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Wittung-Stafshede, Pernilla
Publications (10 of 69) Show all publications
Good, J. A. D., Andersson, C., Hansen, S., Wall, J., Krishnan, S., Begum, A., . . . Johansson, J. (2016). Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA. Cell chemical biology, 23(3), 404-414
Open this publication in new window or tab >>Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA
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2016 (English)In: Cell chemical biology, ISSN 2451-9448, Vol. 23, no 3, p. 404-414Article in journal (Refereed) Published
Abstract [en]

The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes infectivity by reducing the expression of virulence genes, without compromising bacterial growth. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds within a hydrophobic pocket, located between the C- and N-terminal domains of PrfA, and interacts with residues important for PrfA activation. This indicates that these inhibitors maintain the DNA-binding helix-turn-helix motif of PrfA in a disordered state, thereby preventing a PrfA:DNA interaction. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-114083 (URN)10.1016/j.chembiol.2016.02.013 (DOI)000381508300013 ()26991105 (PubMedID)
Note

Originally published in manuscipt form in thesis.

Available from: 2016-01-12 Created: 2016-01-12 Last updated: 2018-06-07Bibliographically approved
Mondol, T., Ådén, J. & Wittung-Stafshede, P. (2016). Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B. Biochemical and Biophysical Research Communications - BBRC, 470(3), 663-669
Open this publication in new window or tab >>Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B
2016 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 470, no 3, p. 663-669Article in journal (Refereed) Published
Abstract [en]

Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle.

Place, publisher, year, edition, pages
Elsevier, 2016
Keywords
Protein-protein interactions, Metalloenzymes, Copper transport, NMR, Circular dichroism, Conformational changes
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-118252 (URN)10.1016/j.bbrc.2016.01.085 (DOI)000370467100030 ()26797276 (PubMedID)
Available from: 2016-03-17 Created: 2016-03-14 Last updated: 2018-06-07Bibliographically approved
Kahra, D., Kovermann, M. & Wittung-Stafshede, P. (2016). The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1. Biophysical Journal, 110(1), 95-102
Open this publication in new window or tab >>The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1
2016 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 1, p. 95-102Article in journal (Refereed) Published
Abstract [en]

Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, K-D, of similar to 10(-14) M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones.

National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-114887 (URN)10.1016/j.bpj.2015.11.016 (DOI)000367783900032 ()26745413 (PubMedID)
Available from: 2016-04-27 Created: 2016-01-29 Last updated: 2018-06-07Bibliographically approved
Chorell, E., Andersson, E., Evans, M. L., Jain, N., Götheson, A., Åden, J., . . . Wittung-Stafshede, P. (2015). Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts. PLoS ONE, 10(10), 1-11, Article ID e0140194.
Open this publication in new window or tab >>Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 10, p. 1-11, article id e0140194Article in journal (Refereed) Published
Abstract [en]

Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson’s disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-110343 (URN)10.1371/journal.pone.0140194 (DOI)000363183100087 ()26465894 (PubMedID)
Available from: 2015-10-21 Created: 2015-10-21 Last updated: 2018-06-07Bibliographically approved
Perdersen, M. N., Fodera, V., Horvath, I., van Maarschalkerweerd, A., Toft, K. N., Weise, C., . . . Vestergaard, B. (2015). Direct Correlation Between Ligand-Induced alpha-Synuclein Oligomers and Amyloid-like Fibril Growth. Scientific Reports, 5, Article ID 10422.
Open this publication in new window or tab >>Direct Correlation Between Ligand-Induced alpha-Synuclein Oligomers and Amyloid-like Fibril Growth
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 10422Article in journal (Refereed) Published
Abstract [en]

Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075: protein ratios, the oligomers appear to be on-pathway and modeling supports an 'oligomer stacking model' for alpha-synuclein fibril elongation.

Place, publisher, year, edition, pages
Springer, 2015
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-106505 (URN)10.1038/srep10422 (DOI)000355546300001 ()
Available from: 2015-07-16 Created: 2015-07-14 Last updated: 2019-03-14Bibliographically approved
Niemiec, M. S., Dingeldein, A. P. G. & Wittung-Stafshede, P. (2015). Enthalpy-entropy compensation at play in human copper ion transfer. Scientific Reports, 5, Article ID 10518.
Open this publication in new window or tab >>Enthalpy-entropy compensation at play in human copper ion transfer
2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 10518Article in journal (Refereed) Published
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.

Keywords
copper chaperone, Atox1, Wilson disease protein, metal transport, size exclusion chromatography, thermodynamics, calorimetry
National Category
Biophysics Biochemistry and Molecular Biology Inorganic Chemistry
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-106506 (URN)10.1038/srep10518 (DOI)000355537200002 ()26013029 (PubMedID)
Available from: 2015-07-16 Created: 2015-07-14 Last updated: 2018-06-07Bibliographically approved
Kahra, D., Mondol, T., Niemiec, M. S. & Wittung-Stafshede, P. (2015). Human Copper Chaperone Atox1 Translocates to the Nucleus but does not Bind DNA In Vitro. Protein peptide letters, 22(6), 532-538
Open this publication in new window or tab >>Human Copper Chaperone Atox1 Translocates to the Nucleus but does not Bind DNA In Vitro
2015 (English)In: Protein peptide letters, ISSN 0929-8665, E-ISSN 1875-5305, Vol. 22, no 6, p. 532-538Article in journal (Refereed) Published
Abstract [en]

After Ctr1-mediated cell uptake, copper (Cu) is transported by the cytoplasmic Cu chaperone Atox1 to P1B type ATPases ATP7A and ATP7B in the Golgi network, for incorporation into Cudependent enzymes. Atox1 is a small 68-residue protein that binds Cu in a conserved CXXC motif; it delivers Cu to target domains in ATP7A/B via direct protein-protein interactions. Specific transcription factors regulating expression of the human Cu transport proteins have not been reported although Atox1 was recently suggested to have dual functionality such that it, in addition to its cytoplasmic chaperone function, acts as a transcription factor in the nucleus. To examine this hypothesis, here we investigated the localization of Atox1 in HeLa cells using fluorescence imaging in combination with in vitro binding experiments to fluorescently labeled DNA duplexes harboring the proposed promotor sequence. We found that whereas Atox1 is present in the nucleus in HeLa cells, it does not bind to DNA in vitro. It appears that Atox1 mediates transcriptional regulation via additional (unknown) proteins.

