| 1. |
- Boros, S, et al.
(författare)
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Site-specific transamidation and deamidation of the small heat-shock protein Hsp20 by tissue transglutaminase
- 2006
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Ingår i: Proteins. - : Wiley. - 0887-3585. ; 62:4, s. 1044-1052
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Tidskriftsartikel (refereegranskat)abstract
- Crosslinking of small heat-shock proteins (sHsps) by tissue transglutaminase (tTG) is enhanced by stress and under pathological conditions. We here used hexapeptide probes to determine the amine donor (K) and acceptor (Q) sites for tTG in Hsp20. Mass spectrometric peptide mass fingerprinting and peptide fragmentation established that Q(31) and the C-terminal K-162 are involved in inter- and intramolecular crosslinking (transamidation). Q(31) is a conserved glutamine in sHsps where the neighboring residue determines its reactivity. Moreover, we detected highly efficient simultaneous deamidation of Q(66), which suggests that tTG-catalyzed transamidation and deamidation is specific for different glutamine residues.
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| 2. |
- Bundgaard, Louise, et al.
(författare)
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Effective protein extraction combined with data independent acquisition analysis reveals a comprehensive and quantifiable insight into the proteomes of articular cartilage and subchondral bone
- 2022
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Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 30:1, s. 137-146
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: The objectives of this study was to establish a sensitive and reproducible method to map the cartilage and subchondral bone proteomes in quantitative terms, and mine the proteomes for proteins of particular interest in the pathogenesis of osteoarthritis (OA). The horse was used as a model animal.DESIGN: Protein was extracted from articular cartilage and subchondral bone samples from three horses in triplicate by pressure cycling technology or ultrasonication. Digested proteins were analysed by data independent acquisition based mass spectrometry. Data was processed using a pre-established spectral library as reference database (FDR 1%).RESULTS: We identified to our knowledge the hitherto most comprehensive quantitative cartilage (1758 proteins) and subchondral bone (1482 proteins) proteomes in all species presented to date. Both extraction methods were sensitive and reproducible and the high consistency of the identified proteomes (>97% overlap) indicated that both methods preserved the diversity among the extracted proteins. Proteome mining revealed a substantial number of quantifiable cartilage and bone matrix proteins and proteins involved in osteogenesis and bone remodeling, including ACAN, BGN, PRELP, FMOD, COMP, ACP5, BMP3, BMP6, BGLAP, TGFB1, IGF1, ALP, MMP3, and collagens. A number of proteins, including COMP and TNN, were identified in different protein isoforms with potential unique biological roles.CONCLUSION: We have successfully developed two sensitive and reproducible non-species specific workflows enabling a comprehensive quantitative insight into the proteomes of cartilage and subchondral bone. This facilitates the prospect of investigating the molecular events at the osteochondral unit in the pathogenesis of OA in future projects.
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| 3. |
- Elowsson Rendin, Linda, et al.
(författare)
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Matrisome Properties of Scaffolds Direct Fibroblasts in Idiopathic Pulmonary Fibrosis
- 2019
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 20:16
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Tidskriftsartikel (refereegranskat)abstract
- In idiopathic pulmonary fibrosis (IPF) structural properties of the extracellular matrix (ECM) are altered and influence cellular responses through cell-matrix interactions. Scaffolds (decellularized tissue) derived from subpleural healthy and IPF lungs were examined regarding biomechanical properties and ECM composition of proteins (the matrisome). Scaffolds were repopulated with healthy fibroblasts cultured under static stretch with heavy isotope amino acids (SILAC), to examine newly synthesized proteins over time. IPF scaffolds were characterized by increased tissue density, stiffness, ultimate force, and differential expressions of matrisome proteins compared to healthy scaffolds. Collagens, proteoglycans, and ECM glycoproteins were increased in IPF scaffolds, however while specific basement membrane (BM) proteins such as laminins and collagen IV were decreased, nidogen-2 was also increased. Findings were confirmed with histology, clearly showing a disorganized BM. Fibroblasts produced scaffold-specific proteins mimicking preexisting scaffold composition, where 11 out of 20 BM proteins were differentially expressed, along with increased periostin and proteoglycans production. We demonstrate how matrisome changes affect fibroblast activity using novel approaches to study temporal differences, where IPF scaffolds support a disorganized BM and upregulation of disease-associated proteins. These matrix-directed cellular responses emphasize the IPF matrisome and specifically the BM components as important factors for disease progression.
