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Bax and oxidized phospholipids - a deadly complex: Apoptotic protein-lipid assemblies studied by MAS NMR spectroscopy
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mitochondria are renowned for their vital role as cellular powerhouses because they provide ATP via cellular respiration. Additionally, these organelles also play an important role in other physiological processes, such as apoptosis. Apoptosis, or regulated cell death, is an important mechanism that regulates, for example, tissue homeostasis and embryonic development. Malfunctioning apoptosis can cause severe diseases such as various types of cancer and neurodegenerative diseases. The significance of mitochondria for apoptosis is that mitochondria host a variety of apoptogenic factors, such as cytochrome c. The release of these factors after the formation of an apoptotic pore can be regarded as a point of no return in the onset of apoptosis as these factors trigger the activation of caspases and consequently nuclear fragmentation.

The mitochondrial outer membrane (MOM) is essential for deciding the cell’s fate, since the MOM provides an interaction surface for the pro- and anti-apoptotic proteins of the Bcl-2 protein family. Further, oxidized phospholipids (OxPls) within the MOM that are generated under oxidative stress conditions (a potent pro-apoptotic stimulus) can directly affect the equilibrium between pro- and anti-apoptotic proteins at the MOM surface, hence influencing the formation of apoptotic pores.

To characterize the impact of different OxPls on membrane dynamics and organization, several MOM-mimicking systems were studied by solid-state magic angle spinning nuclear magnetic resonance spectroscopy (MAS NMR). These main experiments were accompanied by fluorescence spectroscopy and differential scanning calorimetry (DSC) studies to investigate the impact of OxPls and their interactions with the pro-apoptotic Bax protein at both the macroscopic and molecular levels. By combining several orthogonal methods, we were able to obtain a detailed description of the changes in MOM-mimicking vesicles induced by several types of OxPls. Moreover, we demonstrated how these changes impact the interaction between liposomes and the pro-apoptotic Bax protein.

By using DSC, we were able to determine not only the macroscopic effect of two OxPls – PazePC and PoxnoPC – but also a concentration threshold. Both OxPls disrupted the membrane order such that the melting behavior of the MOM-mimicking vesicles became less cooperative. A decrease in cooperativity was detectable for OxPl concentrations of up to 5 mol%, after which the cooperativity remained constant. The addition of Bax resulted in an observable ordering effect, as some of the membrane disorder was negated by Bax and the melting process became more cooperative again. The ordering effect of Bax was subsequently confirmed by 31P MAS NMR experiments and cross polarization (CP) buildup curves. Analysis of the buildup curves revealed that at the molecular level, Bax enabled more efficient CP transfer, which indicated rigidification of the membranes after Bax insertion. Furthermore, the 31P NMR experiments provided the first molecular evidence of the importance of cardiolipin as a membrane contact site for Bax.

Despite having similar disordering effects when studied with DSC, PoxnoPC and PazePC exhibited opposing effects on the pore formation ability of Bax. In studies with fluorescent dye-based leakage assays, Bax was able to form long-lived, stable pores in PazePC-containing giant unilamellar vesicles (GUVs). However, the observed leakage mechanism in PoxnoPC-containing GUVs could no longer be explained by an all-or-none leakage mechanism due to the brevity of the formed pores, leading to partially leaked vesicles, indicating a graded leakage mechanism instead.

To investigate the possible reasons for the different leakage activities and to obtain mechanistic insights, we conducted 13C MAS NMR experiments. These experiments enabled us to pinpoint specific carbon sites in the different MOM-mimicking systems and to study their dynamic profile as a function of temperature. Though the OxPl-containing multilamellar vesicles (MLVs), compared with non-oxidized systems, also showed drastic dynamic changes, the molecular differences between PazePC- and PoxnoPC-containing vesicles were not significant enough to constitute a structural reason for the opposing leakage activities.

In addition to investigating membrane dynamics, we were able to establish a novel strategy for producing cytotoxic Bax protein. This novel expression and purification strategy increased the obtained yields by an order of magnitude. By deploying a double fusion approach, we were able to inhibit both termini of the protein from exhibiting their disruptive, yield-diminishing interactions with the host cell membranes.

In conclusion, over the course of this thesis we were able to develop fast, yet powerful tools to investigate the dynamic changes of MOM-mimicking vesicles under the influence of OxPls and pro-apoptotic Bax. In the future, these tools could be used to characterize the underlying protein-lipid interactions that are responsible for the opposing leakage activities. Due to the development of a novel Bax production strategy, future research could shift to protein-focused studies with the primary goal of determining the structure of the apoptotic Bax pore.

