| 1. |
- Broman, Axel, et al.
(författare)
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Rapid multinodal acoustic trapping of extracellular vesicles for downstream mass spectrometry analysis
- 2020
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Ingår i: MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - 9781733419017 ; , s. 148-149
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Konferensbidrag (refereegranskat)abstract
- We report the use of multinodal acoustic trapping for high throughput and high capacity capturing of EV's (extracellular vesicles) for quantitative mass spectrometry analysis. The multinode trapping unit was shown to isolate sufficient amount of EV's from dilute biological samples (urine and cell culture supernatant) at flow rates of 500 ul/min within minutes, enabling EV proteome profiling. This was shown by differential protein expression analysis of urine and the urine EV fraction. Differential protein profiling of trapped EVs from stimulated versus non-stimulated platelets also demonstrated an easy access to differential expression in the EV-proteome.
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| 2. |
- 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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| 3. |
- Fehniger, Thomas E, et al.
(författare)
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Exploring the context of the lung proteome within the airway mucosa following allergen challenge.
- 2004
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 3:2, s. 307-320
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Forskningsöversikt (refereegranskat)abstract
- The lung proteome is a dynamic collection of specialized proteins related to pulmonary function. Many cells of different derivations, activation states, and levels of maturity contribute to the changing environment, which produces the lung proteome. Inflammatory cells reacting to environmental challenge, for example from allergens, produce and secrete proteins which have profound effects on both resident and nonresident cells located in airways, alveoli, and the vascular tree which provides blood cells to the parenchyma alveolar bed for gas exchange. In an experimental model of allergic airway inflammation, we have compared control and allergen challenged lung compartments to determine global protein expression patterns using 2D-gel electrophoresis and subsequent spot identification by MS/MS mass spectrometry. We have then specifically isolated the epithelial mucosal layer, which lines conducting airways, from control and allergen challenged lungs, using laser capture technology and performed proteome identification on these selected cell samples. A central component of our investigations has been to contextually relate the histological features of the dynamic pulmonary environment to the changes in protein expression observed following challenge. Our results provide new information of the complexity of the submucosa/epithelium interface and the mechanisms behind the transformation of airway epithelium from normal steady states to functionally activated states.
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| 4. |
- Golden, Gregory J, et al.
(författare)
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Endothelial Heparan Sulfate Mediates Hepatic Neutrophil Trafficking and Injury during Staphylococcus aureus Sepsis
- 2021
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Ingår i: mBio. - 2161-2129. ; 12:5
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Tidskriftsartikel (refereegranskat)abstract
- Hepatic failure is an important risk factor for poor outcome in septic patients. Using a chemical tagging workflow and high-resolution mass spectrometry, we demonstrate that rapid proteome remodeling of the vascular surfaces precedes hepatic damage in a murine model of Staphylococcus aureus sepsis. These early changes include vascular deposition of neutrophil-derived proteins, shedding of vascular receptors, and altered levels of heparin/heparan sulfate-binding factors. Modification of endothelial heparan sulfate, a major component of the vascular glycocalyx, diminishes neutrophil trafficking to the liver and reduces hepatic coagulopathy and organ damage during the systemic inflammatory response to infection. Modifying endothelial heparan sulfate likewise reduces neutrophil trafficking in sterile hepatic injury, reflecting a more general role of heparan sulfate contribution to the modulation of leukocyte behavior during inflammation. IMPORTANCE Vascular glycocalyx remodeling is critical to sepsis pathology, but the glycocalyx components that contribute to this process remain poorly characterized. This article shows that during Staphylococcus aureus sepsis, the liver vascular glycocalyx undergoes dramatic changes in protein composition associated with neutrophilic activity and heparin/heparan sulfate binding, all before organ damage is detectable by standard circulating liver damage markers or histology. Targeted manipulation of endothelial heparan sulfate modulates S. aureus sepsis-induced hepatotoxicity by controlling the magnitude of neutrophilic infiltration into the liver in both nonsterile and sterile injury. These data identify an important vascular glycocalyx component that impacts hepatic failure during nonsterile and sterile injury.
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| 5. |
- Gómez Toledo, Alejandro, et al.
(författare)
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A Systems View of the Heparan Sulfate Interactome
- 2021
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Ingår i: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. - : SAGE Publications. - 0022-1554. ; 69:2, s. 105-119
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate proteoglycans consist of a small family of proteins decorated with one or more covalently attached heparan sulfate glycosaminoglycan chains. These chains have intricate structural patterns based on the position of sulfate groups and uronic acid epimers, which dictate their ability to engage a large repertoire of heparan sulfate-binding proteins, including extracellular matrix proteins, growth factors and morphogens, cytokines and chemokines, apolipoproteins and lipases, adhesion and growth factor receptors, and components of the complement and coagulation system. This review highlights recent progress in the characterization of the so-called "heparan sulfate interactome," with a major focus on systems-wide strategies as a tool for discovery and characterization of this subproteome. In addition, we compiled all heparan sulfate-binding proteins reported in the literature to date and grouped them into a few major functional classes by applying a networking approach.
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| 6. |
- Hallgren, Oskar, et al.
