia coli (E. coli) was employed to produce recombinant protein encoded by an optimized gene sequence. Presence of polyoxyethylene-(20)-cetyl-ether (Brij-58) in the reaction mixture and subsequently in the immobilized metal-affinity purification steps was crucial to keep Bcl-2(2) soluble. The obtained yield was 0.25-0.3mg per ml of cell-free reaction. Far-UV circular dichroism (CD) spectroscopy confirmed the α-helical structure of the purified protein, characteristic for members of the Bcl-2 protein family.

Place, publisher, year, edition, pages
Elsevier Inc, 2011
Keyword
Apoptosis, Bcl-2, Cell-free protein synthesis, Full-length protein, Optimized gene
National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-40403 (URN)10.1016/j.pep.2011.02.003 (DOI)21315822 (PubMedID)
Available from: 2011-03-10 Created: 2011-02-22 Last updated: 2017-12-11Bibliographically approved

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