| 1. |
- Ahlgren, Eva Christina, et al.
(author)
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Iron-induced oligomerization of human FXN81-210 and bacterial CyaY frataxin and the effect of iron chelators
- 2017
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:12
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Journal article (peer-reviewed)abstract
- Patients suffering from the progressive neurodegenerative disease Friedreich’s ataxia have reduced expression levels of the protein frataxin. Three major isoforms of human frataxin have been identified, FXN42-210, FXN56-210 and FXN81-210, of which FXN81-210 is considered to be the mature form. Both long forms, FXN42-210 and FXN56-210, have been shown to spontaneously form oligomeric particles stabilized by the extended N-terminal sequence. The short variant FXN81-210, on other hand, has only been observed in the monomeric state. However, a highly homologous E. coli frataxin CyaY, which also lacks an N-terminal extension, has been shown to oligomerize in the presence of iron. To explore the mechanisms of stabilization of short variant frataxin oligomers we compare here the effect of iron on the oligomerization of CyaY and FXN81-210. Using dynamic light scattering, small-angle X-ray scattering, electron microscopy (EM) and cross linking mass spectrometry (MS), we show that at aerobic conditions in the presence of iron both FXN81-210 and CyaY form oligomers. However, while CyaY oligomers are stable over time, FXN81-210 oligomers are unstable and dissociate into monomers after about 24 h. EM and MS studies suggest that within the oligomers FXN81-210 and CyaY monomers are packed in a head-to-tail fashion in ring-shaped structures with potential iron-binding sites located at the interface between monomers. The higher stability of CyaY oligomers can be explained by a higher number of acidic residues at the interface between monomers, which may result in a more stable iron binding. We also show that CyaY oligomers may be dissociated by ferric iron chelators deferiprone and DFO, as well as by the ferrous iron chelator BIPY. Surprisingly, deferiprone and DFO stimulate FXN81-210 oligomerization, while BIPY does not show any effect on oligomerization in this case. The results suggest that FXN81-210 oligomerization is primarily driven by ferric iron, while both ferric and ferrous iron participate in CyaY oligomer stabilization. Analysis of the amino acid sequences of bacterial and eukaryotic frataxins suggests that variations in the position of the acidic residues in helix 1, β-strand 1 and the loop between them may control the mode of frataxin oligomerization.
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| 2. |
- Al-Eryani, Yusra, et al.
(author)
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Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry
- 2016
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In: Proteins: Structure, Function and Genetics. - : Wiley. - 0887-3585. ; 84:9, s. 1234-1245
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Journal article (peer-reviewed)abstract
- Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol-specific oxidative stress in Bacillus subtilis. Under normal growth conditions, the transcription factor is bound to the adaptor protein and therefore degraded by the AAA+ protease ClpXP. If this binding is alleviated during stress, the transcription factor accumulates and turns on genes encoding stress-alleviating proteins. The adaptor protein YjbH is thus a key player involved in these interactions but its structure is unknown. To gain insight into its structure and interactions we have used chemical crosslinking mass spectrometry. Distance constraints obtained from the crosslinked monomer were used to select and validate a structure model of YjbH and then to probe its interactions with other proteins. The core structure of YjbH is reminiscent of DsbA family proteins. One lysine residue in YjbH (K177), located in one of the α-helices outside the thioredoxin fold, crosslinked to both Spx K99 and Spx K117, thereby suggesting one side of the YjbH for the interaction with Spx. Another lysine residue that crosslinked to Spx was YjbH K5, located in the long and presumably very flexible N-terminal arm of YjbH. Our crosslinking data lend support to a model proposed based on site-directed mutagenesis where the YjbH interaction with Spx can stabilize and present the C-terminal region of Spx for protease recognition and proteolysis. Proteins 2016; 84:1234–1245.
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| 3. |
- Boelt, Sanne Grundvad, et al.
(author)
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Mapping the Ca2 + induced structural change in calreticulin
- 2016
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In: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 142, s. 138-148
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Journal article (peer-reviewed)abstract
- Calreticulin is a highly conserved multifunctional protein implicated in many different biological systems and has therefore been the subject of intensive research. It is primarily present in the endoplasmatic reticulum where its main functions are to regulate Ca2 + homeostasis, act as a chaperone and stabilize the MHC class I peptide-loading complex. Although several high-resolution structures of calreticulin exist, these only cover three-quarters of the entire protein leaving the extended structures unsolved. Additionally, the structure of calreticulin is influenced by the presence of Ca2 +. The conformational changes induced by Ca2 + have not been determined yet as they are hard to study with traditional approaches. Here, we investigated the Ca2 +-induced conformational changes with a combination of chemical cross-linking, mass spectrometry, bioinformatics analysis and modelling in Rosetta. Using a bifunctional linker, we found a large Ca2 +-induced change to the cross-linking pattern in calreticulin. Our results are consistent with a high flexibility in the P-loop, a stabilization of the acidic C-terminal and a relatively close interaction of the P-loop and the acidic C-terminal. Biological significance The function of calreticulin, an endoplasmatic reticulin chaperone, is affected by fluctuations in Ca2 + concentration, but the structural mechanism is unknown. The present work suggests that Ca2 +-dependent regulation is caused by different conformations of a long proline-rich loop that changes the accessibility to the peptide/lectin-binding site. Our results indicate that the binding of Ca2 + to calreticulin may thus not only just be a question of Ca2 + storage but is likely to have an impact on the chaperone activity.