(författare)
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Splicosomal and serine and arginine-rich splicing factors as targets for TGF-β
- 2012
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Ingår i: Fibrogenesis & tissue repair. - : Springer Science and Business Media LLC. - 1755-1536. ; 5:1, s. 6-6
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Transforming growth factor-β1 (TGF-β1) is a potent regulator of cell growth and differentiation. TGF-β1 has been shown to be a key player in tissue remodeling processes in a number of disease states by inducing expression of extracellular matrix proteins. In this study a quantitative proteomic analysis was undertaken to investigate if TGF-β1 contributes to tissue remodeling by mediating mRNA splicing and production of alternative isoforms of proteins.METHODOLOGY/PRINCIPAL FINDINGS: The expression of proteins involved in mRNA splicing from TGF-β1-stimulated lung fibroblasts was compared to non-stimulated cells by employing isotope coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry. A total of 1733 proteins were identified and quantified with a relative standard deviation of 11% +/- 8 from enriched nuclear fractions. Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection. In addition, TGF-β1 was observed to alter the relative expression of splicing proteins that may be important for alternative splicing of fibronectin. Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin. The induction of SRp20 was further confirmed by western blot and immunofluorescence.CONCLUSIONS: The results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts. This may have an impact on the production of alternative isoforms of matrix proteins and can therefore be an important factor in tissue remodeling and disease progression.
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| 7. |
- Hasan, Mahmudul, et al.
(författare)
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The structure of human dermatan sulfate epimerase 1 emphasizes the importance of C5-epimerization of glucuronic acid in higher organisms
- 2021
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 12:5, s. 1869-1885
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Tidskriftsartikel (refereegranskat)abstract
- Dermatan sulfate epimerase 1 (DS-epi1, EC 5.1.3.19) catalyzes the conversion of d-glucuronic acid to l-iduronic acid on the polymer level, a key step in the biosynthesis of the glycosaminoglycan dermatan sulfate. Here, we present the first crystal structure of the catalytic domains of DS-epi1, solved at 2.4 Å resolution, as well as a model of the full-length luminal protein obtained by a combination of macromolecular crystallography and targeted cross-linking mass spectrometry. Based on docking studies and molecular dynamics simulations of the protein structure and a chondroitin substrate, we suggest a novel mechanism of DS-epi1, involving a His/double-Tyr motif. Our work uncovers detailed information about the domain architecture, active site, metal-coordinating center and pattern of N-glycosylation of the protein. Additionally, the structure of DS-epi1 reveals a high structural similarity to proteins from several families of bacterial polysaccharide lyases. DS-epi1 is of great importance in a range of diseases, and the structure provides a necessary starting point for design of active site inhibitors.
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| 8. |
- Jassinskaja, Maria, et al.
(författare)
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Comprehensive Proteomic Characterization of Ontogenic Changes in Hematopoietic Stem and Progenitor Cells
- 2017
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 21:11, s. 3285-3297
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Tidskriftsartikel (refereegranskat)abstract
- Hematopoietic stem and progenitor cells (HSPCs) in the fetus and adult possess distinct molecular landscapes that regulate cell fate and change their susceptibility to initiation and progression of hematopoietic malignancies. Here, we applied in-depth quantitative proteomics to comprehensively describe and compare the proteome of fetal and adult HSPCs. Our data uncover a striking difference in complexity of the cellular proteomes, with more diverse adult-specific HSPC proteomic signatures. The differential protein content in fetal and adult HSPCs indicate distinct metabolic profiles and protein complex stoichiometries. Additionally, adult characteristics include an arsenal of proteins linked to viral and bacterial defense, as well as protection against ROS-induced protein oxidation. Further analyses show that interferon α as well as Neutrophil elastase, has distinct functional effects in fetal and adult HSPCs. This study provides a rich resource aimed toward an enhanced mechanistic understanding of normal and malignant hematopoiesis during fetal and adult life. Jassinskaja et al. describe the proteomic makeup of fetal and adult hematopoietic stem and progenitor cells. They uncover differences in protein complex stoichiometry, defense against pathogens and ROS, and show that IFN-α has distinct functional effects on fetal and adult HSPCs.
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| 9. |
- Karlsson, Christofer, et al.
(författare)
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Analysis of bacterial surface interactions with mass spectrometry-based proteomics
- 2017
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745. ; 1535, s. 17-24
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Bokkapitel (refereegranskat)abstract
- Host–pathogen protein–protein interaction networks are highly complex and dynamic. In this experimental protocol we describe a method to isolate host proteins attached to the bacterial surface followed by quantitative mass spectrometry based proteomics analysis. This technique provides an overview of the host–pathogen interaction network, which can be used to guide directed perturbations of the system, and to select target of specific interest for further studies.
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| 10. |
- Larsen, Kristoffer, et al.
(författare)
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Antiproliferative heparan sulfate inhibiting hyaluronan and transforming growth factor-β expression in human lung fibroblast cells
- 2005
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Ingår i: Clinical Proteomics. - 1559-0275. ; 1:3-4, s. 271-284
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this study was to examine the effects of heparan sulfate (HS) on factors involved in the remodeling of connective tissue observed in patients with fibrotic respiratory disorders such as asthma. A suitable working model is to stimulate human fetal lung fibroblasts in vitro with structurally different forms of HS. Highly sulfated and iduronic acid (IdoUA)-rich HS specifically decreased cell proliferaton, production of jyaluronan (HA), transforming growth factor (TGF)-β1, and TFF-β-induced α-smooth muscle actin but did not affect the overall proteoglycan production in the cells. These repressed factors are suggested to play a critical role in the early stages of remodeling and myofibroblast activation. Low sulfated and IdoUA-poor HS did not display any effects on these factors. Furthermore, analysis of the protein expression pattern by two-dimensional gel electrophoresis revealed a 70% increased expression of annexin II, which has previously been shown to have a high affinity for both heparin and HS. Heat-shock protein 27 and arsenite translocating factor, both involved in actin organization and polymerization, were also increased in the HS-stimulated cells. Thus, the reduced expression of HA and TGF-β1, both important in the development of fibrosis, seems to be mediated by pecific changes in protein expression of the fibroblast. The observed inhibition of cell proliferation, HA, and TGF-β1 allows speculation of highly sulfated HS as a antifibrotic candidate in the early stage of remodeling.
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