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| 4. |
- Furst, Camilla Melin, et al.
(författare)
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Quantitative mass spectrometry to study inflammatory cartilage degradation and resulting interactions with the complement system
- 2016
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 197:8, s. 3415-3424
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Tidskriftsartikel (refereegranskat)abstract
- Joint diseases are often characterized by inflammatory processes that result in pathological changes in joint tissues, including cartilage degradation and release of components into the synovial fluid. The complement system plays a central role in promoting the inflammation. Because several cartilage proteins are known to interact with complement, causing either activation or inhibition of the system, we aimed to investigate these interactions comprehensively. Bovine cartilage explants were cultured with IL-1α to induce cartilage degradation, followed by incubation with human serum. Label-free selected reaction monitoring mass spectrometry was used to specifically quantify complement proteins interacting with the cartilage explant. In parallel, the time-dependent degradation of cartilage was detected using mass spectrometry analysis (liquid chromatography-tandem mass spectrometry). Complement proteins resulting from activation of the classical, alternative, and terminal pathways were detected on IL-1α-stimulated cartilage at time points when clear alterations in extracellular matrix composition had occurred. Increased levels of the complement activation product C4d, as detected by ELISA in serum after incubation with IL-1α-stimulated cartilage, confirmed the selected reaction monitoring results indicating complement activation. Further, typical activated (cleaved) C3 fragments were detected by Western blotting in extracts of IL-1α-stimulated cartilage. No complement activation was triggered by cartilage cultured in the absence of IL-1α. Components released from IL-1α-stimulated cartilage during culture had an inhibitory effect on complement activation. These were released after a longer incubation period with IL-1α and may represent a feedback reaction to cartilage-triggered complement activation observed after a shorter incubation period.
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| 5. |
- Ising, Erik, et al.
(författare)
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Quantification of heat shock proteins in the posterior interosseous nerve among subjects with type 1 and type 2 diabetes compared to healthy controls
- 2023
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Ingår i: Frontiers in Neuroscience. - : FRONTIERS MEDIA SA. - 1662-4548 .- 1662-453X. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Diabetic peripheral neuropathy (DPN) is a common complication of both type 1 (T1D) and type 2 diabetes (T2D). No cure for DPN is available, but several potential targets have been proposed for treatment. Heat shock proteins (HSPs) are known to respond to both hyper- and hypoglycemia. DPN can be diagnosed using electrophysiology and studied using peripheral nerve biopsies.Aim: This study aimed to analyze the presence and patterns of HSPs in peripheral nerve biopsies from subjects with T1D, T2D, and healthy controls.Methods: Posterior interosseous nerves (PIN) from a total of 56 subjects with T1D (n = 9), with T2D (n = 24), and without diabetes (i.e., healthy controls, n = 23) were harvested under local anesthesia and prepared for quantitative mass spectrometry analysis. Protein intensities were associated with electrophysiology data of the ulnar nerve and morphometry of the same PIN, and differences in protein intensities between groups were analyzed.Results: In total, 32 different HSPs were identified and quantified in the nerve specimens. No statistically significant differences were observed regarding protein intensities between groups. Furthermore, protein intensities did not correlate with amplitude or conduction velocity in the ulnar nerve or with the myelinated nerve fiber density of PIN.Conclusion: Quantitative proteomics can be used to study HSPs in nerve biopsies, but no clear differences in protein quantities were observed between groups in this cohort.
