| 1. |
- Al-Eryani, Yusra, et al.
(author)
-
Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry
- 2016
-
In: Proteins: Structure, Function and Genetics. - : Wiley. - 0887-3585. ; 84:9, s. 1234-1245
-
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.
|
|
| 2. |
- Ampah-Korsah, Henry, et al.
(author)
-
The aquaporin splice variant NbXIP1;1α is permeable to boric acid and is Phosphorylated in the N-terminal domain
- 2016
-
In: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 7:JUNE2016
-
Journal article (peer-reviewed)abstract
- Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.
|
|
| 3. |
- Awad, Wael, et al.
(author)
-
Structural and Biophysical Characterization of Human EXTL3 : Domain Organization, Glycosylation, and Solution Structure
- 2018
-
In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 57:7, s. 1166-1177
-
Journal article (peer-reviewed)abstract
- Heparan sulfate proteoglycans are proteins substituted with one or more heparan sulfate (HS) polysaccharides, found in abundance at cell surfaces. HS chains influence the activity of many biologically important molecules involved in cellular communication and signaling. The exostosin (EXT) proteins are glycosyltransferases in the Golgi apparatus that assemble HS chains on HSPGs. The EXTL3 enzyme mainly works as an initiator in HS biosynthesis. In this work, human lumenal N-glycosylated EXTL3 (EXTL3ΔN) was cloned, expressed in human embryonic kidney cells, and purified. Various biophysical and biochemical approaches were then employed to elucidate the N-glycosylation sites and the function of their attached N-glycans. Furthermore, the stability and conformation of the purified EXTL3ΔN protein in solution have been analyzed. Our data show that EXTL3ΔN has N-glycans at least at two positions, Asn290 and Asn592, which seem to be critical for proper protein folding and/or release. EXTL3ΔN is quite stable, as high temperature (∼59 °C) was required for denaturation. Deconvolution of the EXTL3ΔN far-UV CD spectrum revealed a substantial fraction of β sheets (25%) with a minor proportion of α-helices (14%) in the secondary structure. Solution small-angle X-ray scattering and dynamic light scattering revealed an extended structure suggestive of a dimeric arrangement and consisting of two distinct regions, narrow and broad, respectively. This is consistent with bioinformatics analyses suggesting a 3-domain structure with two glycosyltransferase domains and a coiled-coil domain.
|
|
| 4. |
- Hsueh, Ming Feng, et al.
(author)
-
Elucidating the Molecular Composition of Cartilage by Proteomics
- 2016
-
In: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 15:2, s. 374-388
-
Journal article (peer-reviewed)abstract
- Articular cartilage consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. Most prior studies defining the zonal distribution of cartilage have extracted proteins with guanidine-HCl. However, an unextracted collagen-rich residual is left after extraction. In addition, the high abundance of anionic polysaccharide molecules extracted from cartilage adversely affects the chromatographic separation. In this study, we established a method for removing chondrocytes from cartilage sections with minimal extracellular matrix protein loss. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. Almost four-times more collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. These different methods were used to extract cartilage sections from different cartilage layers (superficial, intermediate, and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic), and the extractions were evaluated by quantitative and qualitative proteomic analyses. The results of this study led to the identifications of the potential biomarkers of osteoarthritis (OA), OA progression, and the joint specific biomarkers.
|
|
| 5. |
|
|
| 6. |
- Khan, Ashhar, et al.
(author)
-
Adsorption of unfolded Cu/Zn superoxide dismutase onto hydrophobic surfaces catalyzes its formation of amyloid fibrils
- 2019
-
In: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 32:2, s. 77-85
-
Journal article (peer-reviewed)abstract
- Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area. We compared fibril formation in hydrophilic and hydrophobic sample wells, as a function of denaturant concentration and extraneous hydrophobic surface area. In the presence of hydrophobic surfaces, SOD1 unfolding promotes fibril nucleation. By contrast, in the presence of hydrophilic surfaces, increasing denaturant concentration retards the onset of fibril formation. We conclude that the mechanism of fibril formation depends on the surrounding surfaces and that the nucleating species might correspond to different conformational states of SOD1 depending on the nature of these surfaces.
|
|
| 7. |
- Khan, Ashhar, et al.
