| 1. |
- Hansen, Finja C., et al.
(författare)
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Differential Internalization of Thrombin-Derived Host Defense Peptides into Monocytes and Macrophages
- 2022
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Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 14:5, s. 418-432
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Tidskriftsartikel (refereegranskat)abstract
- Proteolytic cleavage of thrombin generates C-terminal host defense peptides exerting multiple immunomodulatory effects in response to bacterial stimuli. Previously, we reported that thrombin-derived C-terminal peptides (TCPs) are internalized in monocytes and macrophages in a time- and temperature-dependent manner. In this study, we investigated which endocytosis pathways are responsible for the internalization of TCPs. Using confocal microscopy and flow cytometry, we show that both clathrin-dependent and clathrin-independent pathways are involved in the internalization of the prototypic TCP GKY25 in RAW264.7 and human monocyte-derived M1 macrophages, whereas the uptake of GKY25 in monocytic THP-1 cells is mainly dynamin-dependent. Internalized GKY25 was transported to endosomes and finally lysosomes, where it remained detectable for up to 10 h. Comparison of GKY25 uptake with that of the natural occurring TCPs HVF18 and FYT21 indicates that the pathway of TCP endocytosis is not only cell type-dependent but also depends on the length and composition of the peptide as well as the presence of LPS and bacteria. Finally, using neutron reflectometry, we show that the observed differences between HVF18 and the other 2 TCPs may be explained partially by differences in membrane insertion. Taken together, we show that TCPs are differentially internalized into monocytes and macrophages.
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| 2. |
- Luchini, Alessandra, et al.
(författare)
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Structural model of tissue factor (TF) and TF-factor VIIa complex in a lipid membrane : A combined experimental and computational study
- 2022
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier. - 0021-9797 .- 1095-7103. ; 623, s. 294-305
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Tidskriftsartikel (refereegranskat)abstract
- Tissue factor (TF) is a membrane protein involved in blood coagulation. TF initiates a cascade of proteolytic reactions, ultimately leading to the formation of a blood clot. The first reaction consists of the binding of the coagulation factor VII and its conversion to the activated form, FVIIa. Here, we combined experimental, i.e. quartz crystal microbalance with dissipation monitoring and neutron reflectometry, and computational, i.e. molecular dynamics (MD) simulation, methods to derive a complete structural model of TF and TF/FVIIa complex in a lipid bilayer. This model shows that the TF transmembrane domain (TMD), and the flexible linker connecting the TMD to the extracellular domain (ECD), define the location of the ECD on the membrane surface. The average orientation of the ECD relative to the bilayer surface is slightly tilted towards the lipid headgroups, a conformation that we suggest is promoted by phosphatidylserine lipids, and favours the binding of FVIIa. On the other hand, the formation of the TF/FVIIa complex induces minor changes in the TF structure, and reduces the conformational freedom of both TF and FVIIA. Altogether we describe the protein-protein and protein-lipid interactions favouring blood coagulation, but also instrumental to the development of new drugs.
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| 3. |
- Nagy, Bela, et al.
(författare)
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Structure and pH-Induced Swelling of Polymer Films Prepared from Sequentially Grafted Polyelectrolytes
- 2022
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 38:5, s. 1725-1737
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Tidskriftsartikel (refereegranskat)abstract
- We have prepared a series of ampholytic polymer films, using a self-initiated photografting and photopolymerization (SI-PGP) method to sequentially polymerize first anionic (deuterated methacrylic acid (dMAA)) and thereafter cationic (2-aminoethyl methacrylate (AEMA)) monomers to investigate the SI-PGP grafting process. Dry films were investigated by ellipsometry, X-ray, and neutron reflectometry, and their swelling was followed over a pH range from 4.5 to 10.5 with spectroscopic ellipsometry. The deuterated monomer allows us to separate the distributions of the two components by neutron reflectometry. Growth of both polymers proceeds via grafting of solution-polymerized fragments to the surface, and also the second layer is primarily grafted to the substrate and not as a continuation of the existing chains. The polymer films are stratified, with one layer of near 1:1 composition and the other layer enriched in one component and located either above or below the former layer. The ellipsometry results show swelling transitions at low and high pH but with no systematic variation in the pH values where these transitions occur. The results suggest that grafting density in SI-PGP-prepared homopolymers could be increased via repeated polymerization steps, but that this process does not necessarily increase the average chain length.
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