umu.sePublications
Change search
Link to record
Permanent link

Direct link
BETA
Gröbner, Gerhard
Publications (10 of 100) Show all publications
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
Show others...
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
Alam, M. K., Vinklarek, I., Gröbner, G., Johansson, L.-Å. B. & Sachl, R. (2019). Fluorescence Studies of Lipid Distribution in Bilayers under Oxidative Stress. 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 >>Fluorescence Studies of Lipid Distribution in Bilayers under Oxidative Stress
Show others...
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-157777 (URN)10.1016/j.bpj.2018.11.2741 (DOI)000460779802555 ()
Conference
63rd Annual Meeting of the Biophysical-Society, MAR 02-06, 2019, Baltimore, MD
Available from: 2019-04-11 Created: 2019-04-11 Last updated: 2019-04-11Bibliographically 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
Show others...
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
Show others...
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
Sousa, T., Pokorna, S., Castro, R., Bernardes, N., Banerjee, S., Gröbner, G., . . . Fernandes, F. (2019). Towards and understanding of the role of Bax on bile acid-mediated cytoprotection. 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, S177-S177
Open this publication in new window or tab >>Towards and understanding of the role of Bax on bile acid-mediated cytoprotection
Show others...
2019 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 48, p. S177-S177Article in journal, Meeting abstract (Other academic) Published
Abstract [en]

Cytoprotective bile acids such as ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) are known for their ability to inhibit apoptosis at submicellar concentrations in both hepatic and nonhepatic cells. The exact mechanism by which they exert this cytoprotection is not yet entirely clear, but their effect seems to be related with the blockage of processes that converge on mitochondrial damage. Bcl-2-associated X protein (Bax) plays a key role in apoptosis, which is achieved through translocation of the protein to the mitochondria from the cytosol after an apoptotic stimulus and formation of pores in mitochondrial membranes. We show here that both UDCA and TUDCA inhibit the interaction of Bax with activator molecules such as the Bid-BH3 peptide and decrease the affinity of Bax for liposomes mimicking outer mitochondrial membrane composition. Importantly, UDCA and TUDCA are shown to dramatically inhibit Bax-induced permeabilization of model membranes. The direct impact of apoptotic and cytoprotective bile acids on Bax translocation is monitored in HCT116 Bax/Bak DKO cells expressing Bax-GFP. The findings presented here clearly show that at physiologically active submicellar concentrations, bile acids have the ability to inhibit Bax poreforming activity and suggest that the cytoprotective activity of UDCA and TUDCA could be the result of this process.

Place, publisher, year, edition, pages
Springer, 2019
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-161706 (URN)10.1007/s00249-019-01373-4 (DOI)000473420400584 ()
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-396

Available from: 2019-08-05 Created: 2019-08-05 Last updated: 2019-08-05Bibliographically 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
Show others...
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
Bugaytsova, J. A., Björnham, O., Chernov, Y. A., Gideonsson, P., Henriksson, S., Mendez, M., . . . Boren, T. (2017). Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence. Cell Host and Microbe, 21(3), 376-389
Open this publication in new window or tab >>Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence
Show others...
2017 (English)In: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 21, no 3, p. 376-389Article in journal (Refereed) Published
Abstract [en]

The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

Place, publisher, year, edition, pages
CELL PRESS, 2017
National Category
Microbiology in the medical area Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-132788 (URN)10.1016/j.chom.2017.02.013 (DOI)000396375600023 ()28279347 (PubMedID)
Available from: 2017-05-11 Created: 2017-05-11 Last updated: 2019-05-24Bibliographically approved
Koukalova, A., Amaro, M., Aydogan, G., Gröbner, G., Williamson, P. T. F., Mikhalyov, I., . . . Sachl, R. (2017). Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered. Scientific Reports, 7, Article ID 5460.
Open this publication in new window or tab >>Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered
Show others...
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 5460Article in journal (Refereed) Published
Abstract [en]

It is a fundamental question in cell biology and biophysics whether sphingomyelin (SM)-and cholesterol (Chol)-driven nanodomains exist in living cells and in model membranes. Biophysical studies on model membranes revealed SM and Chol driven micrometer-sized liquid-ordered domains. Although the existence of such microdomains has not been proven for the plasma membrane, such lipid mixtures have been often used as a model system for 'rafts'. On the other hand, recent super resolution and single molecule results indicate that the plasma membrane might organize into nanocompartments. However, due to the limited resolution of those techniques their unambiguous characterization is still missing. In this work, a novel combination of Forster resonance energy transfer and Monte Carlo simulations (MC-FRET) identifies directly 10 nm large nanodomains in liquid-disordered model membranes composed of lipid mixtures containing SM and Chol. Combining MC-FRET with solidstate wide-line and high resolution magic angle spinning NMR as well as with fluorescence correlation spectroscopy we demonstrate that these nanodomains containing hundreds of lipid molecules are fluid and disordered. In terms of their size, fluidity, order and lifetime these nanodomains may represent a relevant model system for cellular membranes and are closely related to nanocompartments suggested to exist in cellular membranes.

National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-139014 (URN)10.1038/s41598-017-05539-y (DOI)000405464200097 ()28710349 (PubMedID)
Available from: 2017-09-14 Created: 2017-09-14 Last updated: 2018-06-09Bibliographically approved
Velas, L., Koukalova, A., Amaro, M., Gröbner, G., Williamson, P., Mikhalyov, I., . . . Sachl, R. (2017). Oxidized phospholipids induce formation of inter-leaflet coupled nanodomains in giant lipid vesicles. Paper presented at 19th IUPAB Congress / 11th EBSA Congress, JUL 16-20, 2017, British Biophys Soc, Edinburgh, SCOTLAND. European Biophysics Journal, 46, S128-S128
Open this publication in new window or tab >>Oxidized phospholipids induce formation of inter-leaflet coupled nanodomains in giant lipid vesicles
Show others...
2017 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, p. S128-S128Article in journal, Meeting abstract (Other academic) Published
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-143672 (URN)000416406200263 ()
Conference
19th IUPAB Congress / 11th EBSA Congress, JUL 16-20, 2017, British Biophys Soc, Edinburgh, SCOTLAND
Note

Supplement: 1, Meeting Abstract: P-52

Available from: 2018-01-16 Created: 2018-01-16 Last updated: 2018-06-09Bibliographically approved
Organisations

Search in DiVA

Show all publications