(author)
-
Cu/Zn Superoxide Dismutase Forms Amyloid Fibrils under Near-Physiological Quiescent Conditions : The Roles of Disulfide Bonds and Effects of Denaturant
- 2017
-
In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 8:9, s. 2019-2026
-
Journal article (peer-reviewed)abstract
- Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth.
|
|
| 8. |
- Kjellström, Barbro, et al.
(author)
-
Sex-specific differences and survival in patients with idiopathic pulmonary arterial hypertension 2008-2016
- 2019
-
In: ERJ Open Research. - Lausanne, Switzerland : European Respiratory Society (ERS). - 2312-0541. ; 5:3
-
Journal article (peer-reviewed)abstract
- Background: Women with idiopathic pulmonary arterial hypertension (IPAH) have been found to have a worse haemodynamic status at diagnosis, but better survival than men. Over the past decade, demographics have changed and new treatments have become available. The objective of this study was to investigate sex differences in an incident IPAH population diagnosed between 2008 and 2016.Methods: Differences in clinical characteristics of patients included in the Swedish Pulmonary Arterial Hypertension Register (SPAHR) were analysed at the time of diagnosis. Survival by sex was investigated using Cox proportional hazard regression and Kaplan-Meier curves.Results: The study included 271 patients diagnosed with IPAH, median age was 68 (1st-3rd quartiles 54-74) years and 56% were women. At diagnosis, women were younger, had lower pulmonary vascular resistance and fewer comorbidities and more often received a combination of PAH-targeted therapies than men. Men had worse survival rates than women (hazard ratio 1.49; CI 1.02-2.18; p=0.038), but this difference did not remain after adjustment for age (hazard ratio 1.30; CI 0.89-1.90; p=0.178).Conclusions: Men with incident IPAH have worse crude survival than women. This is due to women being younger with a less pronounced comorbidity burden than men at the time of diagnosis.
|
|
| 9. |
- Linares-Pastén, Javier A., et al.
(author)
-
Three-dimensional structures and functional studies of two GH43 arabinofuranosidases from Weissella sp. strain 142 and Lactobacillus brevis
- 2017
-
In: The FEBS Journal. - : Wiley. - 1742-464X. ; 284:13, s. 2019-2036
-
Journal article (peer-reviewed)abstract
- Arabinofuranosidases degrade arabinose-containing oligo and polysaccharides, releasing l-arabinose, which is a potentially useful sugar, shown to reduce glycemic response under certain conditions. Arabinofuranosidases (Arafs) are frequently found in GH43, one of the most common GH-families encoded in genomes in gut microbiota, and hence it is of interest to increase understanding of the function of these enzymes in species occurring in the gut. Here we have produced, characterized and solved the three-dimensional structures, at 1.9 and 2.0 Å resolution respectively, of two homologous GH43 enzymes, classified under subfamily 26, from Lactobacillus brevis DSM1269 (LbAraf43) and Weissella strain 142 (WAraf43), respectively. The enzymes, with 74% sequence identity to each other, are composed of a single catalytic module with a β-propeller structure typical of GH43, and an active-site pocket with three identifiable subsites (−1, +1, and +2). According to size exclusion chromatography, native WAraf43 is a dimer, while LbAraf43 is a tetramer in solution. Both of them show activity with similar catalytic efficiency on 1,5-α-l-arabinooligosaccharides with a degree of polymerization (DP) of 2–3. Activity is restricted to substrates of low DP, and the reason for this is believed to be an extended loop at the entrance to the active site, creating interactions in the +2 subsite. Database: Structural data are available in the PDB under the accession numbers 5M8B (LbAraf43) and 5M8E (WAraf43).
|
|
| 10. |
- Mehmeti, Meliha, et al.
(author)
-
Wnt5a is a TLR2/4-ligand that induces tolerance in human myeloid cells
- 2019
-
In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 2
-
Journal article (peer-reviewed)abstract
- Innate immune responses are rapid, dynamic and highly regulated to avoid overt reactions. This regulation is executed by innate immune tolerance mechanisms that remain obscure. Wnt5a is a signalling protein mainly involved in developmental processes and cancer. The effect of Wnt5a on inflammatory myeloid cells is controversial. Here, we combine primary cell cultures, in vitro binding studies, mass spectrometry and Drosophila protein modelling to show that Wnt5a is a direct ligand of toll-like receptor (TLR) 2 and 4. The binding promotes a MyD88-non-canonical nuclear factor of kappa B (NFκB) and AP-1 signalling cascade, with contradictory profiles in mouse (pro-inflammatory) and human (anti-inflammatory) myeloid immune cells. These data reveal that the true nature of Wnt5a in inflammatory cells, is to regulate TLR signals, and in human myeloid cells it acts as an endogenous, tolerance-associated molecular pattern (TAMP), inducing IL-10 and innate immune tolerance.
|
|
| 11. |
- Möller, Mattias, et al.