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| 6. |
- Ising, Erik, et al.
(författare)
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Quantitative proteomic analysis of human peripheral nerves from subjects with type 2 diabetes
- 2021
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Ingår i: Diabetic Medicine: A journal of the British Diabetic Association. - : Wiley. - 1464-5491. ; 38:11
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Tidskriftsartikel (refereegranskat)abstract
- AIMS: Diabetic peripheral neuropathy (DPN) is a common and severe complication to type 2 diabetes (T2D). The pathogenesis of DPN is not fully known, but several pathways and gene polymorphisms contributing to DPN are described. DPN can be studied using nerve biopsies, but studies on the proteome of the nerve itself, and its surrounding tissue as a whole, are lacking. Studies on the posterior interosseous nerve (PIN) have proposed PIN a useful indicator of DPN.METHODS: A quantitative mass spectrometry-based proteomics analysis was made of peripheral nerves from age- and gender-matched living human male tissue donors; nine T2D subjects, with decreased sural nerve action potentials indicating DPN, and six controls without T2D, with normal electrophysiology results.RESULTS: A total of 2617 proteins were identified. Linear regression was used to discover which proteins were differentially expressed between T2D and controls. Only soft signals were found. Therefore, clustering of the 500 most variable proteins were made in order to find clusters of similar proteins in T2D subjects and healthy controls.CONCLUSIONS: This feasibility study shows, for the first time, that the use of quantitative mass spectrometry enables quantification of proteins from nerve biopsies from subjects with and without T2D, which may aid in finding biomarkers of importance to DPN development.
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| 7. |
- Kadefors, Måns, et al.
(författare)
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CD105+CD90+CD13+ identifies a clonogenic subset of adventitial lung fibroblasts
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal cells are important components of specified niches in the lung, and can mediate a wide range of processes including tissue regeneration and repair. Dysregulation of these processes can lead to improper remodeling of tissue as observed in several lung diseases. The mesenchymal cells responsible remain poorly described, partially due to the heterogenic nature of the mesenchymal compartment and the absence of appropriate markers. Here, we describe that CD105+CD90+ mesenchymal cells can be divided into two populations based on their expression of CD13/aminopeptidase N (CD105+CD90+CD13− and CD105+CD90+CD13+). By prospective isolation using FACS, we show that both these populations give rise to clonogenic fibroblast-like cells, but with an increased clonogenic and proliferative capacity of CD105+CD90+CD13+ cells. Transcriptomic and spatial analysis pinpoints an adventitial fibroblast subset as the origin of CD105+CD90+CD13+ clonogenic mesenchymal cells in human lung.
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| 8. |
- Lambert, W, et al.
(författare)
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Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21
- 2011
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 20:2, s. 291-301
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Tidskriftsartikel (refereegranskat)abstract
- Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25 degrees in relation to each other, suggesting a role for global dynamics in dodecamer function.
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| 9. |
- Müller, Catharina, et al.
(författare)
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Protein signatures of remodeled airways in transplanted lungs with Bronchiolitis Obliterans Syndrome obtained using laser capture microdissection
- 2021
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Ingår i: American Journal of Pathology. - : Elsevier BV. - 1525-2191 .- 0002-9440. ; 191:8, s. 1398-1411
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Tidskriftsartikel (refereegranskat)abstract
- Bronchiolitis obliterans syndrome (BOS), a common form of chronic lung allograft dysfunction, is the major limitation to long-term survival after lung transplantation. The histological correlate is progressive, fibrotic occlusion of small airways, obliterative bronchiolitis lesions, ultimately leading to organ failure. The molecular composition of these lesions is unknown. By laser-capture microdissection and optimized sample preparation protocols for mass spectrometry the protein composition of the lesions in explanted lungs from four end-stage BOS patients were analysed. Immunohistochemistry and immunofluorescence were used to determine the spatial distribution of commonly identified proteins on the tissue level, protein signatures for in total 14 OB-lesions were established. A set of 39 proteins identified in more than 75% of lesions included distinct structural proteins (collagen type IV and VI) and cellular components (actins, vimentin, tryptase). Each respective lesion exhibited a unique composition of proteins (on average n=66 proteins), thereby mirroring the morphological variation of the lesions. Antibody-based staining confirmed these MS-based findings. The 14 analyzed OB-lesions showed variations in their protein content, but also common features. This study provides molecular and morphological insights into the development of chronic rejection after lung transplantation. The protein patterns in the lesions were correlated to pathways of extracellular matrix organization, tissue development and wound healing processes.
