Deposits of protein misfolding and/or aggregates are a pathological hallmark of amyloid-related diseases. For instance, insulin amyloid fibril deposits have been observed in patients with ...insulin-dependent diabetes mellitus after insulin administration. Here, we report on the use of AuNPs functionalized with linear- (i.e. dextrin and chitosan) and branched- (i.e. dextran-40 and dextran-10) biopolymers as potential agents to inhibit insulin fibril formation. Our dynamic light scattering analyses showed a size decrease of the amyloid fibrils in the presence of functionalized AuNPs. Circular dichroism spectroscopy as well as enzyme-linked immunosorbent assay data demonstrated that the secondary structural transition from α-helix to β-sheet (which is characteristic for insulin amyloid fibril formation) was significantly suppressed by all biopolymer-coated AuNPs, and in particular, by those functionalized with linear biopolymers. Both transmission electron microscopy and atomic force microscopy analyses showed that the long thick amyloid fibrils formed by insulin alone become shorter, thinner or cluster when incubated with biopolymer-coated AuNPs. Dextrin- and chitosan-coated AuNPs were found to be the best inhibitors of the fibril formation. Based on these results, we propose a mechanism for the inhibition of insulin amyloid fibrils: biopolymer-coated AuNPsstrongly interact with the insulin monomers and inhibit the oligomer formation as well as elongation of the protofibrils.Moreover, cytotoxicity experiments showed that AuNP-insulin amyloid fibrils are less toxic compared to insulin amyloid fibrils alone. Our results suggest that both dextrin- and chitosan-AuNPs could be used as therapeutic agents for the treatment of amyloid-related disorders.
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Antibodies recognizing complexes of the chemokine platelet factor 4 (PF4/CXCL4) and polyanions (P) opsonize PF4-coated bacteria hereby mediating bacterial host defense. A subset of these antibodies ...may activate platelets after binding to PF4/heparin complexes, causing the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). In autoimmune-HIT, anti-PF4/P-antibodies activate platelets in the absence of heparin. Here we show that antibodies with binding forces of approximately 60-100 pN activate platelets in the presence of polyanions, while a subset of antibodies from autoimmune-HIT patients with binding forces ≥100 pN binds to PF4 alone in the absence of polyanions. These antibodies with high binding forces cluster PF4-molecules forming antigenic complexes which allow binding of polyanion-dependent anti-PF4/P-antibodies. The resulting immunocomplexes induce massive platelet activation in the absence of heparin. Antibody-mediated changes in endogenous proteins that trigger binding of otherwise non-pathogenic (or cofactor-dependent) antibodies may also be relevant in other antibody-mediated autoimmune disorders.
In the 1980s, Helmuth Möhwald studied lipid monolayers at the air/water interface to understand the thermodynamically characterized phases at the molecular level. In collaboration with Jens ...Als-Nielsen, X-ray reflectometry was used and further developed to determine the electron density profile perpendicular to the water surface. Using a slab model, parameters such as thickness and density of the individual molecular regions, as well as the roughness of the individual interfaces, were determined. Later, X-ray and neutron reflectometry helped to understand the coverage and conformation of anchored and adsorbed polymers. Nowadays, they resolve molecular properties in emerging topics such as liquid metals and ionic liquids. Much is still to be learned about buried interfaces (e.g., liquid/liquid interfaces). In this Article, a historical and theoretical background of X-ray reflectivity is given, recent developments of X-ray and neutron reflectometry for polymers at interfaces and thin layers are highlighted, and emerging research topics involving these techniques are emphasized.
