| 1. |
- Aevarsson, Arnthór, et al.
(författare)
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Going to extremes - a metagenomic journey into the dark matter of life
- 2021
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968. ; 368:12
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Forskningsöversikt (refereegranskat)abstract
- The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.
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| 2. |
- Abdillahi, Suado M., et al.
(författare)
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Collagen VI Contains Multiple Host Defense Peptides with Potent In Vivo Activity
- 2018
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Ingår i: Journal of Immunology. - : AMER ASSOC IMMUNOLOGISTS. - 0022-1767 .- 1550-6606. ; 201:3, s. 1007-1020
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Tidskriftsartikel (refereegranskat)abstract
- Collagen VI is a ubiquitous extracellular matrix component that forms extensive microfibrillar networks in most connective tissues. In this study, we describe for the first time, to our knowledge, that the collagen VI von Willebrand factor type A like domains exhibit a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria in human skin infections in vivo. In silico sequence and structural analysis of VWA domains revealed that they contain cationic and amphipathic peptide sequence motifs, which might explain the antimicrobial nature of collagen VI. In vitro and in vivo studies show that these peptides exhibited significant antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa through membrane disruption. Our findings shed new light on the role of collagen VI derived peptides in innate host defense and provide templates for development of peptide-based antibacterial therapies.
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| 3. |
- Anderson, Lissa C., et al.
(författare)
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Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)
- 2017
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Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305. ; 28:9, s. 1796-1804
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Tidskriftsartikel (refereegranskat)abstract
- Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a ~45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM. [Figure not available: see fulltext.].
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| 4. |
- Chen, Yihong, et al.
(författare)
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Identification of an osteopontin-derived peptide that binds neuropilin-1 and activates vascular repair responses and angiogenesis
- 2024
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Ingår i: Pharmacological Research. - 1096-1186. ; 205
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Tidskriftsartikel (refereegranskat)abstract
- The osteopontin-derived peptide FOL-005 stimulates hair growth. Using ligand-receptor glyco-capture technology we identified neuropilin-1 (NRP-1), a known co-receptor for vascular endothelial growth factor (VEGF) receptors, as the most probable receptor for FOL-005 and the more stable analogue FOL-026. X-ray diffraction and microscale thermophoresis analysis revealed that FOL-026 shares binding site with VEGF in the NRP-1 b1-subdomain. Stimulation of human umbilical vein endothelial cells with FOL-026 resulted in phosphorylation of VEGFR-2, ERK1/2 and AKT, increased cell growth and migration, stimulation of endothelial tube formation and inhibition of apoptosis in vitro. FOL-026 also promoted angiogenesis in vivo as assessed by subcutaneous Matrigel plug and hind limb ischemia models. NRP-1 knock-down or treatment of NRP-1 antagonist EG00229 blocked the stimulatory effects of FOL-026 on endothelial cells. Exposure of human coronary artery smooth muscle cells to FOL-026 stimulated cell growth, migration, inhibited apoptosis, and induced VEGF gene expression and VEGFR-2/AKT phosphorylation by an NRP-1-dependent mechanism. RNA sequencing showed that FOL-026 activated pathways involved in tissue repair. These findings identify NRP-1 as the receptor for FOL-026 and show that its biological effects mimic that of growth factors binding to the VEGF receptor family. They also suggest that FOL-026 may have therapeutical potential in conditions that require vascular repair and/or enhanced angiogenesis.
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| 5. |
- Dahlén, Eva, et al.
(författare)
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Development of interleukin-1 receptor antagonist mutants with enhanced antagonistic activity in vitro and improved therapeutic efficacy in collagen-induced arthritis.
- 2008
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Ingår i: Journal of Immunotoxicology. - : Informa UK Limited. - 1547-6901 .- 1547-691X. ; 5:2, s. 189-199
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Tidskriftsartikel (refereegranskat)abstract
- Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring inhibitor of the pro-inflammatory interleukin-1-mediated activation of the interleukin-1 receptor (IL-1R). Although wild-type IL-1Ra is used for treatment of inflammatory diseases, its effect is moderate and/or short-lived. The objective of this study was to generate IL-1Ra mutants with enhanced antagonistic activity for potential therapeutic use. Using a directed evolution approach in which libraries of IL-1Ra gene mutants were generated and screened in functional assays, mutants with desired properties were identified. Initially, diversity was introduced into the IL-1Ra using random mutagenesis. Mutations resulting in enhanced antagonistic activity were identified by screening in a reporter cell assay. To further enhance the antagonistic activity, selected mutations were recombined using the DNA recombination technology Fragment-INduced Diversity (FIND). Following three rounds of FIND recombination, several mutants with up to nine times enhanced antagonistic activity (mean IC50 +/- SEM value: 0.78 +/- 0.050 vs. 6.8 +/- 1.1 ng/ml for mutant and wild-type, respectively) were identified. Sequence analysis identified the mutations D47N, E52R and E90Y as being most important for this effect, however, the mutations P38Y, H54R, Q129L and M136N further enhanced the antagonistic function. Analysis of identified mutations in protein models based on the crystal structure of the IL-1Ra/IL-1R complex suggested that mutations found to enhance the antagonistic activity had a stabilizing effect on the IL-1Ra mutants or increased the affinity for the IL-1R. Finally, the therapeutic effect of one mutant was compared to that of wild-type IL-1Ra in collagen-induced arthritis in mice. Indeed, the enhanced antagonistic effect of the mutants observed in vitro was also seen in vivo. In conclusion, these results demonstrate that directed evolution of IL-1Ra is an effective means of generating highly potent therapeutic proteins.
