| 1. |
- Bernfur, Katja, et al.
(författare)
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The chloroplast-localized small heat shock protein Hsp21 associates with the thylakoid membranes in heat-stressed plants
- 2017
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Ingår i: Protein Science. - : Wiley. - 0961-8368. ; 26:9, s. 1773-1784
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Tidskriftsartikel (refereegranskat)abstract
- The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity of sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps with a single conserved β-sandwich fold domain defining the family, the α-crystallin domain, whereas the N-terminal and C-terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non-metazoan sHsp expressed in the chloroplasts in green plants which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat-stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic 15N-isotope labeling of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat-stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered.
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| 2. |
- Gisladottir, Rosa S, et al.
(författare)
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Sequence Variants in TAAR5 and Other Loci Affect Human Odor Perception and Naming.
- 2020
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Ingår i: Current biology : CB. - : Elsevier BV. - 1879-0445 .- 0960-9822. ; 30:23
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Tidskriftsartikel (refereegranskat)abstract
- Olfactory receptor (OR) genes in humans form a special class characterized by unusually high DNA sequence diversity, which should give rise to differences in perception and behavior. In the largest genome-wide association study to date based on olfactory testing, we investigated odor perception and naming with smell tasks performed by 9,122 Icelanders, with replication in a separate sample of 2,204 individuals. We discovered an association between a low-frequency missense variant in TAAR5 and reduced intensity rating of fish odor containing trimethylamine (p.Ser95Pro, pcombined= 5.6× 10-15). We demonstrate that TAAR5 genotype affects aversion to fish odor, reflected by linguistic descriptions of the odor and pleasantness ratings. We also discovered common sequence variants in two canonical olfactory receptor loci that associate with increased intensity and naming of licorice odor (trans-anethole: lead variant p.Lys233Asn in OR6C70, pcombined= 8.8× 10-16 and pcombined= 1.4× 10-9) and enhanced naming of cinnamon (trans-cinnamaldehyde; intergenic variant rs317787-T, pcombined= 5.0× 10-17). Together, our results show that TAAR5 genotype variation influences human odor responses and highlight that sequence diversity in canonical OR genes can lead to enhanced olfactory ability, in contrast to the view that greater tolerance for mutations in the human OR repertoire leads to diminished function.
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| 3. |
- Lambert, Wietske, et al.
(författare)
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Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry.
- 2012
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Ingår i: Cell Stress & Chaperones. - : Springer Science and Business Media LLC. - 1466-1268 .- 1355-8145.
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Tidskriftsartikel (refereegranskat)abstract
- Small heat-shock protein chaperones are important players in the protein quality control system of the cell, because they can immediately respond to partially unfolded proteins, thereby protecting the cell from harmful aggregates. The small heat-shock proteins can form large polydisperse oligomers that are exceptionally dynamic, which is implicated in their function of protecting substrate proteins from aggregation. Yet the mechanism of substrate recognition remains poorly understood, and little is known about what parts of the small heat-shock proteins interact with substrates and what parts of a partially unfolded substrate protein interact with the small heat-shock proteins. The transient nature of the interactions that prevent substrate aggregation rationalize probing this interaction by crosslinking mass spectrometry. Here, we used a workflow with lysine-specific crosslinking and offline nano-liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry to explore the interaction between the plant small heat-shock protein Hsp21 and a thermosensitive model substrate protein, malate dehydrogenase. The identified crosslinks point at an interaction between the disordered N-terminal region of Hsp21 and the C-terminal presumably unfolding part of the substrate protein.
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| 4. |
- Lambert, Wietske, et al.
(författare)
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Thiol-exchange in DTSSP crosslinked peptides is proportional to cysteine content and precisely controlled in crosslink detection by two-step LC-MALDI MSMS
- 2011
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 20:10, s. 1682-1691
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Tidskriftsartikel (refereegranskat)abstract
- The lysine-specific crosslinker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) is commonly used in the structural characterization of proteins by chemical crosslinking and mass spectrometry and we here describe an efficient two-step LC-MALDI-TOF/TOF procedure to detect crosslinked peptides. First MS data are acquired, and the properties of isotope-labeled DTSSP are used in data analysis to identify candidate crosslinks. MSMS data are then acquired for a restricted number of precursor ions per spot for final crosslink identification. We show that the thiol-catalyzed exchange between crosslinked peptides, which is due to the disulfide bond in DTSSP and known to possibly obscure data, can be precisely quantified using isotope-labeled DTSSP. Crosslinked peptides are recognized as 8 Da doublet peaks and a new isotopic peak with twice the intensity appears in the middle of the doublet as a consequence of the thiol-exchange. False-positive crosslinks, formed exclusively by thiol-exchange, yield a 1:2:1 isotope pattern, whereas true crosslinks, formed by two lysine residues within crosslinkable distance in the native protein structure, yield a 1:0:1 isotope pattern. Peaks with a 1:X:1 isotope pattern, where 0 < X < 2, can be trusted as true crosslinks, with a defined proportion of the signal [2X/(2 + X)] being noise from the thiol-exchange. The thiol-exchange was correlated with the protein cysteine content and was minimized by shortening the trypsin incubation time, and for two molecular chaperone proteins with known structure all crosslinks fitted well to the structure data. The thiol-exchange can thus be controlled and isotope-labeled DTSSP safely used to detect true crosslinks between lysine residues in proteins.
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| 5. |
- Peterson Wulff, Ragna, et al.
