Open Access

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B–BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.

Unlike the native surface of the implant material (Ti6Al4V), oxidation with H2O2 leads to increased binding of the effective antimicrobial agent poly(hexamethylene) biguanide [PHMB]. However, treating with NaOH instead results in an even higher PHMB mass coverage. After oxidation with H2O2, strong differences in the PHMB adsorption capability between polished and corundum-blasted surfaces appear, indicating a roughness dependence. After NaOH treatment, no such effect was observed. The wetting properties of specimens treated with either H2O2 or NaOH prior to PHMB exposure clearly varied. To unravel the nature of this interaction, widespread in silico and in vitro experiments were performed. Methods: By X-ray photoelectron spectroscopy, scanning electron microscopy, water contact angle measurements and MD simulations, we characterized the interplay between the polycationic antimicrobial agent and the implant surface. A theoretical model for PHMB micelles is tested for its wetting properties and compared to carbon contaminated TiO2. In addition, quantitation of anionic functional group equivalents, the binding properties of PHMB with blocked amino end-group, and the ability to bind chlorhexidine digluconate (CHG) were investigated. Ultimately, the capability of osteoblasts to build calcium apatite, and the activity of alkaline phosphatase on PHMB coated specimens, were determined. Results: Simulated water contact angles on carbon contaminated TiO2 surfaces and PHMB micelle models reveal little influence of PHMB on the wetting properties and point out the major influence of remaining and recovering contamination from ambient air. Testing PHMB adsorption beyond the critical micelle concentration and subsequent staining reveals an island-like pattern with H2O2 as compared to an evenly modified surface with NaOH. Both CHG and PHMB, with blocked amino end groups, were adsorbed on the treated surfaces, thus negating the significant influence of PHMB’s terminal groups. The ability of osteoblasts to produce calcium apatite and alkaline phosphatase is not negatively impaired for PHMB mass coverages up to 8 μg/specimen. Conclusion: Differences in PHMB adsorption are triggered by the number of anionic groups and carbon contaminants, both of which depend on the specimen pre-treatment. With more PHMB covering, the implant surface is protected against the capture of new contamination from the ambient air, thus building a robust antimicrobial and biocompatible surface coating.

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 (BCL11B42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B42-94 in E. coli 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-BCL11B42-94 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.

One of the most common mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene is the N34S variant which is strongly associated with chronic pancreatitis. Although it is assumed that N34S mutation constitutes a high-risk factor, the underlying pathologic mechanism is still unknown. In the present study, we investigated the impact of physiological stress factors on SPINK1 protein structure and trypsin inhibitor function using biophysical methods. Our circular dichroism spectroscopy data revealed differences in the secondary structure of SPINK1 and N34S mutant suggesting protein structural changes induced by the mutation as an impairment that could be disease-relevant. We further confirmed that both SPINK1 (KD of 0.15 ± 0.06 nM) and its N34S variant (KD of 0.08 ± 0.02 nM) have similar binding affinity and inhibitory effect towards trypsin as shown by surface plasmon resonance and trypsin inhibition assay studies, respectively. We found that stress conditions such as altered ion concentrations (i.e. potassium, calcium), temperature shifts, as well as environmental pH lead to insignificant differences in trypsin inhibition between SPINK1 and N34S mutant. However, we have shown that the environmental pH induces structural changes in both SPINK1 constructs in a different manner. Our findings suggest protein structural changes in the N34S variant as an impairment of SPINK1 and environmental pH shift as a trigger that could play a role in disease progression of pancreatitis.

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.