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Dingeldein, Artur P. G.
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Publications (10 of 17) Show all publications
Ådén, J., Ul Mushtaq, A., Dingeldein, A. P. G., Wallgren, M. & Gröbner, G. (2020). A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-2. Protein Expression and Purification, 172, Article ID 105628.
Open this publication in new window or tab >>A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-2
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2020 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 172, article id 105628Article in journal (Refereed) Published
Abstract [en]

Programmed cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of harmful cells. Besides an extrinsic pathway, an intrinsic (mitochondrial) apoptotic pathway exists where mitochondria are actively involved in cellular clearance in response to internal stress signals. Pro-apoptotic (death) and anti-apoptotic (survival) members of the B cell CLL/lymphoma-2 (Bcl-2) protein family meet at the mitochondrion's surface where they accurately regulate apoptosis. Overexpression of the anti-apoptotic Bcl-2 protein is a hallmark for many types of cancers and in particular for many treatment resistant tumors. Bcl-2 is a membrane protein residing in the mitochondrial outer membrane. Due to its typical membrane protein features including very limited solubility, it is difficult to express and to purify. Therefore, most biophysical and structural studies have used truncated, soluble versions. However, to understand its membrane-coupled function and structure, access to sufficient amount of full-length human Bcl-2 protein is a necessity. Here, we present a novel, E. coli based approach for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)), solubilized in detergent micelles, which allows for easy exchange of the detergent.

Place, publisher, year, edition, pages
Elsevier, 2020
Keywords
Apoptosis, Pro-survival Bcl-2 protein, High-yield expression, Full-length membrane protein, Detergent exchange
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology Cell Biology Biophysics
Identifiers
urn:nbn:se:umu:diva-169249 (URN)10.1016/j.pep.2020.105628 (DOI)
Available from: 2020-03-27 Created: 2020-03-27 Last updated: 2020-04-02Bibliographically approved
Ådén, J., Ul Mushtaq, A., Sparrman, T., Dingeldein, A. P., Wacklin, H. P., Wacklin, H., . . . Gröbner, G. (2020). Mitochondrial Membranes Involved in Apoptosis: the Bcl-2 Proteins. Paper presented at 64th Annual Meeting of the Biophysical-Society, FEB 15-19, 2020, San Diego, CA. Biophysical Journal, 118(3), 394A-394A
Open this publication in new window or tab >>Mitochondrial Membranes Involved in Apoptosis: the Bcl-2 Proteins
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2020 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 118, no 3, p. 394A-394AArticle in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Cell Press, 2020
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-169079 (URN)10.1016/j.bpj.2019.11.2242 (DOI)000513023202469 ()
Conference
64th Annual Meeting of the Biophysical-Society, FEB 15-19, 2020, San Diego, CA
Note

Supplement: 1

Meeting Abstract: 1934-Pos

Available from: 2020-03-23 Created: 2020-03-23 Last updated: 2020-03-23Bibliographically approved
Dingeldein, A. P. G., Lindberg, M. J., Ådén, J., Zhong, X., Stoll, R. & Gröbner, G. (2019). Bax to the future – A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies. Protein Expression and Purification, 158, 20-26
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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2019 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 158, p. 20-26Article in journal (Refereed) Published
Abstract [en]

Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is crucial for the onset of apoptosis by perforating the mitochondrial outer membrane (MOM). This process can be seen as point of no return, since disintegration of the MOM leads to the release of apotogenic factors such as cytochrome c into the cytosol triggering the activation of caspases and subsequent apoptotic steps. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli. In this study, we present a novel approach to express and purify full-length Bax with significantly increased yields, when compared to the commonly applied strategy. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we were able to render both Bax termini inactive and prevent disruptive interactions from occurring during gene expression. By deploying an Intein-CBD-tag at the C-terminus we were further able to avoid 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 robustness of the purification procedure. We report yields for pure, full-length Bax protein that are increased by an order of magnitude, when compared to commonly used Bax expression protocols.