Abstract [sv]

Mitokondrierna är kända för sin viktiga roll som cellens energikälla då de levererar ATP genom cellandning. Denna organell har även en viktig funktion i andra fysiologiska processer, till exempel apoptos. Apoptos, eller reglerad celldöd, är en viktig mekanism som styr till exempel homeostas i vävnad samt fostrets utveckling under graviditeten. Uppstår problem i denna process så kan detta leda till allvarliga sjukdomar, till exempel olika former av cancer, och även neurodegenerativa sjukdomar. Mitokondriens viktiga roll för apoptos beror på att den är värd för en stor del apoptosregulerande faktorer såsom cytokrom c. Frigörande av dessa faktorer efter permeabilisering av mitokondriens membran anses vara ett tillstånd då cellen är dömd att genomgå apoptos eftersom detta även aktiverar andra enzymer såsom caspaser, vilket leder slutligen till fragmentering av hela cellen.

Mitokondriens yttermembran (MOM) spelar en ytterst viktig roll för kontroll av apoptos eftersom det tillhandahåller en interaktionsyta för både antiapoptotiska och apoptotiska proteiner, tillhörande en proteinfamilj kallad Bcl-2. Även oxiderade fosfolipider (OxPl) inom MOM som genereras under oxidativ stress (som är ett kraftfullt stimuli för apoptos) kan direkt påverka jämvikten mellan de anti- och apoptotiska proteinerna, som i sin tur då påverkar hela apoptosmekanismen.

För att karaktärisera betydelsen av oxiderade lipiders dynamik och organisation har vi använt ett modellsystem som efterliknar MOM och undersökt detta med kärnmagnetisk resonansspektroskopi i fast fas (MAS-NMR). Vi kompletterade dessa experiment med fluorescensspektroskopi och differentialkalorimetri (DSC) för att undersöka betydelsen av OxPl och tittade även på interaktionen mellan det apoptosinducerade proteinet Bax och OxPl på både makroskopisk och molekylär nivå. Genom att kombinera flera olika metoder ger detta en detaljerad beskrivning över de processer som sker hos vesiklar som påverkas av olika typer av OxPl. Vi kunde också visa hur dessa förändringar påverkar interaktionen mellan liposomer och proteinet Bax.

Vi undersökte även den makroskopiska effekten av två OxPl – PacePC och PoxnoPC med DSC, samt bestämde den lägsta koncentration då dessa fortfarande påverkar membranets struktur. Vi upptäckte att båda dessa OxPl förändrade strukturen hos MOM så att vesiklarnas termiska egenskaper förändrades. En reducering av kooperativiteten kunde observeras upp till en koncentration av fem mol-procent OxPl, därefter var minskningen försumbar. När vi tillsatte Bax så strukturerades MOM, och även smälttemperaturen blev mer kooperativ. Denna observation bekräftades ytterligare med hjälp av NMR-tekniker som 31P MAS och korspolariserings experiment. Tillsats av Bax gav en effektivare signalöverföring, vilket indikerar att membranet är mer kompakt i närvaro av Bax. Dessa experiment ger en indikation att kardiolipin kan vara en första bindningskontakt för Bax.

Trots både PoxnoPC och PazePC hade en effekt på membranets strukur då de studerades med DSC så hade de olika effekt i närvaro av Bax. Med fluorescensspektroskopi undersökte vi dess förmåga att skapa hål i MOM, och det visade sig att Bax skapade stabila porer (monolamella vesiklar) i närvaro av PazePC. Däremot med PoxnoPC så fann vi att dess mekanism ej är identisk då porerna skapades långsammare. För att fördjupa oss i mekanismen hos apoptos så använde vi oss också av NMR kombinerat med 13C MAS. Dessa experiment gör det möjligt att titta på specifika kolatomer i ett modellsystem (MLVs) där vi imiterat MOM, och även studera hur temperaturen styr dess dess dynamik. OxPl visade här än en gång stora dynamiska förändringar i jämförelse med ickeoxiderade system. Skillnaderna i data mellan PazePC och PoxnoPC-innehållande vesiklar var dock ej stora nog för att förklara vad som händer på strukturell nivå.

Sammanfattningsvis så har vi utvecklat nya snabba och kraftfulla verktyg för att undersöka vesiklar som efterliknar MOM samt tittat på dynamiska förändringar i närvaro av OxPl och Bax. Dessa observationer öppnar nya möjligheter för att studera de protein-lipid-interaktioner som leder till permiabilisering av mitokontriens membran. Slutligen, genom att även ha utvecklat en ny och effektivare produktion av Bax har vi också skapat grund för framtida forskning där fokus ligger på att studera strukturen av mitokontriens membran i närvaro av Bax; studier som kan ge värdefull insikt i hur apoptos sker.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2018. , p. 125
Keywords [en]
NMR spectroscopy, mitochondrial membranes, apoptosis, membrane dynamics and organization, Bcl-2 family proteins, Bax protein
National Category
Biophysics
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-147502ISBN: 978-91-7601-893-4 (print)OAI: oai:DiVA.org:umu-147502DiVA, id: diva2:1203705
Public defence
2018-06-01, KB.E3.01 (Lilla hörsalen), Kemiskt Biologiskt Centrum (KBC), Linnaeus väg 10, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2018-05-09 Created: 2018-05-04 Last updated: 2018-06-09Bibliographically approved
List of papers
1. 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, p. 1288-1297Article 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.

