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Lindberg, Mikael J.
Publications (4 of 4) 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
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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
Karlberg, T., Hornyak, P., Pinto, A. F., Milanova, S., Ebrahimi, M., Lindberg, M. J., . . . Schüler, H. (2018). 14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface. Nature Communications, 9, Article ID 3785.
Open this publication in new window or tab >>14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface
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2018 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 3785Article in journal (Refereed) Published
Abstract [en]

Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3 beta: ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Cell and Molecular Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-152249 (URN)10.1038/s41467-018-06194-1 (DOI)000444757900003 ()30224724 (PubMedID)
Funder
Swedish Foundation for Strategic Research , SB12-0022Swedish Research Council, 2012-2802Swedish Research Council, 2015-4200Swedish Research Council, 2015-4603Wenner-Gren Foundations
Available from: 2018-10-04 Created: 2018-10-04 Last updated: 2018-10-04Bibliographically approved
Zhang, J., Grundström, C., Brännström, K., Iakovleva, I., Lindberg, M. J., Olofsson, A., . . . Sauer-Eriksson, A. E. (2018). Interspecies variation between fish and human transthyretins in their binding of thyroid-disrupting chemicals. Environmental Science and Technology, 52(20), 11865-11874
Open this publication in new window or tab >>Interspecies variation between fish and human transthyretins in their binding of thyroid-disrupting chemicals
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2018 (English)In: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 52, no 20, p. 11865-11874Article in journal (Refereed) Published
Abstract [en]

Thyroid-disrupting chemicals (TDCs) are xenobiotics that can interfere with the endocrine system and cause adverse effects in organisms and their offspring. TDCs affect both the thyroid gland and regulatory enzymes associated with thyroid hormone homeostasis. Transthyretin (TTR) is found in the serum and cerebrospinal fluid of vertebrates, where it transports thyroid hormones. Here, we explored the interspecies variation in TDC binding to human and fish TTR (exemplified by Gilthead seabream (Sparus aurata)). The in vitro binding experiments showed that TDCs bind with equal or weaker affinity to seabream TTR than to the human TTR, in particular, the polar TDCs (>500-fold lower affinity). Crystal structures of the seabream TTR TDC complexes revealed that all TDCs bound at the thyroid binding sites. However, amino acid substitution of Ser117 in human TTR to Thr117 in seabream prevented polar TDCs from binding deep in the hormone binding cavity, which explains their low affinity to seabream TTR Molecular dynamics and in silico alanine scanning simulation also suggested that the protein backbone of seabream TTR is more rigid than the human one and that Thr117 provides fewer electrostatic contributions than Ser117 to ligand binding. This provides an explanation for the weaker affinities of the ligands that rely on electrostatic interactions with Thr117. The lower affinities of TDCs to fish TTR, in particular the polar ones, could potentially lead to milder thyroid-related effects in fish.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-153704 (URN)10.1021/acs.est.8b03581 (DOI)000447816100046 ()30226982 (PubMedID)
Funder
Swedish Research Council Formas, 210-2012-131Swedish Research Council, 521-2011-6427Swedish Research Council, 2015-03607
Available from: 2018-12-05 Created: 2018-12-05 Last updated: 2018-12-05Bibliographically approved
Hall, M., Grundström, C., Begum, A., Lindberg, M. J., Sauer, U. H., Almqvist, F., . . . Sauer-Eriksson, A. E. (2016). Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria. Proceedings of the National Academy of Sciences of the United States of America, 113(51), 14733-14738
Open this publication in new window or tab >>Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria
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2016 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 51, p. 14733-14738Article in journal (Refereed) Published
Abstract [en]

Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.

Keywords
Listeria, PrfA, activation, glutathione, virulence
National Category
Organic Chemistry Medical Genetics
Identifiers
urn:nbn:se:umu:diva-128915 (URN)10.1073/pnas.1614028114 (DOI)000390044900062 ()
Available from: 2016-12-19 Created: 2016-12-19 Last updated: 2018-06-09Bibliographically approved
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