Place, publisher, year, edition, pages
Elsevier, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-156983 (URN)10.1016/j.pep.2019.02.004 (DOI)000462805100004 ()30738180 (PubMedID)2-s2.0-85061543221 (Scopus ID)
Funder
Swedish Research CouncilSwedish Cancer SocietyThe Kempe FoundationsKnut and Alice Wallenberg Foundation
Available from: 2019-03-04 Created: 2019-03-04 Last updated: 2019-04-15Bibliographically approved
Dingeldein, A. P. G., Sparrman, T., Ådén, J., Wacklin, H. P., Clifton, L. A. & Gröbner, G. (2019). Mitochondrial Membrane Organization under Oxidative Stress: Insight by Solid-State NMR and Neutron Reflectometry. Paper presented at 63rd Annual Meeting of the Biophysical-Society, MAR 02-06, 2019, Baltimore, MD. Biophysical Journal, 116(3), 508A-508A
Open this publication in new window or tab >>Mitochondrial Membrane Organization under Oxidative Stress: Insight by Solid-State NMR and Neutron Reflectometry
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2019 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 116, no 3, p. 508A-508AArticle in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
CELL PRESS, 2019
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-157775 (URN)10.1016/j.bpj.2018.11.2742 (DOI)000460779802556 ()
Conference
63rd Annual Meeting of the Biophysical-Society, MAR 02-06, 2019, Baltimore, MD
Available from: 2019-04-10 Created: 2019-04-10 Last updated: 2019-04-10Bibliographically approved
Koukalova, A., Pokorna, S., Lidman, M., Dingeldein, A. P. G., Hof, M., Gröbner, G. & Sachl, R. (2019). Oxidative stress as a modulator of BAX apoptotic activity. Paper presented at Joint 12th EBSA European Biophysics Congress / 10th IUPAP International Conference on Biological Physics (ICBP), Madrid, Spain, July 20-24, 2019. European Biophysics Journal, 48, S170-S170
Open this publication in new window or tab >>Oxidative stress as a modulator of BAX apoptotic activity
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2019 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 48, p. S170-S170Article in journal, Meeting abstract (Other academic) Published
Abstract [en]

Apoptosis is a regulated process of cell death that plays an important role in embryonic development, regulation of tissue homeostasis or removal of harmful cells in multicellular organisms. A failure in apoptosis can lead to severe pathological disorders including cancer, autoimmunity or neurodegenerative diseases. Mitochondria play a key role in the intrinsic apoptotic pathway, which is tightly regulated by Bcl-2 protein family. In response to cytotoxic stress, these proteins initiate cell death by permeabilization of the mitochondrial outer membrane (MOM) followed by the release of apoptotic factors, such as cytochrome c, which represents a point of no return in apoptotic progression. BAX protein, a member of the pro-apoptotic Bcl-2 family with a pore forming activity, is a critical effector of the mitochondrial cell death pathway. Upon interaction with BH3-only proteins, cytosolic BAX undergoes conformational activation and translocation resulting in MOM permeabilization. However, the underlying mechanism controlling this membrane-associated BAX action is still poorly understood. Recent findings suggest that the activity of BAX protein is significantly regulated by the lipid composition of the MOM. In particular, oxidized phospholipids (OxPL) that are generated under oxidative stress conditions seem to directly influence BAX membrane penetration and its activity [1,2].

By employing single molecule fluorescence spectroscopy techniques and various dye leakage assays we study the BAX membrane interactions and its activity in in vitro system that makes use of synthetic giant unilamellar vesicles (GUVs) and large unilamellar vesicles (LUVs) doped with varying amounts of OxPL species, which mimics oxidative stress conditions. Dye leakage assays revealed that BAX activity is sensitive to the type and concentration of different OxPL species in the MOM-mimicking vesicles. Higher levels of oxidized phospholipids in MOM mimicking model lipid bilayer significantly enhance membrane affinity and partial penetration of full length BAX. In addition, solid state NMR studies and calorimetric experiments on the lipid vesicles revealed that the presence of OxPL disrupts the membrane organization enabling BAX to penetrate into the membrane.

