Structural Elucidation of a Metagenomic Urethanase and Its Engineering Towards Enhanced Hydrolysis Profiles.

While plastics like polyethylene terephthalate can already be degraded efficiently by the activity of hydrolases, other synthetic polymers like polyurethanes (PUs) and polyamides (PAs) largely resist biodegradation. In this study, we solved the first crystal structure of the metagenomic urethanase UMG-SP-1, identified highly flexible loop regions to comprise active site residues, and targeted a total of 20 potential hot spots by site-saturation mutagenesis. Engineering campaigns yielded variants with single mutations, exhibiting almost 3- and 8-fold improved activity against highly stable N-aryl urethane and amide bonds, respectively. Furthermore, we demonstrated the release of the corresponding monomers from a thermoplastic polyester-PU and a PA (nylon 6) by the activity of a single, metagenome-derived urethanase after short incubation times. Thereby, we expanded the hydrolysis profile of UMG-SP-1 beyond the reported low-molecular weight carbamates. Together, these findings promise advanced strategies for the bio-based degradation and recycling of plastic materials and waste, aiding efforts to establish a circular economy for synthetic polymers.

Impact of Protein Corona Formation and Polystyrene Nanoparticle Functionalisation on the Interaction with Dynamic Biomimetic Membranes Comprising of Integrin.

Plastics, omnipresent in the environment, have become a global concern due to their durability and limited biodegradability, especially in the form of microparticles and nanoparticles. Polystyrene (PS), a key plastic type, is susceptible to fragmentation and surface alterations induced by environmental factors or industrial processes. With widespread human exposure through pollution and diverse industrial applications, understanding the physiological impact of PS, particularly in nanoparticle form (PS-NPs), is crucial. This study focuses on the interaction of PS-NPs with model blood proteins, emphasising the formation of a protein corona, and explores the subsequent contact with platelet membrane mimetics using experimental and theoretical approaches. The investigation involves αIIbß3-expressing cells and biomimetic membranes, enabling real-time and label-free nanoscale precision. By employing quartz-crystal microbalance with dissipation monitoring studies, the concentration-dependent cytotoxic effects of differently functionalised ~210 nm PS-NPs on HEK293 cells overexpressing αIIbß3 are evaluated in detail. The study unveils insights into the molecular details of PS-NP interaction with supported lipid bilayers, demonstrating that a protein corona formed in the presence of exemplary blood proteins offers protection against membrane damage, mitigating PS-NP cytotoxicity.

Liposomal formulation of model pentathiepin improves solubility and stability toward glutathione while preserving anticancer activity.

The biological properties of pentathiepins have been attracting increased attention in recent years. Experiments have shown a wide range of effects of pentathiepins in vitro, such as induction of apoptosis and alteration of mitochondrial membrane potential in cancer cells, and inhibition of antioxidant enzymes, for example, glutathione peroxidase 1 (GPx1). Biological evaluation is sometimes limited due to low aqueous solubility, high lipophilicity, and poor stability toward thiols, for example, glutathione (GSH). To assess whether liposomes are suitable as drug carriers to overcome these drawbacks, a model pentathiepin was formulated in a liposomal preparation. The success of loading liposomes with pentathiepins was evaluated by using ultraviolet-visible light (UV-Vis) spectroscopy, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC). Through inclusion into 100-nm-sized 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes, the aqueous solubility of a representative pentathiepin could be increased by several orders of magnitude to ca. 400 µM. The stability of the pentathiepin in the presence of GSH was increased fourfold as determined by UV-Vis spectroscopy. In antiproliferation experiments with two human cancer cell lines, no decrease in potency in the liposomal loaded pentathiepin compared to the free pentathiepin was found. In conclusion, liposomes are a suitable carrier for pentathiepins and improve both solubility and stability in the presence of thiols without compromising anticancer activity.

Inhibitory Effect of Epigallocatechin Gallate-Silver Nanoparticles and Their Lysozyme Bioconjugates on Biofilm Formation and Cytotoxicity.