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IJS, KILJ, NUK, PNG, UL, UM
Quantum dot (QD) based micro-/nanopatterned arrays are of broad interest in applications ranging from electronics, photonics, to sensor devices for biomedical purposes. Here, we report on a rapid, ...physico-chemically mild approach to generate high fidelity micropattern arrays of prefunctionalized water-soluble quantum dots using electron beam lithography. We show that such patterns retain their fluorescence and bioaffinity upon electron beam lithography and, based on the streptavidin–biotin interaction, allow for detection of proteins, colloidal gold nanoparticles and magnetic microparticles. Furthermore, we demonstrate the applicability of QD based microarray patterns differing in their shape (circles, squares, grid-like), size (from 1 to 10 μm) and pitch distance to study the adhesion, spreading and migration of human blood derived neutrophils. Using live cell confocal fluorescence microscopy, we show that pattern geometry and pitch distance influence the adhesion, spreading and migratory behavior of neutrophils. Research reported in this work paves the way for producing QD microarrays with multiplexed functionalities relevant for applications in analyte sensing and cellular dynamics.
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Abstract
Understanding the nanoparticle-cell interactions in physiological media is vital in determining the biological fate of the nanoparticles (NPs). These interactions depend on the ...physicochemical properties of the NPs and their colloidal behavior in cell culture media (CCM). Furthermore, the impact of the bioconjugates made by nanoparticle with proteins from CCM on the mechanical properties of cells upon interaction is unknown. Here, we analyzed the time dependent stability of gold nanoparticles (AuNPs) functionalized with citrate, dextran-10, dextrin and chitosan polymers in protein poor- and protein rich CCM. Further, we implemented the high-throughput technology real-time deformability cytometry (RT-DC) to investigate the impact of AuNP-bioconjugates on the cell mechanics of HL60 suspension cells. We found that dextrin-AuNPs form stable bioconjugates in both CCM and have a little impact on cell mechanics, ROS production and cell viability. In contrast, positively charged chitosan-AuNPs were observed to form spherical and non-spherical aggregated conjugates in both CCM and to induce increased cytotoxicity. Citrate- and dextran-10-AuNPs formed spherical and non-spherical aggregated conjugates in protein rich- and protein poor CCM and induced at short incubation times cell stiffening. We anticipate based on our results that dextrin-AuNPs can be used for therapeutic purposes as they show lower cytotoxicity and insignificant changes in cell physiology.
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Neutrophils are immune cells that engage in a suicidal pathway leading to the release of partially decondensed chromatin, or neutrophil extracellular traps (NETs). NETs behave as a double edged ...sword; they can bind to pathogens thereby ensnaring them and limiting their spread during infection; however, they may bind to host circulating materials and trigger thrombotic events, and are associated with autoimmune disorders. Despite the fundamental role of NETs as part of an immune system response, there is currently a very poor understanding of how their nanoscale properties are reflected in their macroscopic impact. In this work, using a combination of fluorescence and atomic force microscopy, we show that NETs appear as a branching filament network that results in a substantially organized porous structure with openings with 0.03 ± 0.04 μm(2) on average and thus in the size range of small pathogens. Topological profiles typically up to 3 ± 1 nm in height are compatible with a "beads on a string" model of nucleosome chromatin. Typical branch lengths of 153 ± 103 nm appearing as rigid rods and height profiles of naked DNA in NETs of 1.2 ± 0.5 nm are indicative of extensive DNA supercoiling throughout NETs. The presence of DNA duplexes could also be inferred from force spectroscopy and the occurrence of force plateaus that ranged from ∼65 pN to 300 pN. Proteolytic digestion of NETs resulted in widespread disassembly of the network structure and considerable loss of mechanical properties. Our results suggest that the underlying structure of NETs is considerably organized and that part of its protein content plays an important role in maintaining its mesh architecture. We anticipate that NETs may work as microscopic mechanical sieves with elastic properties that stem from their DNA-protein composition, which is able to segregate particles also as a result of their size. Such a behavior may explain their participation in capturing pathogens and their association with thrombosis.
Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. ...B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B
) for in vitro studies. First, we expressed fluorescently tagged BCL11B
in
and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B
was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.