Place, publisher, year, edition, pages
Bentham Science, 2015
Keywords
Atox1, Copper chaperone, fluorescence microscopy, fluorescence spectroscopy, transcription factor
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-105277 (URN)000355192800008 ()25962064 (PubMedID)
Available from: 2015-06-22 Created: 2015-06-22 Last updated: 2018-06-07Bibliographically approved
Petzoldt, S., Kahra, D., Kovermann, M., Dingeldein, A. P., Niemiec, M. S., Ådén, J. & Wittung-Stafshede, P. (2015). Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro. Biometals, 28(3), 577-585
Open this publication in new window or tab >>Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro
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2015 (English)In: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 28, no 3, p. 577-585Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Springer, 2015
Keywords
Human copper transport, Atox1, Copper chaperone for superoxide dismutase, (SOD), Size exclusion chromatography, Proton-NMR
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-100334 (URN)10.1007/s10534-015-9832-1 (DOI)000354273900014 ()25673218 (PubMedID)
Available from: 2015-03-01 Created: 2015-03-01 Last updated: 2018-06-07Bibliographically approved
Öhrvik, H. & Wittung-Stafshede, P. (2015). Identification of New Potential Interaction Partners for Human Cytoplasmic Copper Chaperone Atox1: Roles in Gene Regulation?. International Journal of Molecular Sciences, 16(8), 16728-16739
Open this publication in new window or tab >>Identification of New Potential Interaction Partners for Human Cytoplasmic Copper Chaperone Atox1: Roles in Gene Regulation?
2015 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 16, no 8, p. 16728-16739Article in journal (Refereed) Published
Abstract [en]

The human copper (Cu) chaperone Atox1 delivers Cu to P-1B type ATPases in the Golgi network, for incorporation into essential Cu-dependent enzymes. Atox1 homologs are found in most organisms; it is a 68-residue ferredoxin-fold protein that binds Cu in a conserved surface-exposed Cys-X-X-Cys (CXXC) motif. In addition to its well-documented cytoplasmic chaperone function, in 2008 Atox1 was suggested to have functionality in the nucleus. To identify new interactions partners of Atox1, we performed a yeast two-hybrid screen with a large human placenta library of cDNA fragments using Atox1 as bait. Among 98 million fragments investigated, 25 proteins were found to be confident interaction partners. Nine of these were uncharacterized proteins, and the remaining 16 proteins were analyzed by bioinformatics with respect to cell localization, tissue distribution, function, sequence motifs, three-dimensional structures and interaction networks. Several of the hits were eukaryotic-specific proteins interacting with DNA or RNA implying that Atox1 may act as a modulator of gene regulation. Notably, because many of the identified proteins contain CXXC motifs, similarly to the Cu transport reactions, interactions between these and Atox1 may be mediated by Cu.

Place, publisher, year, edition, pages
MPDI, 2015
Keywords
copper chaperone, Atox1, transcription factor, two-hybrid screen, bioinformatics
National Category
Microbiology in the medical area Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-114650 (URN)10.3390/ijms160816728 (DOI)000366826100002 ()26213915 (PubMedID)
Available from: 2016-01-25 Created: 2016-01-25 Last updated: 2018-06-07Bibliographically approved
Sharma, S. K., Chorell, E. & Wittung-Stafshede, P. (2015). Insulin-degrading enzyme is activated by the C-terminus of alpha-synuclein. Biochemical and Biophysical Research Communications - BBRC, 466(2), 192-195
Open this publication in new window or tab >>Insulin-degrading enzyme is activated by the C-terminus of alpha-synuclein
2015 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 466, no 2, p. 192-195Article in journal (Refereed) Published
Abstract [en]

The insulin-degrading enzyme (IDE) plays a key role in type-2 diabetes and typically degrades small peptides such as insulin, amyloid beta and islet amyloid polypeptide. We recently reported a novel non-proteolytical interaction in vitro between IDE and the Parkinson's disease 140-residue protein alpha-synuclein that resulted in dual effects: arrested alpha-synuclein oligomers and, simultaneously, increased IDE proteolysis activity. Here we demonstrate that these outcomes arise due to IDE interactions with the C-terminus of alpha-synuclein. Whereas a peptide containing the first 97 residues of alpha-synuclein did not improve IDE activity and its aggregation was not blocked by IDE, a peptide with the C-terminal 44 residues of alpha-synuclein increased IDE proteolysis to the same degree as full-length alpha-synuclein. Because the alpha-synuclein C-terminus is acidic, the interaction appears to involve electrostatic attraction with IDE's basic exosite, known to be involved in activation.

Keywords
Insulin degrading enzyme, Parkinson's disease, alpha-Synuclein, Amyloid, Proteolysis
National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-110991 (URN)10.1016/j.bbrc.2015.09.002 (DOI)000362610800008 ()26343304 (PubMedID)
Available from: 2015-11-18 Created: 2015-11-02 Last updated: 2018-06-07Bibliographically approved
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