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| 6. |
- Fritzson, Ingela, et al.
(författare)
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Inhibition of Human DHODH by 4-Hydroxycoumarins, Fenamic Acids, and N-(Alkylcarbonyl)anthranilic Acids Identified by Structure-Guided Fragment Selection
- 2010
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Ingår i: ChemMedChem. - : Wiley. - 1860-7187 .- 1860-7179. ; 5:4, s. 608-617
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Tidskriftsartikel (refereegranskat)abstract
- A strategy that combines virtual screening and structureguided selection of fragments was used to identify three unexplored classes of human DHODH inhibitor compounds: 4-hydroxycoumarins, fenamic acids, and N-(alkylcarbonyl)anthranilic acids. Structure-guided selection of fragments targeting the inner subsite of the DHODH ubiquinone binding site made these findings possible with screening of fewer than 300 fragments in a DHODH assay. Fragments from the three inhibitor classes identified were subsequently chemically expanded to target an additional subsite of hydrophobic character. All three classes were found to exhibit distinct structure–activity relationships upon expansion. The novel N-(alkylcarbonyl anthranilic acid class shows the most promising potency against human DHODH, with IC50 values in the low nanomolar range. The structure of human DHODH in complex with an inhibitor of this class is presented.
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| 7. |
- Gustafsson, Erika, et al.
(författare)
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Directed evolution of chemotaxis inhibitory protein of Staphylococcus aureus generates biologically functional variants with reduced interaction with human antibodies
- 2010
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 23:2, s. 91-101
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Tidskriftsartikel (refereegranskat)abstract
- Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a protein that binds and blocks the C5a receptor (C5aR) and formylated peptide receptor, thereby inhibiting the immune cell recruitment associated with inflammation. If CHIPS was less reactive with existing human antibodies, it would be a promising anti-inflammatory drug candidate. Therefore, we applied directed evolution and computational/rational design to the CHIPS gene in order to generate new CHIPS variants displaying lower interaction with human IgG, yet retaining biological function. The optimization was performed in four rounds: one round of random mutagenesis to add diversity into the CHIPS gene and three rounds of DNA recombination by Fragment INduced Diversity (FIND((R))). Every round was screened by phage selection and/or ELISA for decreased interaction with human IgG and retained C5aR binding. The mean binding of human anti-CHIPS IgG decreased with every round of evolution. For further optimization, new amino acid substitutions were introduced by rational design, based on the mutations identified during directed evolution. Finally, seven CHIPS variants with low interaction with human IgG and retained C5aR blocking capacity could be identified.
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| 8. |
- Gustafsson, Erika, et al.
(författare)
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Identification of conformational epitopes for human IgG on chemotaxis inhibitory protein of Staphylococcus aureus
- 2009
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Ingår i: BMC Immunology. - : Springer Science and Business Media LLC. - 1471-2172. ; 10:13
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Tidskriftsartikel (refereegranskat)abstract
- Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31-121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusions Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.
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| 9. |
- Hassan, Mujtaba, et al.
(författare)
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Benzimidazole–galactosides bind selectively to the Galectin-8 N-Terminal domain : Structure-based design and optimisation
- 2021
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Ingår i: European Journal of Medicinal Chemistry. - : Elsevier BV. - 0223-5234. ; 223
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Tidskriftsartikel (refereegranskat)abstract
- We have obtained the X-ray crystal structure of the galectin-8 N-terminal domain (galectin-8N) with a previously reported quinoline–galactoside ligand at a resolution of 1.6 Å. Based on this X-ray structure, a collection of galactosides derivatised at O3 with triazole, benzimidazole, benzothiazole, and benzoxazole moieties were designed and synthesised. This led to the discovery of a 3-O-(N-methylbenzimidazolylmethyl)–galactoside with a Kd of 1.8 μM for galectin-8N, the most potent selective synthetic galectin-8N ligand to date. Molecular dynamics simulations showed that benzimidazole–galactoside derivatives bind the non-conserved amino acid Gln47, accounting for the higher selectivity for galectin-8N. Galectin-8 is a carbohydrate-binding protein that plays a key role in pathological lymphangiogenesis, modulation of the immune system, and autophagy. Thus, the benzimidazole-derivatised galactosides represent promising compounds for studies of the pathological implications of galectin-8, as well as a starting point for the development of anti-tumour and anti-inflammatory therapeutics targeting galectin-8.
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| 10. |
- Kasetty, Gopinath, et al.
(författare)
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The C-Terminal Sequence of Several Human Serine Proteases Encodes Host Defense Functions.
- 2011
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Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 3:5, s. 471-482
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Tidskriftsartikel (refereegranskat)abstract
- Serine proteases of the S1 family have maintained a common structure over an evolutionary span of more than one billion years, and evolved a variety of substrate specificities and diverse biological roles, involving digestion and degradation, blood clotting, fibrinolysis and epithelial homeostasis. We here show that a wide range of C-terminal peptide sequences of serine proteases, particularly from the coagulation and kallikrein systems, share characteristics common with classical antimicrobial peptides of innate immunity. Under physiological conditions, these peptides exert antimicrobial effects as well as immunomodulatory functions by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, selected peptides are protective against lipopolysaccharide-induced shock. Moreover, these S1-derived host defense peptides exhibit helical structures upon binding to lipopolysaccharide and also permeabilize liposomes. The results uncover new and fundamental aspects on host defense functions of serine proteases present particularly in blood and epithelia, and provide tools for the identification of host defense molecules of therapeutic interest.
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