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| 10. |
- Rolandsson Enes, Sara, et al.
(författare)
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Quantitative proteomic characterization of lung-MSC and bone marrow-MSC using DIA-mass spectrometry
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
-
Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stromal cells (MSC) are ideal candidates for cell therapies, due to their immune-regulatory and regenerative properties. We have previously reported that lung-derived MSC are tissue-resident cells with lung-specific properties compared to bone marrow-derived MSC. Assessing relevant molecular differences between lung-MSC and bone marrow-MSC is important, given that such differences may impact their behavior and potential therapeutic use. Here, we present an in-depth mass spectrometry (MS) based strategy to investigate the proteomes of lung-MSC and bone marrow-MSC. The MS-strategy relies on label free quantitative data-independent acquisition (DIA) analysis and targeted data analysis using a MSC specific spectral library. We identified several significantly differentially expressed proteins between lung-MSC and bone marrow-MSC within the cell layer (352 proteins) and in the conditioned medium (49 proteins). Bioinformatics analysis revealed differences in regulation of cell proliferation, which was functionally confirmed by decreasing proliferation rate through Cytochrome P450 stimulation. Our study reveals important differences within proteome and matrisome profiles between lung- and bone marrow-derived MSC that may influence their behavior and affect the clinical outcome when used for cell-therapy.
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| 11. |
- Rosmark, Oskar, et al.
(författare)
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Quantifying extracellular matrix turnover in human lung scaffold cultures
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- Remodelling of the extracellular matrix is accomplished by altering the balance between matrix macromolecule production and degradation. However, it is not well understood how cells balance production of new matrix molecules and degradation of existing ones during tissue remodelling and regeneration. In this study, we used decellularized lung scaffolds repopulated with allogenic lung fibroblasts cultured with stable isotope labelled amino acids to quantify the balance between matrix production and degradation at a proteome-wide scale. Specific temporal dynamics of different matrisome proteins were found to correspond to the proliferative activity of the repopulating cells and the degree of extracellular deposition. The remodeling of the scaffold was characterized by an initial phase with cell proliferation and high production of cell adhesion proteins such as emilin-1 and fibronectin. Extended culture time resulted in increased levels of core matrisome proteins. In a comparison with monolayer cultures on plastic, culture in lung scaffolds lead to a pronounced accumulation of proteoglycans, such as versican and decorin, resulting in regeneration of an extracellular matrix with greater resemblance to native lung tissue compared to standard monolayer cultures. Collectively, the study presents a promising technique for increasing the understanding of cell- extracellular matrix interactions under healthy and diseased conditions.
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| 12. |
- Rucci, Nadia, et al.
