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| 2. |
- Hafstrand, Ida, et al.
(author)
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Successive crystal structure snapshots suggest the basis for MHC class I peptide loading and editing by tapasin
- 2019
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In: Proceedings of the National Academy of Sciences of the United States of America. - : NATL ACAD SCIENCES. - 0027-8424 .- 1091-6490. ; 116:11, s. 5055-5060
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Journal article (peer-reviewed)abstract
- MHC-I epitope presentation to CD8(+) T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptide-loading complex, the peptide editor tapasin is key to the selection of MHC-I-bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles.
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| 3. |
- Hjelm, Linnea C., et al.
(author)
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Lysis of Staphylococcal Cells by Modular Lysin Domains Linked via a n-covalent Barnase-Barstar Interaction Bridge
- 2019
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In: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 10
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Journal article (peer-reviewed)abstract
- Bacteriophage endolysins and bacterial exolysins are capable of enzymatic degradation of the cell wall peptidoglycan layer and thus show promise as a new class of antimicrobials. Both exolysins and endolysins often consist of different modules, which are responsible for enzymatic functions and cell wall binding, respectively. Individual modules from different endo- or exolysins with different binding and enzymatic activities, can via gene fusion technology be re-combined into novel variants for investigations of arrangements of potential clinical interest. The aim of this study was to investigate if separately produced cell wall binding and enzyme modules could be assembled into a functional lysin via a non-covalent affinity interaction bridge composed of the barnase ribonuclease from Bacillus amyloliquefaciens and its cognate inhibitor barstar, known to form a stable heterodimeric complex. In a proof-of-principle study, using surface plasmon resonance, flow cytometry and turbidity reduction assays, we show that separately produced modules of a lysin cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) from Staphylococcus aureus bacteriophage K endolysin (LysK) fused to barnase and a cell wall binding Src homology 3 domain (SH3b) from the S. simulans exolysin lysostaphin fused to barstar can be non-covalently assembled into a functional lysin showing both cell wall binding and staphylolytic activity. We hypothesize that the described principle for assembly of functional lysins from separate modules through appended hetero-dimerization domains has a potential for investigations of also other combinations of enzymatically active and cell wall binding domains for desired applications.
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| 5. |
- Lundqvist, Magnus, et al.
(author)
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Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion
- 2019
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In: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
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Journal article (peer-reviewed)abstract
- Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1-10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1-10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1-10 in analyses. The pre-maturated GFP 1-10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1-10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1-10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.
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| 7. |
- Norhammar, Anna, et al.
(author)
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Undetected Dysglycemia Is an Important Risk Factor for Two Common Diseases, Myocardial Infarction and Periodontitis : A Report From the PAROKRANK Study
- 2019
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In: Diabetes Care. - : NLM (Medline). - 0149-5992 .- 1935-5548. ; 42:8, s. 1504-1511
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Journal article (peer-reviewed)abstract
- OBJECTIVE: Information on the relationship among dysglycemia (prediabetes or diabetes), myocardial infarction (MI), and periodontitis (PD) is limited. This study tests the hypothesis that undetected dysglycemia is associated with both conditions. RESEARCH DESIGN AND METHODS: The PAROKRANK (Periodontitis and Its Relation to Coronary Artery Disease) study included 805 patients with a first MI and 805 matched control subjects. All participants without diabetes (91%) were examined with an oral glucose tolerance test. Abnormal glucose tolerance (AGT) (impaired glucose tolerance or diabetes) was categorized according to the World Health Organization. Periodontal status was categorized from dental X-rays as healthy (≥80% remaining alveolar bone height), moderate (79-66%), or severe (<66%) PD. Odds ratios (ORs) and 95% CIs were calculated by logistic regression and were adjusted for age, sex, smoking, education, marital status, and explored associated risks of dysglycemia to PD and MI, respectively. RESULTS: AGT was more common in patients than in control subjects (32% vs. 19%; P < 0.001) and was associated with MI (OR 2.03; 95% CI 1.58-2.60). Undetected diabetes was associated with severe PD (2.50; 1.36-4.63) and more strongly in patients (2.35; 1.15-4.80) than in control subjects (1.80; 0.48-6.78), but not when categorized as AGT (total cohort: 1.07; 0.67-1.72). Severe PD was most frequent in subjects with undetected diabetes, and reversely undetected diabetes was most frequent in patients with severe PD. CONCLUSIONS: In this large case-control study previously undetected dysglycemia was independently associated to both MI and severe PD. In principal, it doubled the risk of a first MI and of severe PD. This supports the hypothesis that dysglycemia drives two common diseases, MI and PD.
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| 8. |
- Ståhl, Stefan, et al.
(author)
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Affibody Molecules in Biotechnological and Medical Applications
- 2017
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In: Trends in Biotechnology. - : Elsevier. - 0167-7799 .- 1879-3096. ; 35:8, s. 691-712
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Research review (peer-reviewed)abstract
- Affibody molecules are small (6.5-kDa) affinity proteins based on a three-helix bundle domain framework. Since their introduction 20 years ago as an alternative to antibodies for biotechnological applications, the first therapeutic affibody molecules have now entered clinical development and more than 400 studies have been published in which affibody molecules have been developed and used in a variety of contexts. In this review, we focus primarily on efforts over the past 5 years to explore the potential of affibody molecules for medical applications in oncology, neurodegenerative, and inflammation disorders, including molecular imaging, receptor signal blocking, and delivery of toxic payloads. In addition, we describe recent examples of biotechnological applications, in which affibody molecules have been exploited as modular affinity fusion partners.
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| 9. |
- Subramanian, Karthik, et al.
(author)
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Pneumolysin binds to the mannose receptor C type 1 (MRC-1) leading to anti-inflammatory responses and enhanced pneumococcal survival
- 2019
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In: Nature Microbiology. - : Nature Publishing Group. - 2058-5276. ; 4:1, s. 62-70
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Journal article (peer-reviewed)abstract
- Streptococcus pneumoniae (the pneumococcus) is a major cause of mortality and morbidity globally, and the leading cause of death in children under 5 years old. The pneumococcal cytolysin pneumolysin (PLY) is a major virulence determinant known to induce pore-dependent pro-inflammatory responses. These inflammatory responses are driven by PLY-host cell membrane cholesterol interactions, but binding to a host cell receptor has not been previously demonstrated. Here, we discovered a receptor for PLY, whereby pro-inflammatory cytokine responses and Toll-like receptor signalling are inhibited following PLY binding to the mannose receptor C type 1 (MRC-1) in human dendritic cells and mouse alveolar macrophages. The cytokine suppressor SOCS1 is also upregulated. Moreover, PLY-MRC-1 interactions mediate pneumococcal internalization into non-lysosomal compartments and polarize naive T cells into an interferon-gamma(low), interleukin-4(high) and FoxP3(+) immunoregulatory phenotype. In mice, PLY-expressing pneumococci colocalize with MRC-1 in alveolar macrophages, induce lower pro-inflammatory cytokine responses and reduce neutrophil infiltration compared with a PLY mutant. In vivo, reduced bacterial loads occur in the airways of MRC-1-deficient mice and in mice in which MRC-1 is inhibited using blocking antibodies. In conclusion, we show that pneumococci use PLY-MRC-1 interactions to downregulate inflammation and enhance bacterial survival in the airways. These findings have important implications for future vaccine design.
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