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| 4. |
- Christiansen, Maja, et al.
(author)
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Protein chemical characterization of Gc globulin (vitamin D-binding protein) isoforms; Gc-1f, Gc-1s and Gc-2.
- 2007
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In: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434. ; 1774:4
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Journal article (peer-reviewed)abstract
- Gc globulin, also called vitamin D-binding protein, is a plasma protein involved in the extracellular actin-scavenger system, vitamin D transport and possibly also other biological activities. Low levels of Gc globulin have been found to correlate with multiple organ failure and non-survival of patients with fulminant hepatic failure and trauma. Here, we characterize the dominant isoforms of plasma-derived Gc globulin from Cohn fraction IV paste with respect to amino acid sequence and posttranslational modifications. Gc globulin was purified in large scale and the isoforms separated by ion exchange chromatography. The separated isoforms and several commercial preparations of individual isoforms were characterized by mass spectrometry. This revealed that the major isoforms were non-glycosylated. Compared to the Gc-1f isoform the other dominating isoforms represented an Asp/Glu substitution (Gc-1s) and a Thr/Lys substitution (Gc-2) in agreement with DNA sequencing studies. The commercial preparations were found to represent mainly one or two isoforms. An O-linked glycan with a mass of 656 Da and terminating with a sialic acid residue was detected on a minor proportion of Gc globulin molecules.
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| 5. |
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| 6. |
- Pannee, Josef, 1979, et al.
(author)
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A selected reaction monitoring (SRM)-based method for absolute quantification of Aβ38, Aβ40, and Aβ42 in cerebrospinal fluid of Alzheimer's Disease patients and healthy controls.
- 2013
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In: Journal of Alzheimer's Disease. - 1387-2877. ; 33:4, s. 1021-32
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Journal article (peer-reviewed)abstract
- Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in research centers, clinical trials, and clinical settings. However, their broad-scale use is hampered by lack of standardization across analytical platforms and by interference from binding of amyloid-β (Aβ) to matrix proteins as well as self-aggregation. Here, we report on a matrix effect-resistant method for the measurement of the AD-associated 42 amino acid species of Aβ (Aβ42), together with Aβ40 and Aβ38 in human CSF based on mass spectrometric quantification using selected reaction monitoring (SRM). Samples were prepared by solid-phase extraction and quantification was performed using stable-isotope labeled Aβ peptides as internal standards. The diagnostic performance of the method was evaluated on two independent clinical materials with research volunteers who were cognitively normal and AD patients with mild to moderate dementia. Analytical characteristics of the method include a lower limit of quantification of 62.5 pg/mL for Aβ42 and coefficients of variations below 10%. In a pilot study on AD patients and controls, we verified disease-association with decreased levels of Aβ42 similar to that obtained by ELISA and even better separation was obtained using the Aβ42/Aβ40 ratio. The developed assay is sensitive and is not influenced by matrix effects, enabling absolute quantification of Aβ42, Aβ40, and Aβ38 in CSF, while it retains the ability to distinguish AD patients from controls. We suggest this SRM-based method for Aβ peptide quantification in human CSF valuable for clinical research and trials.
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| 7. |
- Rutsdottir, Gudrun, et al.
(author)
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Structural model of dodecameric heat-shock protein Hsp21 : Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity
- 2017
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In: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 292:19, s. 8103-8121
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Journal article (peer-reviewed)abstract
- Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.
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| 8. |
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| 9. |
- Sun, Jianmin, et al.
(author)
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XK-related protein 5 (XKR5) is a novel negative regulator of KIT/D816V-mediated transformation
- 2018
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In: Oncogenesis. - : Springer Science and Business Media LLC. - 2157-9024. ; 7:6
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Journal article (peer-reviewed)abstract
- In order to investigate the molecular mechanisms by which the oncogenic mutant KIT/D816V causes transformation of cells, we investigated proteins that selectively bind KIT/D816V, but not wild-type KIT, as potential mediators of transformation. By mass spectrometry several proteins were identified, among them a previously uncharacterized protein denoted XKR5 (XK-related protein 5), which is related to the X Kell blood group proteins. We could demonstrate that interaction between XKR5 and KIT/D816V leads to phosphorylation of XKR5 at Tyr 369, Tyr487, and Tyr 543. Tyrosine phosphorylated XKR5 acts as a negative regulator of KIT signaling, which leads to downregulation of phosphorylation of ERK, AKT, and p38. This led to reduced proliferation and colony forming capacity in semi-solid medium. Taken together, our data demonstrate that XKR5 is a novel type of negative regulator of KIT-mediated transformation.
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