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The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Gerhard Gröbner)ORCID iD: 0000-0003-2432-8118
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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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.

Place, publisher, year, edition, pages
2016. Vol. 1858, no 6, p. 1288-1297
Keyword [en]
Apoptosis, Bax-protein, Calorimetry, Membranes, NMR, Oxidized phospholipids, Leakage
National Category
Chemical Sciences
Research subject
Biochemistry; Physical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-110699DOI: 10.1016/j.bbamem.2016.03.003ISI: 000375356900023OAI: oai:DiVA.org:umu-110699DiVA, id: diva2:864257
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
In thesis
1. 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. p. 68
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: 2018-06-07Bibliographically approved
2. Bax and oxidized phospholipids - a deadly complex: Apoptotic protein-lipid assemblies studied by MAS NMR spectroscopy
Open this publication in new window or tab >>Bax and oxidized phospholipids - a deadly complex: Apoptotic protein-lipid assemblies studied by MAS NMR spectroscopy
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
Keyword
NMR spectroscopy, mitochondrial membranes, apoptosis, membrane dynamics and organization, Bcl-2 family proteins, Bax protein
National Category
Biophysics
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-147502 (URN)978-91-7601-893-4 (ISBN)
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

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Lidman, MartinDingeldein, Artur P. G.Sparrman, TobiasWallgren, MarcusGröbner, Gerhard

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