(författare)
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The glycosaminoglycan-binding domain of PRELP acts as a cell type-specific NF-kappa B inhibitor that impairs osteoclastogenesis
- 2009
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 187:5, s. 669-683
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Tidskriftsartikel (refereegranskat)abstract
- Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a glycosaminoglycan (GAG)- and collagen-binding anchor protein highly expressed in cartilage, basement membranes, and developing bone. We observed that PRELP inhibited in vitro and in vivo mouse osteoclastogenesis through its GAG-binding domain ((PRELP)-P-hbd), involving (a) cell internalization through a chondroitin sulfate-and annexin II-dependent mechanism, (b) nuclear translocation, (c) interaction with p65 nuclear factor. B (NF-kappa B) and inhibition of its DNA binding, and (d) impairment of NF-kappa B transcriptional activity and reduction of osteoclast-specific gene expression. hbdPRELP does not disrupt the mitogen-activated protein kinase signaling nor does it impair cell survival. hbdPRELP activity is cell type specific, given that it is internalized by the RAW264.7 osteoclast-like cell line but fails to affect calvarial osteoblasts, bone marrow macrophages, and epithelial cell lines. In vivo, hbdPRELP reduces osteoclast number and activity in ovariectomized mice, underlying its physiological and/or pathological importance in skeletal remodeling.
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| 13. |
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| 14. |
- Sundby, Cecilia, et al.
(författare)
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Conserved methionines in chloroplasts
- 2005
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639. ; 1703:2, s. 191-202
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Forskningsöversikt (refereegranskat)abstract
- Heat shock proteins counteract heat and oxidative stress. In chloroplasts, a small heat shock protein (Hsp21) contains a set of conserved methionines, which date back to early in the emergence of terrestrial plants. Methionines M49, M52, M55, M59, M62, M67 are located on one side of an amphipathic helix, which may fold back over two other conserved methionines (M97 and M101), to form a binding groove lined with methionines, for sequence-independent recognition of peptides with an overall hydrophobic character. The sHsps protect other proteins from aggregation by binding to their hydrophobic surfaces, which become exposed under stress. Data are presented showing that keeping the conserved methionines in Hsp21 in a reduced form is a prerequisite to maintain such binding. The chloroplast generates reactive oxygen species under both stress and unstressed conditions, but this organelle is also a highly reducing cellular compartment. Chloroplasts contain a specialized isoform of the enzyme, peptide methionine sulfoxide reductase, the expression of which is light-induced. Recombinant proteins were used to measure that this reductase can restore Hsp21 methionines after sulfoxidation. This paper also describes how methionine sulfoxidation-reduction can be directly assessed by mass spectrometry, how methionine-to-leucine substitution affects Hsp21, and discusses the possible role for an Hsp21 methionine sulfoxidation-reduction cycle in quenching reactive oxygen species. (C) 2004 Elsevier B.V. All rights reserved.
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| 15. |
- Weitoft, Maria, et al.
(författare)
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Comparison of Normal and Metaplastic Epithelium in Patients with Stable versus Persistently Symptomatic Severe Asthma Using Laser-Capture Microdissection and Data-Independent Acquisition–Mass Spectrometry
- 2019
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Ingår i: American Journal of Pathology. - : Elsevier BV. - 0002-9440. ; 189:12, s. 2358-2365
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Tidskriftsartikel (refereegranskat)abstract
- A proportion of patients with severe asthma (SA) show poor responses to traditional asthma medications; however, it remains unknown why some patients remain persistently symptomatic. Our objective was to explore the use of laser-capture microdissection of specific epithelial structures combined with quantitative data-independent acquisition mass spectrometry to elucidate differences in protein composition in patients with SA with varying symptom control. Unbiased label-free quantitative proteome analyses were performed on laser-capture–microdissected areas of specific epithelial structures from patients with SA with varying degrees of symptom control. A total of 1993 stable SA and 1652 symptomatic SA proteins in normal epithelium and 1458 stable SA and 1647 symptomatic SA proteins in metaplastic epithelium were quantified. When comparing proteome profiles based on symptom control, 33 proteins in patients with stable SA (≥twofold change; P ≤ 0.05) and 13 proteins in patients with persistently symptomatic SA (≥twofold change; P ≤ 0.05) were enriched significantly. When comparing proteome profiles based on epithelial status, 21 proteins in normal epithelium (≥twofold change; P ≤ 0.05) and 6 proteins in metaplastic epithelium (≥twofold change; P ≤ 0.05) were enriched significantly. New treatment strategies are needed for patients with severe asthma and exploratory studies of unbiased nature such as this may help when searching for new mechanisms and potential targets involved in the disease pathology.
