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  • 1.
    Dingeldein, Artur
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bax and oxidized phospholipids - a deadly complex: Apoptotic protein-lipid assemblies studied by MAS NMR spectroscopy2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    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.

  • 2.
    Dingeldein, Artur P G
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lidman, Martin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pokorna, Sarka
    Hof, Martin
    Pedersen, Anders
    Karlsson, Göran
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    BCL-2 Family Proteins Effect on Mitochondrial-Mimicking Membrane Structure by Solid State NMR2015Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, nr 2, s. 251A-252AArtikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    Mitochondria are not only the cells' powerhouse, but also involved in their suicide via apoptosis. Key regulators of this pathway are members of the Bcl-2 protein family which interact with the outer mitochondrial membrane to modulate permeability and enable the release of apoptotic stimuli like cytochrome c. For a long time the mitochondrial membrane forming lipids have been seen as merely structural building units with proteins doing the actual work. This view changed in recent years, since lipids were shown to be also directly involved in apoptotic events e.g. under intracellular oxidative stress. Oxidized phospholipids (OxPls) generated under these stress conditions might trigger mitochondria-mediated apoptosis. Their presence in mitochondrial membranes can severely alter the properties of these membranes with yet unknown consequences regarding the formation of pores through membrane-mediated interplay with apoptotic Bax protein. We therefore devised a model system that embodies oxidative stress conditions by incorporating OxPls into mitochondria mimicking model membranes composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) to study the impact of OxPls on apoptotic Bax-membrane interactions. To obtain molecular insight into hydrophobic fatty acid regions of membranes and their hydrophilic interface which is responsible for first protein-membrane contacts, we used differential scanning calorimetry (DSC) and solid state NMR spectroscopy. Upon incorporating OxPls with carboxyl (PoxnoPC) or aldehyde (PazePC) groups at their truncated sn-2-chains into our mitochondria model membranes, calorimetric and NMR measurements showed dramatic changes. 31P NMR experiments revealed major perturbation effects in these membranes; an effect which presumably elevates the membrane binding of apoptotic Bax to the charged membranes and its partial penetration, being a prerequisite for its final formation of pores which enable cytochrome c release from the mitochondrial interior. Currently structural studies of various Bax-lipid assemblies are ongoing.

  • 3.
    Dingeldein, Artur P. G.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lidman, Martin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dynamical and Structural Alterations withing Lipid-Protein Assemblies Control Apoptotic Pore Formation - A Solid State NMR Study2016Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, nr 3, s. 59A-60AArtikel i tidskrift (Övrigt vetenskapligt)
  • 4.
    Dingeldein, Artur P. G.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lindberg, Mikael J.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Zhong, Xueyin
    Stoll, Raphael
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bax to the future – A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies2019Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 158, s. 20-26Artikel i tidskrift (Refereegranskat)
    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.

  • 5.
    Dingeldein, Artur P. G.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Oxidatively stressed mitochondria-mimicking membranes: a molecular insight into their organization during apoptosis2018Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, nr 12, s. 2644-2654Artikel i tidskrift (Refereegranskat)
    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.

  • 6.
    Dingeldein, Artur P. G.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wacklin, Hanna P.
    Clifton, Luke A.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Mitochondrial Membrane Organization under Oxidative Stress: Insight by Solid-State NMR and Neutron Reflectometry2019Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 116, nr 3, s. 508A-508AArtikel i tidskrift (Övrigt vetenskapligt)
  • 7.
    Dingeldein, Artur Peter Günther
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lindberg, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Zhong, Xueyin
    Ruhr University of Bochum, Biomolecular NMR Spectroscopy.
    Stoll, Raphael
    Ruhr University of Bochum, Biomolecular NMR Spectroscopy.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bax to the future: A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studiesIngår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279Artikel i tidskrift (Refereegranskat)
    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.

  • 8.
    Dingeldein, Artur Peter Günther
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pokorna, Sarka
    Lidman, Martin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sachl, Radek
    Hof, Martin
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Apoptotic Bax at Oxidatively Stressed Mitochondrial Membranes: Lipid Dynamics and Permeabilization2017Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, nr 10, s. 2147-2158Artikel i tidskrift (Refereegranskat)
    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.

  • 9.
    Dingeldein, Artur Peter Günther
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Oxidatively stressed mitochondrial membranes: A molecular insight into their organization and function during apoptosisIngår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086Artikel i tidskrift (Refereegranskat)
    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.

  • 10. Koukalova, A.
    et al.
    Pokorna, S.
    Lidman, Martin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dingeldein, Artur P. G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Hof, M.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sachl, R.
    Oxidative stress as a modulator of BAX apoptotic activity2019Ingår i: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 48, s. S170-S170Artikel i tidskrift (Övrigt vetenskapligt)
    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.