Place, publisher, year, edition, pages
Springer, 2019
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-161702 (URN)10.1007/s00249-019-01373-4 (DOI)000473420400554 ()
Conference
Joint 12th EBSA European Biophysics Congress / 10th IUPAP International Conference on Biological Physics (ICBP), Madrid, Spain, July 20-24, 2019
Note

Supplement: 1

Meeting Abstract: P-366

Available from: 2019-08-05 Created: 2019-08-05 Last updated: 2019-08-05Bibliographically approved
Dingeldein, A. (2018). Bax and oxidized phospholipids - a deadly complex: Apoptotic protein-lipid assemblies studied by MAS NMR spectroscopy. (Doctoral dissertation). Umeå: Umeå University
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
Keywords
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
Dingeldein, A. P. G., Sparrman, T. & Gröbner, G. (2018). Oxidatively stressed mitochondria-mimicking membranes: a molecular insight into their organization during apoptosis. Biochimica et Biophysica Acta - Biomembranes, 1860(12), 2644-2654
Open this publication in new window or tab >>Oxidatively stressed mitochondria-mimicking membranes: a molecular insight into their organization during apoptosis
2018 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 12, p. 2644-2654Article in journal (Refereed) Published
Abstract [en]

Mitochondria are crucially involved in the removal of eukaryotic cells by the intrinsic pathway of programmed cell death (apoptosis). The mitochondrion's outer membrane (MOM) is the platform where this pathway takes place. Upon oxidative stress triggering apoptotic action, the MOM undergoes permeabilization and release of cytochrome c, ultimately causing cell death. This membrane perforation is regulated not only by opposing members of the Bcl-2 protein family meeting at the MOM but also actively the membrane itself. Upon oxidative damage, the membrane undergoes severe reorganization causing an increase in cell death-causing apoptotic Bcl-2 proteins. To understand the active role of MOM, we provided a detailed molecular view of its structural and dynamic reorganization upon oxidative stress by solid-state C-13 MAS NMR (magic angle spinning nuclear magnetic resonance) accompanied by calorimetric studies. By focusing on MOM-like vesicles doped with oxidized lipid species, direct polarization C-13 MAS NMR provided a quantitative overview and identification of all lipid moieties across the membrane. H-1-C-1(3) cross polarization and insensitive nuclei enhanced by polarization transfer MAS NMR generated a dynamic - mobile versus restricted - membrane profile. Oxidized phospholipids significantly perturb the structural membrane organization and increase membrane dynamics. These perturbations are not uniformly distributed as the hydrophobic core is reflecting the melting of lipid chains and increase in molecular disorder directly, whereas the interface and headgroup region undergo complex dynamical changes, reflecting increased intra-molecular flexibility of these moieties. These changes are potentially crucial in augmenting pro-apoptotic action of proteins like Bax.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Apoptosis, Oxidized lipids, Mitochondrial membranes, Solid-state NMR, Membrane dynamics, Membrane order
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-154934 (URN)10.1016/j.bbamem.2018.10.007 (DOI)000449898900017 ()30296415 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer SocietyThe Kempe FoundationsKnut and Alice Wallenberg Foundation
Available from: 2019-01-07 Created: 2019-01-07 Last updated: 2019-01-07Bibliographically approved
Dingeldein, A. P., Pokorna, S., Lidman, M., Sparrman, T., Sachl, R., Hof, M. & Gröbner, G. (2017). Apoptotic Bax at Oxidatively Stressed Mitochondrial Membranes: Lipid Dynamics and Permeabilization. Biophysical Journal, 112(10), 2147-2158
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
Dingeldein, A. P. G., Lidman, M., Sparrman, T. & Gröbner, G. (2016). Dynamical and Structural Alterations withing Lipid-Protein Assemblies Control Apoptotic Pore Formation - A Solid State NMR Study. Paper presented at 60th Annual Meeting of the Biophysical-Society, FEB 27-MAR 02, 2016, Los Angeles, CA. Biophysical Journal, 110(3), 59A-60A
Open this publication in new window or tab >>Dynamical and Structural Alterations withing Lipid-Protein Assemblies Control Apoptotic Pore Formation - A Solid State NMR Study
2016 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 3, p. 59A-60AArticle in journal, Meeting abstract (Other academic) Published
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-121605 (URN)000375093500308 ()
Conference
60th Annual Meeting of the Biophysical-Society, FEB 27-MAR 02, 2016, Los Angeles, CA
Available from: 2016-06-21 Created: 2016-06-03 Last updated: 2018-06-07Bibliographically approved
Lidman, M., Pokorná, Š., Dingeldein, A. P. G., Sparrman, T., Wallgren, M., Šachl, R., . . . Gröbner, G. (2016). The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax. Biochimica et Biophysica Acta - Biomembranes, 1858(6), 1288-1297
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
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