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| 16. |
- Westergren-Thorsson, Gunilla, et al.
(författare)
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Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis
- 2017
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Ingår i: International Journal of Biochemistry & Cell Biology. - : Elsevier BV. - 1357-2725 .- 1878-5875. ; 83, s. 27-38
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Tidskriftsartikel (refereegranskat)abstract
- Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape. (C) 2016 The Authors. Published by Elsevier Ltd.
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| 17. |
- Åhrman, Emma, et al.
(författare)
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Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase
- 2007
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 16:7, s. 1464-1478
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Tidskriftsartikel (refereegranskat)abstract
- The molecular mechanism whereby the small heat-shock protein ( sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase ( CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,39-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross- linker captured Hsp21 in dodecameric form, even at increased temperature ( 47 C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21-Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21-CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21.
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| 18. |
- Åhrman, Emma
(författare)
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Interactions Between Small Heat Shock Proteins and Substrate Proteins Mass Spectrometric Detection of Crosslinked Peptides to Map Protein-Protein Interactions
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The small heat shock proteins (sHsps) are a widespread class of molecular chaperones existing in all organisms. During heat stress the sHsps protect other proteins from unfolding and aggregation. In plants the sHsps play an especially prominent part of the stress response. In this thesis chemical crosslinking and mass spectrometric mapping of crosslinked peptides has been used to investigate the interaction between sHsps and substrate proteins. The focus has been on Hsp21, a chloroplast-localized sHsp which increases the stress resistance of Arabidopsis thaliana plants, and a thermosensitive model substrate protein, citrate synthase (CS), using the chemical crosslinker DTSSP (3,3?-dithiobis[sulfosuccinimidylpropionate]), for covalent crosslinking between lysine residues or other primary amines. In peptide array screening, binding of sHsps occurred to a peptide located in a stem-like structure protruding from the CS dimer, where the C-terminus from one CS monomer interacts with the N-terminal part of the other monomer. Mass spectrometric mapping of crosslinked Hsp21-CS peptides further confirmed interactions between Hsp21 and lysine residues (K16, K22, K432, K437) located in this stem-like structure of CS. We propose that sHsps bind to this region, which is absent in thermostable CS forms, to stabilize the Nand C-terminae and thereby prevent CS dimer dissociation and aggregation. The crosslinked Hsp21-CS peptides indicated that the substrate-binding region in the N-terminal arm in Hsp21 interacted with the thermosensitive part of CS. Similar mass spectrometric peptide mapping was also used to show how this N-terminal arm region of a mammalian sHsp, Hsp20, is involved in transglutaminase-catalyzed crosslinking of sHsps to various substrate proteins in cellular aggregates. This was studied using a peptide probe which could be crosslinked by transglutaminase to the conserved Q31 in the Nterminal arm of mammalian sHsps. Whereas the N-terminus of Hsp21 was detected in crosslinked Hsp21-Hsp21 and Hsp21-CS peptides under conditions where the Hsp21 dodecamer was intact, the C-terminal tail and a C-terminal binding groove was detected in crosslinked Hsp21-CS peptides only under conditions where the Hsp21 dodecamer dissociated. The crosslinked Hsp21-CS peptides mapped onto several sites on the CS surface, indicating that several weak and short-lived interactions between Hsp21 and CS occur already at normal temperatures. Single particle reconstruction EM of crosslinked Hsp21 in the presence of CS indicated that the crosslinker could capture the Hsp21 dodecamers in an ?activated? conformation induced by such weak and short-lived interactions with CS.
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| 19. |
- Åhrman, Emma, et al.
(författare)
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Novel COMP Neoepitopes Identified in Synovial Fluids from Patients with Joint Diseases using Affinity Chromatography and Mass Spectrometry.