  • 11.
    Lidman, Martin
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dingeldein, Artur
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pokorná, Šárka
    Šachl, Radek
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Hof, Martin
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    The Role of Lipids in Regulation of Programmed Cell Death2016Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, nr 3, s. 473A-473AArtikel i tidskrift (Refereegranskat)
  • 12.
    Lidman, Martin
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pokorná, Šárka
    Dingeldein, Artur P. G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wallgren, Marcus
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Šachl, Radek
    Hof, Martin
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax2016Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, nr 6, s. 1288-1297Artikel i tidskrift (Refereegranskat)
    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.

  • 13.
    Niemiec, Moritz S.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dingeldein, Artur P. G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Enthalpy-entropy compensation at play in human copper ion transfer2015Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, artikel-id 10518Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Copper (Cu) is an essential trace element but toxic in free form. After cell uptake, Cu is transferred, via direct protein-protein interactions, from the chaperone Atox1 to the Wilson disease protein (WD) for incorporation into Cu-dependent enzymes. Cu binds to a conserved C1XXC2 motif in the chaperone as well as in each of the cytoplasmic metal-binding domains of WD. Here, we dissect mechanism and thermodynamics of Cu transfer from Atox1 to the fourth metal binding domain of WD. Using chromatography and calorimetry together with single Cys-to-Ala variants, we demonstrate that Cu-dependent protein heterocomplexes require the presence of C-1 but not C-2. Comparison of thermodynamic parameters for mutant versus wild type reactions reveals that the wild type reaction involves strong entropy-enthalpy compensation. This property is explained by a dynamic inter-conversion of Cu-Cys coordinations in the wild type ensemble and may provide functional advantage by protecting against Cu mis-ligation and bypassing enthalpic traps.

  • 14.
    Niemiec, Moritz S.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dingeldein, Artur P. G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    T versus D in the MTCXXC motif of copper transport proteins plays a role in directional metal transport2014Ingår i: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 19, nr 6, s. 1037-1047Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To avoid toxicity and control levels of metal ions, organisms have developed specific metal transport systems. In humans, the cytoplasmic Cu chaperone Atox1 delivers Cu to metal-binding domains of ATP7A/B in the Golgi, for incorporation into Cu-dependent proteins. The Cu-binding motif in Atox1, as well as in target Cu-binding domains of ATP7A/B, consists of a MX1CXXC motif where X-1 = T. The same motif, with X-1 = D, is found in metal-binding domains of bacterial zinc transporters, such as ZntA. The Asp is proposed to stabilize divalent over monovalent metals in the binding site, although metal selectivity in vivo appears predominantly governed by protein-protein interactions. To probe the role of T versus D at the X-1 position for Cu transfer in vitro, we created MDCXXC variants of Atox1 and the fourth metal-binding domain of ATP7B, WD4. We find that the mutants bind Cu like the wild-type proteins, but when mixed, in contrast to the wild-type pair, the mutant pair favors Cu-dependent hetero-dimers over directional Cu transport from Atox1 to WD4. Notably, both wild-type and mutant proteins can bind Zn in the absence of competing reducing agents. In presence of zinc, hetero-complexes are strongly favored for both protein pairs. We propose that T is conserved in this motif of Cu-transport proteins to promote directional metal transfer toward ATP7B, without formation of energetic sinks. The ability of both Atox1 and WD4 to bind zinc ions may not be a problem in vivo due to the presence of specific transport chains for Cu and Zn ions.

  • 15.
    Petzoldt, Svenja
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Center of Life and Food Sciences, Technische Universität München, Freising, Germany.
    Kahra, Dana
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kovermann, Michael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dingeldein, Artur PG
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Niemiec, Moritz S.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro2015Ingår i: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 28, nr 3, s. 577-585Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P-1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.

  • 16.
    Ådén, Jörgen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ul Mushtaq, Ameeq
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dingeldein, Artur P. G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wallgren, Marcus
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-22020Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 172, artikel-id 105628Artikel i tidskrift (Refereegranskat)
    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.

  • 17.
    Ådén, Jörgen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ul Mushtaq, Ameeq
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dingeldein, Artur P.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wacklin, Hanna P.
    Wacklin, Hanna
    Clifton, Luke A.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Mitochondrial Membranes Involved in Apoptosis: the Bcl-2 Proteins2020Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 118, nr 3, s. 394A-394AArtikel i tidskrift (Övrigt vetenskapligt)
1 - 17 av 17
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