(author)
-
Disruption of a GATA1-binding motif upstream of XG/PBDX abolishes Xga expression and resolves the Xg blood group system
- 2018
-
In: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 132:3, s. 334-338
-
Journal article (peer-reviewed)abstract
- The Xga blood group is differentially expressed on erythrocytes from men and women. The underlying gene, PBDX, was identified in 1994, but the molecular background for Xga expression remains undefined. This gene, now designated XG, partly resides in pseudoautosomal region 1 and encodes a protein of unknown function from the X chromosome. By comparing calculated Xga allele frequencies in different populations with 2612 genetic variants in the XG region, rs311103 showed the strongest correlation to the expected distribution. The same single-nucleotide polymorphism (SNP) had the most significant impact on XG transcript levels in whole blood (P 5 2.0 3 10222). The minor allele, rs311103C, disrupts a GATA-binding motif 3.7 kb upstream of the transcription start point. This silences erythroid XG messenger RNA expression and causes the Xg(a2) phenotype, a finding corroborated by SNP genotyping in 158 blood donors. Binding of GATA1 to biotinylated oligonucleotide probes with rs311103G but not rs311103C was observed by electrophoretic mobility shift assay and proven by mass spectrometry. Finally, a luciferase reporter assay indicated this GATA motif to be active for rs311103G but not rs311103C in HEL cells. By using an integrated bioinformatic and molecular biological approach, we elucidated the underlying genetic basis for the last unresolved blood group system and made Xga genotyping possible.
|
|
| 12. |
|
|
| 13. |
- Saravanan, Rathi, et al.
(author)
-
Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo
- 2017
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
-
Journal article (peer-reviewed)abstract
- The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.
|
|
| 14. |
- Saravanan, Rathi, et al.
(author)
-
Structural basis for endotoxin neutralisation and anti-inflammatory activity of thrombin-derived C-terminal peptides
- 2018
-
In: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 9
-
Journal article (peer-reviewed)abstract
- Thrombin-derived C-terminal peptides (TCPs) of about 2 kDa are present in wounds, where they exert anti-endotoxic functions. Employing a combination of nuclear magnetic resonance spectroscopy (NMR), biophysical, mass spectrometry and cellular studies combined with in silico multiscale modelling, we here determine the bound conformation of HVF18 (HVFRLKKWIQKVIDQFGE), a TCP generated by neutrophil elastase, in complex with bacterial lipopolysaccharide (LPS) and define a previously undisclosed interaction between TCPs and human CD14. Further, we show that TCPs bind to the LPS-binding hydrophobic pocket of CD14 and identify the peptide region crucial for TCP interaction with LPS and CD14. Taken together, our results demonstrate the role of structural transitions in LPS complex formation and CD14 interaction, providing a molecular explanation for the previously observed therapeutic effects of TCPs in experimental models of bacterial sepsis and endotoxin shock.
|
|
| 15. |
- Sime, Wondossen, et al.
(author)
-
BAP1 induces cell death via interaction with 14-3-3 in neuroblastoma article
- 2018
-
In: Cell Death and Disease. - : Springer Science and Business Media LLC. - 2041-4889. ; 9:5
-
Journal article (peer-reviewed)abstract
- BRCA1-associated protein 1 (BAP1) is a nuclear deubiquitinating enzyme that is associated with multiprotein complexes that regulate key cellular pathways, including cell cycle, cellular differentiation, cell death, and the DNA damage response. In this study, we found that the reduced expression of BAP1 pro6motes the survival of neuroblastoma cells, and restoring the levels of BAP1 in these cells facilitated a delay in S and G2/M phase of the cell cycle, as well as cell apoptosis. The mechanism that BAP1 induces cell death is mediated via an interaction with 14-3-3 protein. The association between BAP1 and 14-3-3 protein releases the apoptotic inducer protein Bax from 14-3-3 and promotes cell death through the intrinsic apoptosis pathway. Xenograft studies confirmed that the expression of BAP1 reduces tumor growth and progression in vivo by lowering the levels of pro-survival factors such as Bcl-2, which in turn diminish the survival potential of the tumor cells. Patient data analyses confirmed the finding that the high-BAP1 mRNA expression correlates with a better clinical outcome. In summary, our study uncovers a new mechanism for BAP1 in the regulation of cell apoptosis in neuroblastoma cells.
|
|
| 16. |
- Sonesson, Andreas, et al.