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The chemokine platelet factor 4 (PF4) undergoes conformational changes when complexing with polyanions. This can induce the antibody-mediated adverse drug effect of heparin-induced thrombocytopenia ...(HIT). Understanding why the endogenous protein PF4 becomes immunogenic when complexing with heparin is important for the development of other negatively charged drugs and may also hint toward more general mechanisms underlying the induction of autoantibodies to other proteins. By circular dichroism spectroscopy, atomic force microscopy, and isothermal titration calorimetry we characterized the interaction of PF4 with unfractionated heparin (UFH), its 16-, 8-, and 6-mer subfractions, low-molecular-weight heparin (LMWH), and the pentasaccharide fondaparinux. To bind anti-PF4/heparin antibodies, PF4/heparin complexes require (1) an increase in PF4 antiparallel β-sheets exceeding ∼30% (achieved by UFH, LMWH, 16-, 8-, 6-mer), (2) formation of multimolecular complexes (UFH, 16-, 8-mer), and (3) energy (needed for a conformational change), which is released by binding of ≥11-mer heparins to PF4, but not by smaller heparins. These findings may help to synthesize safer heparins. Beyond PF4 and HIT, the methods applied in the current study may be relevant to unravel mechanisms making other endogenous proteins more vulnerable to undergo conformational changes with little energy requirement (eg, point mutations and post-translational modifications) and thereby predisposing them to become immunogenic.
•Besides clustering, platelet factor 4/polyanion complexes require input of energy to become immunogenic.•Minute differences in chain length determine the induction of antigenicity of PF4.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Once introduced into the human body, nanoparticles often interact with blood proteins, which in turn undergo structural changes upon adsorption. Although protein corona formation is a widely studied ...phenomenon, the structure of proteins adsorbed on nanoparticles is far less understood. We propose a model to describe the interaction between human serum albumin (HSA) and nanoparticles (NPs) with arbitrary coatings. Our model takes into account the competition between protonated and unprotonated polymer ends and the curvature of the NPs. To this end, we explored the effects of surface ligands (citrate, PEG-OMe, PEG-NH
2
, PEG-COOH, and glycan) on gold nanoparticles (AuNPs) and the pH of the medium on structural changes in the most abundant protein in blood plasma (HSA), as well as the impact of such changes on cytotoxicity and cellular uptake. We observed a counterintuitive effect on the
ζ
-potential upon binding of negatively charged HSA, while circular dichroism spectroscopy at various pH values showed an unexpected pattern in the reduction of α-helix content, as a function of surface chemistry and curvature. Our model qualitatively reproduces the decrease in α-helix content, thereby offering a rationale based on particle curvature. The simulations quantitatively reproduce the charge inversion measured experimentally through the
ζ
-potential of the AuNPs in the presence of HSA. Finally, we found that AuNPs with adsorbed HSA display lower toxicity and slower cell uptake rates, compared to functionalized systems in the absence of protein. Our study allows examining and explaining the conformational dynamics of blood proteins triggered by NPs and corona formation, thereby opening new avenues toward designing safer NPs for drug delivery and nanomedical applications.
Gold nanoparticles with various functionalities interact with the human serum albumin (HSA) leading to protein structural changes. HSA-nanoparticles bioconjugates display lower toxicity and slower cell uptake rates, compared to nanoparticles in the absence of protein.
Beta-2-glycoprotein I (β2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). β2GPI exists mainly in closed or open conformations and comprises of ...11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide bond at domain V has been associated with different cysteine redox states. The role of this disulfide bond in conformational dynamics of this protein has not been investigated so far. Here, we report on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine; TCEP) of β2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry analyses confirming majority targeting to the fifth domain of β2GPI. Atomic force microscopy images suggested that reduced β2GPI shows a slightly higher proportion of open conformation and is more flexible compared to the untreated protein as confirmed by modelling studies. We have determined a strong increase in the binding of pathogenic APS autoantibodies to reduced β2GPI as demonstrated by ELISA. Our study is relevant for understanding the effect of β2GPI reduction on the protein structure and its implications for antibody binding in APS patients.
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