Keywords
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: 2018-06-07Bibliographically approved
2. Apoptotic Bax at Oxidatively Stressed Mitochondrial Membranes: Lipid Dynamics and Permeabilization
Open this publication in new window or tab >>Apoptotic Bax at Oxidatively Stressed Mitochondrial Membranes: Lipid Dynamics and Permeabilization
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2017 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 10, p. 2147-2158Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Cell Biology
Identifiers
urn:nbn:se:umu:diva-136334 (URN)10.1016/j.bpj.2017.04.019 (DOI)000402119300015 ()28538152 (PubMedID)
Available from: 2017-06-20 Created: 2017-06-20 Last updated: 2018-06-09Bibliographically approved
3. Oxidatively stressed mitochondrial membranes: A molecular insight into their organization and function during apoptosis
Open this publication in new window or tab >>Oxidatively stressed mitochondrial membranes: A molecular insight into their organization and function during apoptosis
(English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086Article in journal (Refereed) Submitted
Abstract [en]

Mitochondria are crucially involved in the removal of eukaryotic cells by theintrinsic pathway of programmed cell death (apoptosis). The mitochondrial outermembrane (MOM) is the platform where this pathway is regulated. Uponoxidative stress that triggers apoptotic action, the MOM undergoespermeabilization and releases cytochrome c, ultimately causing cell death. Notonly is this membrane perforation regulated by opposing members of the Bcl-2protein family meeting at the MOM but also is it regulated by the membrane itselfthat plays an active role. Upon oxidative damage, the membrane undergoessevere reorganization causing an imbalance towards cell death-causing apoptoticBcl-2 proteins. To understand the active role of MOM, we provided a detailedmolecular view of its structural and dynamic reorganization by solid-state13C MAS NMR (magic angle spinning nuclear magnetic resonance) accompaniedby calorimetric studies. By focusing on MOM-like vesicles doped with oxidizedlipid species, direct polarization 13C MAS NMR provided a quantitative overviewand identification of all lipid moieties across the membrane. 1H-13C crosspolarization and insensitive nuclei enhanced by polarization transfer MAS NMRgenerated a dynamic - mobile versus restricted – membrane profile. Evidently,oxidized phospholipids significantly perturb the structural membraneorganization and increase membrane dynamics. However, these perturbationsare not observed uniformly as the hydrophobic core is reflecting the melting ofthe lipid chains and the increase in lipid molecule disorder directly, whereas theinterface and headgroup region undergo complex changes in their dynamics,which are coupled to increased segmental disorder. These changes are potentiallycrucial in augmenting the pro-apoptotic action of proteins like Bax there.

National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-147496 (URN)
Available from: 2018-05-04 Created: 2018-05-04 Last updated: 2018-06-09
4. Bax to the future: A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies
Open this publication in new window or tab >>Bax to the future: A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies
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(English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279Article in journal (Refereed) Submitted
Abstract [en]

Mitochondria-mediated apoptosis (programmed cell death) is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. The members of this family can be divided into pro- and anti-apoptotic functioning proteins. One of the most important members for executing apoptosis is the cytosolic apoptotic Bax protein. It is crucial in facilitating onset of apoptosis by disrupting and forming pores within the mitochondrial outer membrane (MOM). This pore formation is commonly viewed as a point of no return, since it releases apotogenic factors such as cytochrome c into the cytosol who trigger irreversibly cell death by caspase activation and nuclear fragmentation. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli due to the strong similarities between both membrane-active segments. Here, we present a novel approach to express and purify full-length Bax with significantly increased yields compared to the commonly applied strategy with very bad yields. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we rendered both Bax termini inactive and prevented disruptive interactions during gene expression. By deploying an Intein-CBD-tag at the C-terminus we avoided even the introduction of any artificial residues, hence ensuring the native like activity of the membrane-penetrating C-terminus of Bax. Further, by engineering a His6-tag to the C-terminus of the CBD-tag we greatly improved the purification procedure. Yields for pure, full-length Bax protein are increased by an order of magnitude, compared to commonly used Bax expression protocols.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-147497 (URN)
Available from: 2018-05-04 Created: 2018-05-04 Last updated: 2018-06-09

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