Singlet-Oxygen-Induced Phospholipase A2 Inhibition: A Major Role for Interfacial Tryptophan Dioxidation

Several studies have revealed that various diseases such as cancer have been associated with elevated phospholipase A2 (PLA2) activity. Therefore, the regulation of PLA2 catalytic activity is undoubtedly vital. In this study, effective inactivation of PLA2 due to reactive species produced from cold physical plasma as a source to model oxidative stress is reported. We found singlet oxygen to be the most relevant active agent in PLA2 inhibition. A more detailed analysis of the plasma-treated PLA2 identified tryptophan 128 as a hot spot, rich in double oxidation. The significant dioxidation of this interfacial tryptophan resulted in an N-formylkynurenine product via the oxidative opening of the tryptophan indole ring. Molecular dynamics simulation indicated that the efficient interactions between the tryptophan residue and phospholipids are eliminated following tryptophan dioxidation. As interfacial tryptophan residues are predominantly involved in the attaching of membrane enzymes to the bilayers, tryptophan dioxidation and indole ring opening leads to the loss of essential interactions for enzyme binding and, consequently, enzyme inactivation. © 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH Gmb

Antibacterial mesoporous titania films with embedded gentamicin and surface modified with bone morphogenetic protein 2 to promote osseointegration in bone implants

Novel approaches are needed to avoid bacterial infections following implant surgery. Here the use of mesoporous titania films (MTFs) for gentamicin loading and delivery and the surface functionalization of MFTs with human recombinant bone morphogenetic protein 2 (hrBMP-2) are discussed. Gentamicin is incorporated into the MTF pores by immersion of the porous materials in gentamicin solution while hrBMP-2 is adsorbed on top of the MTF. Contact angle and X-ray photoelectron spectroscopy measurements are performed to prove gentamicin loading and hrBMP-2 functionalization. An initial burst release of gentamicin takes place in physiological media followed by a prolonged release that lasts weeks. Such a release profile is highly appealing for bone implants where a high concentration of antibiotics is necessary during implant surgery while a lower antibiotic concentration is needed until tissue is regenerated. The MTFs loaded with gentamicin and functionalized with hrBMP-2 are effective against Staphylococcus aureus colonization, and the presence of hrBMP-2 enhances MC3T3-E1 preosteoblastic cell attachment, proliferation, and differentiation.Fil: Escobar, Ane. Centro de Investigación Cooperativa en Biomateriales; EspañaFil: Muzzio, Nicolás Eduardo. Centro de Investigación Cooperativa en Biomateriales; España. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Coy, Emerson. Adam Mickiewicz University; PoloniaFil: Liu, Hui. Chinese Academy of Sciences; República de ChinaFil: Bindini, Elisa. Centro de Investigación Cooperativa en Biomateriales; EspañaFil: Andreozzi, Patrizia. Centro de Investigación Cooperativa en Biomateriales; EspañaFil: Wang, Guocheng. Chinese Academy of Sciences; República de ChinaFil: Angelome, Paula Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; ArgentinaFil: Delcea, Mihaela. University of Greifswald; AlemaniaFil: Grzelczak, Marek. Donostia International Physic Center; . Basque Foundation for Science; EspañaFil: Moya, Sergio E.. Centro de Investigación Cooperativa en Biomateriales; Españ

Biophysical tools to assess the interaction of PF4 with polyanions

The antigen in heparin-induced thrombocytopenia (HIT) is expressed on platelet factor 4 (PF4) when PF4 complexes with polyanions. In recent years, biophysical tools (e.g. circular dichroism spectroscopy, atomic force microscopy, isothermal titration calorimetry, x-ray crystallography, electron microscopy) have gained an important role to complement immunological and functional assays for better understanding the interaction of heparin with PF4. This allowed identification of those features that make PF4 immunogenic (e.g. a certain conformational change induced by the polyanion, a threshold energy of the complexes, the existence of multimeric complexes, a certain number of bonds formed by PF4 with the polyanion) and to characterize the morphology and thermal stability of complexes formed by the protein with polyanions. These findings and methods can now be applied to test new drugs for their potential to induce the HIT-like adverse drug effect by preclinical in vitro testing. The methods and techniques applied to characterize the antigen in HIT may also be helpful to better understand the mechanisms underlying other antibody-mediated disorders in thrombosis and hemostasis (e.g. acquired hemophilia, thrombotic thrombocytopenic purpura). Furthermore, understanding the mechanisms making the endogenous protein PF4 immunogenic may help to understand the mechanisms underlying other autoimmune disorders

Lysine residues control the conformational dynamics of beta 2-glycoprotein I

One of the major problems in the study of the dynamics of proteins is the visualization of changing conformations that are important for processes ranging from enzyme catalysis to signaling. A protein exhibiting conformational dynamics is the soluble blood protein beta 2-glycoprotein I (beta2GPI), which exists in two conformations: the closed (circular) form and the open (linear) form. It is hypothesized that an increased proportion of the open conformation leads to the autoimmune disease antiphospholipid syndrome (APS). A characteristic feature of beta2GPI is the high content of lysine residues. However, the potential role of lysine in the conformational dynamics of beta2GPI has been poorly investigated. Here, we report on a strategy to permanently open up the closed protein conformation by chemical acetylation of lysine residues using acetic acid N-hydroxysuccinimide ester (NHS-Ac). Specific and complete acetylation was demonstrated by the quantification of primary amino groups with fluoraldehyde o-phthalaldehyde (OPA) reagent, as well as western blot analysis with an anti-acetylated lysine antibody. Our results demonstrate that acetylated beta2GPI preserves its secondary and tertiary structures, as shown by circular dichroism spectroscopy. We found that after lysine acetylation, the majority of proteins are in the open conformation as revealed by atomic force microscopy high-resolution images. Using this strategy, we proved that the electrostatic interaction of lysine residues plays a major role in stabilizing the beta2GPI closed conformation, as confirmed by lysine charge distribution calculations. We foresee that our approach will be applied to other lysine-rich proteins (e.g. histones) undergoing conformational transitions. For instance, conformational dynamics can be triggered by environmental conditions (e.g. pH, ion concentration, post-translational modifications, and binding of ligands). Therefore, our study may be relevant for investigating the equilibrium of protein conformations causing diseases

Left Ventricular noncompaction in a patient with systemic lupus erythematosus

Left ventricular noncompaction is a rare structural cardiomyopathy that can occur isolated or in relationship with other conditions, mainly with musculoskeletal diseases or congenital heart defects. The association of left ventricular noncompaction and connective tissue disorders, including systemic lupus erythematosus, was scarcely described in the literature. Reported cases are, more likely, cardiomyopathies mimicking left ventricular noncompaction or transient left ventricular noncompaction with ventricular function improving after appropriate treatment

Anti-platelet factor 4/polyanion antibodies mediate a new mechanism of autoimmunity

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 Z100 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

The impact of cell culture media on the interaction of biopolymer-functionalized gold nanoparticles with cells: mechanical and toxicological properties

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