(author)
-
Identification of bacterial biofilm and the Staphylococcus aureus derived protease, staphopain, on the skin surface of patients with atopic dermatitis
- 2017
-
In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
-
Journal article (peer-reviewed)abstract
- Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by an impaired epidermal barrier, dysregulation of innate and adaptive immunity, and a high susceptibility to bacterial colonization and infection. In the present study, bacterial biofilm was visualized by electron microscopy at the surface of AD skin. Correspondingly, Staphylococcus aureus (S. aureus) isolates from lesional skin of patients with AD, produced a substantial amount of biofilm in vitro. S. aureus biofilms showed less susceptibility to killing by the antimicrobial peptide LL-37 when compared with results obtained using planktonic cells. Confocal microscopy analysis showed that LL-37 binds to the S. aureus biofilms. Immuno-gold staining of S. aureus biofilm of AD skin detected the S. aureus derived protease staphopain adjacent to the bacteria. In vitro, staphopain B degraded LL-37 into shorter peptide fragments. Further, LL-37 significantly inhibited S. aureus biofilm formation, but no such effects were observed for the degradation products. The data presented here provide novel information on staphopains present in S. aureus biofilms in vivo, and illustrate the complex interplay between biofilm and LL-37 in skin of AD patients, possibly leading to a disturbed host defense, which facilitates bacterial persistence.
|
|
| 17. |
|
|
| 18. |
- van Der Plas, Mariena, et al.
(author)
-
Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses
- 2016
-
In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
-
Journal article (peer-reviewed)abstract
- Pseudomonas aeruginosa is an opportunistic pathogen known for its immune evasive abilities amongst others by degradation of a large variety of host proteins. Here we show that digestion of thrombin by P. aeruginosa elastase leads to the release of the C-terminal thrombin-derived peptide FYT21, which inhibits pro-inflammatory responses to several pathogen-associated molecular patterns in vitro and in vivo by preventing toll-like receptor dimerization and subsequent activation of down-stream signalling pathways. Thus, P. aeruginosa 'hijacks' an endogenous anti-inflammatory peptide-based mechanism, thereby enabling modulation and circumvention of host responses.
|
|
| 19. |
- Westman, Julia S., et al.
(author)
-
Allele-selective RUNX1 binding regulates P1 blood group status by transcriptional control of A4GALT
- 2018
-
In: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 131:14, s. 1611-1616
-
Journal article (peer-reviewed)abstract
- P1 and Pk are glycosphingolipid antigens synthesized by the A4GALT-encoded α1,4-galactosyltransferase, using paragloboside and lactosylceramide as acceptor substrates, respectively. In addition to the compatibility aspects of these histo-blood group molecules, both constitute receptors for multiple microbes and toxins. Presence or absence of P1 antigen on erythrocytes determines the common P1 (P1+Pk+) and P2 (P1-Pk+weak) phenotypes. A4GALT transcript levels are higher in P1 individuals and SNPs in non-coding regions of A4GALT, particularly rs5751348, correlate with P1/P2 status. Despite these recent findings, the molecular mechanism underlying these phenotypes remains elusive. The In(Lu) phenotype is caused by KLF1 haploinsufficiency and shows decreased P1 levels on erythrocytes. We therefore hypothesized KLF1 to regulate A4GALT expression. Intriguingly, P1 -specific sequences including rs5751348 revealed potential binding sites for several hematopoietic transcription factors, including KLF1. However, KLF1 binding did not explain P1-specific EMSA shifts and siRNA silencing of KLF1 did not affect A4GALT transcript levels. Instead, protein pull-down experiments using P1 but not P2 oligonucleotide probes identified RUNX1 by mass spectrometry. Furthermore, RUNX1 binds P1 alleles selectively and knockdown of RUNX1 significantly decreased A4GALT transcription. These data indicate that RUNX1 regulates A4GALT and thereby the expression of clinically important glycosphingolipids implicated in blood-group incompatibility and host-pathogen interactions.
|
|