(författare)
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The activity of barley NADPH-dependent thioredoxin reductase C is independent of the oligomeric state of the protein: tetrameric structure determined by cryo-electron microscopy
- 2011
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 50:18, s. 3713-3723
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Tidskriftsartikel (refereegranskat)abstract
- Thioredoxin and thioredoxin reductase can regulate cell metabolism through redox regulation of disulfide bridges or through removal of H(2)O(2). These two enzymatic functions are combined in NADPH-dependent thioredoxin reductase C (NTRC), which contains an N-terminal thioredoxin reductase domain fused with a C-terminal thioredoxin domain. Rice NTRC exists in different oligomeric states, depending on the absence or presence of its NADPH cofactor. It has been suggested that the different oligomeric states may have diverse activity. Thus, the redox status of the chloroplast could influence the oligomeric state of NTRC and thereby its activity. We have characterized the oligomeric states of NTRC from barley (Hordeum vulgare L.). This also includes a structural model of the tetrameric NTRC derived from cryo-electron microscopy and single-particle reconstruction. We conclude that the tetrameric NTRC is a dimeric arrangement of two NTRC homodimers. Unlike that of rice NTRC, the quaternary structure of barley NTRC complexes is unaffected by addition of NADPH. The activity of NTRC was tested with two different enzyme assays. The N-terminal part of NTRC was tested in a thioredoxin reductase assay. A peroxide sensitive Mg-protoporphyrin IX monomethyl ester (MPE) cyclase enzyme system of the chlorophyll biosynthetic pathway was used to test the catalytic ability of both the N- and C-terminal parts of NTRC. The different oligomeric assembly states do not exhibit significantly different activities. Thus, it appears that the activities are independent of the oligomeric state of barley NTRC.
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| 6. |
- Rutsdottir, Gudrun
(författare)
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Combined structural methods to characterize an oligomeric small heat shock protein Hsp21
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Small heat shock proteins (sHsps) are found in all organisms and evolved early in the history of life, prior to the divergence of the three domains of life (Archaea, Bacteria, and Eukarya). Their malfunction is connected to several different diseases in humans, such as Alzheimer’s and Parkinson’s disease. sHsps are especially abundant in plants, where most are highly upregulated as the first line of defence against heat and other types of stress. sHsps are ATP-independent chaperones that contain a conserved α -crystallin domain with a characteristic β-sandwich fold, flanked by a flexible N-terminal region (NTR), which varies in length between different sHsps and a short C-terminal region (CTR), which contains a conserved I/V-X-I/V-motif important for oligomerization. Since sHsps are highly dynamic, only a few high-resolution structures are available. Most structures have either truncated or unresolved NTR. Hsp21, a dodecameric sHsp found in the chloroplasts of Arabidopsis thaliana and all green plants, has a long NTR with a predicted amphipathic α-helix containing highly conserved methionines. In this thesis, combined structural methods—cryo-electron microscopy, crosslinking mass spectrometry, small angle X-ray scattering, nuclear magnetic resonance spectroscopy, and limited proteolysis—were applied to characterize Hsp21. A structural model for Hsp21 was generated that suggests that oligomerization is maintained by CTR tail-to-tail interactions and that the NTR arms are partly on the outside of the oligomer. The protein-protein interactions between Hsp21 and model substrate proteins were probed by small angle X-ray scattering and crosslinking mass spectrometry showing the NTR in Hsp21 interacts with specific parts of the protected client proteins, presumably the parts that start to unfold under destabilizing conditions. A non-dodecameric mutational variant of Hsp21, Hsp21V181A, which is mainly dimeric, was found to have decreased chaperone activity and was unable to form client-chaperone complexes to the same extent as the oligomeric Hsp21. This emphasizes that oligomerization is an intrinsic property required for chaperone activity in sHsps. Mass spectrometric quantification was used to explore another possible role of Hsp21, namely interaction with membranes. This study concludes that Hsp21 can translocate into the thylakoid membrane during heat stress, in vivo and in vitro. This thesis points at several new findings concerning the structure and function of Hsp21, a chloroplast-localized chaperone, which is of large importance for adaptation of plants to temperature variations and stress.
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| 7. |
- Rutsdottir, Gudrun, et al.
(författare)
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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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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 292:19, s. 8103-8121
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Tidskriftsartikel (refereegranskat)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. |
- Söderberg, Christopher A.G., et al.
(författare)
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Structural modelling of the DNAJB6 oligomeric chaperone shows a peptide-binding cleft lined with conserved S/T-residues at the dimer interface
- 2018
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- = The remarkably efficient suppression of amyloid fibril formation by the DNAJB6 chaperone is dependent on a set of conserved S/T-residues and an oligomeric structure, features unusual among DNAJ chaperones. We explored the structure of DNAJB6 using a combination of structural methods. Lysine-specific crosslinking mass spectrometry provided distance constraints to select a homology model of the DNAJB6 monomer, which was subsequently used in crosslink-assisted docking to generate a dimer model. A peptide-binding cleft lined with S/T-residues is formed at the monomer-monomer interface. Mixed isotope crosslinking showed that the oligomers are dynamic entities that exchange subunits. The purified protein is well folded, soluble and composed of oligomers with a varying number of subunits according to small-angle X-ray scattering (SAXS). Elongated particles (160 x 120 angstrom) were detected by electron microscopy and single particle reconstruction resulted in a density map of 20 angstrom resolution into which the DNAJB6 dimers fit. The structure of the oligomer and the S/T-rich region is of great importance for the understanding of the function of DNAJB6 and how it can bind aggregation-prone peptides and prevent amyloid diseases.
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| 10. |
- Söderberg, Christopher, et al.
(författare)
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Detection of Crosslinks within and between Proteins by LC-MALDI-TOFTOF and the Software FINDX to Reduce the MSMS-Data to Acquire for Validation.
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:6
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Tidskriftsartikel (refereegranskat)abstract
- Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using (14)N/(15)N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers.
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