Biofilms are multicellular communities of microbial cells that grow on natural and synthetic surfaces. They have become the major cause for hospital-acquired infections because once they form, they are very difficult to eradicate. Nanotechnology offers means to fight biofilm-associated infections. Here, we report on the synthesis of silver nanoparticles (AgNPs) with the antibacterial ligand epigallocatechin gallate (EGCG) and the formation of a lysozyme protein corona on AgNPs, as shown by UV-vis, dynamic light scattering, and circular dichroism analyses. We further tested the activity of EGCG-AgNPs and their lysozyme bioconjugates on the viability of Bacillus subtilis cells and biofilm formation. Our results showed that, although EGCG-AgNPs presented no antibacterial activity on planktonic B. subtilis cells, they inhibited B. subtilis biofilm formation at concentrations larger than 40 nM, and EGCG-AgNP-lysozyme bioconjugates inhibited biofilms at concentrations above 80 nM. Cytotoxicity assays performed with human cells showed a reverse trend, where EGCG-AgNPs barely affected human cell viability while EGCG-AgNP-lysozyme bioconjugates severely hampered viability. Our results therefore demonstrate that EGCG-AgNPs may be used as noncytotoxic antibiofilm agents.

Reconstitution of Functional Integrin αIIbß3 and Its Activation in Plasma Membrane-Mimetic Lipid Environments.

The study of the platelet receptor integrin αIIbß3 in a membrane-mimetic environment without interfering signalling pathways is crucial to understand protein structure and dynamics. Our understanding of this receptor and its sequential activation steps has been tremendously progressing using structural and reconstitution approaches in model membranes, such as liposomes or supported-lipid bilayers. For most αIIbß3 reconstitution approaches, saturated short-chain lipids have been used, which is not reflecting the native platelet cell membrane composition. We report here on the reconstitution of label-free full-length αIIbß3 in liposomes containing cholesterol, sphingomyelin, and unsaturated phosphatidylcholine mimicking the plasma membrane that formed supported-lipid bilayers for quartz-crystal microbalance with dissipation (QCM-D) experiments. We demonstrate the relevance of the lipid environment and its resulting physicochemical properties on integrin reconstitution efficiency and its conformational dynamics. We present here an approach to investigate αIIbß3 in a biomimetic membrane system as a useful platform do dissect disease-relevant integrin mutations and effects on ligand binding in a lipid-specific context, which might be applicable for drug screening.

Interaction of fibrinogen-magnetic nanoparticle bioconjugates with integrin reconstituted into artificial membranes.

Magnetic nanoparticles have a broad spectrum of biomedical applications including cell separation, diagnostics and therapy. One key issue is little explored: how do the engineered nanoparticles interact with blood components after injection? The formation of bioconjugates in the bloodstream and subsequent reactions are potentially toxic due to the ability to induce an immune response. The understanding of the underlying processes is of major relevance to design not only efficient, but also safe nanoparticles for e.g. targeted drug delivery applications. In this study, we report on maghemite nanoparticles functionalized with citrate-, dextran- and polyethylene glycol coatings and their interaction with the clotting protein fibrinogen. Further, we investigate using biophysical tools (e.g. dynamic light scattering, circular dichroism spectroscopy and quartz crystal microbalance) the interaction of the magnetic nanoparticles-fibrinogen bioconjugates with artificial cell membranes as a model system for blood platelets. We found that fibrinogen corona formation provides colloidal stability to maghemite nanoparticles. In addition, bioconjugates of fibrinogen with dextran- and citrate-coated NPs interact with integrin-containing lipid bilayer, especially upon treatment with divalent ions, whereas PEG-coating reveals minor interaction. Our study at the interface of protein-conjugated nanoparticles and artificial cell membranes is essential for engineering safe nanoparticles for drug delivery applications.

Biopolymer-coated gold nanoparticles inhibit human insulin amyloid fibrillation.

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 ß-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.

Drug-induced activation of integrin alpha IIb beta 3 leads to minor localized structural changes.