- 2014
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 289:30, s. 20908-20916
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Tidskriftsartikel (refereegranskat)abstract
- To identify patients at risk for progressive joint damage there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open for novel treatment strategies. Disease specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis (RA), osteoarthritis (OA) or acute trauma (AT). Enriched COMP fragments were separated by SDS-PAGE followed by in-gel digestion and mass spectrometric identification and characterization. Using the enzymes trypsin, chymotrypsin and Asp-N for the digestions an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, S77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope S77 were released into the culture medium of cytokine (TNF-α and IL-6/sIL-6R) stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pin-point disease progression, evaluation methods for therapy and means to elucidate disease mechanisms will be provided.
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| 20. |
- Åhrman, Emma, et al.
(författare)
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Quantitative proteomic characterization of the lung extracellular matrix in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis
- 2018
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 189, s. 23-33
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Tidskriftsartikel (refereegranskat)abstract
- Remodeling of the extracellular matrix (ECM) is a common feature in lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Here, we applied a sequential tissue extraction strategy to describe disease-specific remodeling of human lung tissue in disease, using end-stages of COPD and IPF. Our strategy was based on quantitative comparison of the disease proteomes, with specific focus on the matrisome, using data-independent acquisition and targeted data analysis (SWATH-MS). Our work provides an in-depth proteomic characterization of human lung tissue during impaired tissue remodeling. In addition, we show important quantitative and qualitative effects of the solubility of matrisome proteins. COPD was characterized by a disease-specific increase in ECM regulators, metalloproteinase inhibitor 3 (TIMP3) and matrix metalloproteinase 28 (MMP-28), whereas for IPF, impairment in cell adhesion proteins, such as collagen VI and laminins, was most prominent. For both diseases, we identified increased levels of proteins involved in the regulation of endopeptidase activity, with several proteins belonging to the serpin family. The established human lung quantitative proteome inventory and the construction of a tissue-specific protein assay library provides a resource for future quantitative proteomic analyses of human lung tissues. Significance: We present a sequential tissue extraction strategy to determine changes in extractability of matrisome proteins in end-stage COPD and IPF compared to healthy control tissue. Extensive quantitative analysis of the proteome changes of the disease states revealed altered solubility of matrisome proteins involved in ECM regulators and cell-ECM communication. The results highlight disease-specific remodeling mechanisms associated with COPD and IPF.
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| 21. |
- Åhrman, Emma, et al.
(författare)
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Small heat shock proteins prevent aggregation of citrate synthase and bind to the N-terminal region which is absent in thermostable forms of citrate synthase
- 2007
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Ingår i: Extremophiles. - : Springer Science and Business Media LLC. - 1433-4909 .- 1431-0651. ; 11:5, s. 659-666
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Tidskriftsartikel (refereegranskat)abstract
- Citrate synthase (CS) is often used in chaperone assays since this thermosensitive enzyme aggregates at moderately increased temperatures. Small heat shock proteins (sHsps) are molecular chaperones specialized in preventing the aggregation of other proteins, termed substrate proteins, under conditions of transient heat stress. To investigate the mechanism whereby sHsps bind to and stabilize a substrate protein, we here used peptide array screening covering the sequence of porcine CS (P00889). Strong binding of sHsps was detected to a peptide corresponding to the most N-terminal alpha-helix in CS (amino acids Leu(13) to Gln(27)). The N-terminal alpha-helices in the CS dimer intertwine with the C-terminus in the other subunit and together form a stem-like structure which is protruding from the CS dimer. This stem-like structure is absent in thermostable forms of CS from thermophilic archaebacteria like Pyrococcus furiosus and Sulfolobus solfatacarium. These data therefore suggest that thermostabilization of thermosensitive CS by sHsps is achieved by stabilization of the C- and N-terminae in the protruding thermosensitive softspot, which is absent in thermostable forms of the CS dimer.
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