Integrins are transmembrane proteins involved in hemostasis, wound healing, immunity and cancer. In response to intracellular signals and ligand binding, integrins adopt different conformations: the bent (resting) form; the intermediate extended form; and the ligand-occupied active form. An integrin undergoing such conformational dynamics is the heterodimeric platelet receptor αIIbß3. Although the dramatic rearrangement of the overall structure of αIIbß3 during the activation process is potentially related to changes in the protein secondary structure, this has not been investigated so far in a membrane environment. Here we examine the Mn2+- and drug-induced activation of αIIbß3 and the impact on the structure of this protein reconstituted into liposomes. By quartz crystal microbalance with dissipation monitoring and activation assays we show that Mn2+ induces binding of the conformation-specific antibody PAC-1, which only recognizes the extended, active integrin. Circular dichroism spectroscopy reveals, however, that Mn2+-treatment does not induce major secondary structural changes of αIIbß3. Similarly, we found that treatment with clinically relevant drugs (e.g. quinine) led to the activation of αIIbß3 without significant changes in protein secondary structure. Molecular dynamics simulation studies revealed minor local changes in the beta-sheet probability of several extracellular domains of the integrin. Our experimental setup represents a new approach to study transmembrane proteins, especially integrins, in a membrane environment and opens a new way for testing drug binding to integrins under clinically relevant conditions.

Identification of conserved amino acids in pUL34 which are critical for function of the pseudorabies virus nuclear egress complex.

UNLABELLED: Nuclear egress of herpesvirus capsids is mediated by a conserved heterodimeric complex of two viral proteins, designated pUL34 and pUL31 in herpes simplex virus and pseudorabies virus (PrV). pUL34, a tail-anchored membrane protein, is targeted to the nuclear envelope and recruits pUL31 to the inner nuclear membrane (INM) to provide the docking and envelopment machinery for the nascent capsid. While the less conserved C-terminal part of pUL34 is required for correct positioning of the nuclear egress complex (NEC) at the INM, the conserved N-terminal part functions as a docking site for pUL31. Since no crystal structure of NEC is available yet, structure-function studies depend on mutational analyses, with several approaches already being performed for different herpesvirus NECs. Here, we extended our studies on PrV pUL34 and identified two asparagine residues (N75, N103) and a dileucine motif (LL166/167), adjacent to an endoplasmic reticulum retention signal, which are absolutely required for NEC function. While the pUL34-N75A substitution mutant is unable to interact with pUL31, the pUL34-N103A mutant is nonfunctional, despite continuing complex formation. Surprisingly, mutant pUL34-G77A, which does not efficiently recruit pUL31 to the nuclear rim after cotransfection, nonetheless complements a UL34 deletion mutant, indicating that the NEC may be stabilized by additional viral factors during infection. IMPORTANCE: In the absence of a crystal structure of the nuclear egress complex (NEC) required for herpesvirus maturation, site-directed mutagenesis studies provide important information on critical amino acid residues. Here, we identify conserved amino acid residues in the membrane-bound component of the NEC which are relevant for its function.

Karyotyping of human chondrocytes in scaffold-assisted cartilage tissue engineering.

Scaffold-assisted autologous chondrocyte implantation (ACI) is an effective clinical procedure for cartilage repair. The aim of our study was to evaluate the chromosomal stability of human chondrocytes subjected to typical cell culture procedures needed for regenerative approaches in polymer-scaffold-assisted cartilage repair. Chondrocytes derived from post mortem donors and from donors scheduled for ACI were expanded, cryopreserved and re-arranged in polyglycolic acid (PGA)-fibrin scaffolds for tissue culture. Chondrocyte redifferentiation was analyzed by electron microscopy, histology and gene expression analysis. Karyotyping was performed using GTG banding and fluorescence in situ hybridization on a single cell basis. Chondrocytes showed de- and redifferentiation accompanied by the formation of extracellular matrix and induction of typical chondrocyte marker genes like type II collagen in PGA-fibrin scaffolds. Post mortem chondrocytes showed up to 1.7% structural and high numbers of numerical (up to 26.7%) chromosomal aberrations, while chondrocytes from living donors scheduled for ACI showed up to 1.8% structural and up to 1.3% numerical alterations. Cytogenetically, cell culture procedures and PGA-fibrin scaffolds did not significantly alter chromosomal integrity of the chondrocyte genome. Human chondrocytes derived from living donors subjected to regenerative medicine cell culture procedures like cell expansion, cryopreservation and culture in resorbable polymer-based scaffolds show normal chromosomal integrity and normal karyotypes.