Modular bioengineered kinase sensors via scaffold protein-mediated split-luciferase complementation.

Phosphorylation is a key regulation event in cellular signaling. Sensing the underlying kinase activity is of crucial importance for its fundamental understanding and for drug development. For this, modular kinase activity sensing concepts are urgently needed. We engineered modular serine kinase sensors based on complementation of split NanoBiT luciferase on protein assembly platforms generated from the scaffold protein 14-3-3. The bioengineered platforms are modular and easy adaptable as exemplary shown using novel sensors for the kinases PKA, PKB, and CHK1. Two designs were conceptualized, both relying on binding of defined mono- or bivalent kinase recognition motifs to the 14-3-3 platform upon phosphorylation, resulting in reconstitution of active split-luciferase. Especially the design based on double phosphorylation and bivalent 14-3-3 binding exhibits high efficiency for signal amplification (>1000-fold) and sensitivity to specific kinases, including in cellular lysates.

Proximity-induced caspase-9 activation on a DNA origami-based synthetic apoptosome.

Living cells regulate key cellular processes by spatial organisation of catalytically active proteins in higher-order signalling complexes. These act as organising centres to facilitate proximity-induced activation and inhibition of multiple intrinsically weakly associating signalling components, which makes elucidation of the underlying protein-protein interactions challenging. Here we show that DNA origami nanostructures provide a programmable molecular platform for the systematic analysis of signalling proteins by engineering a synthetic DNA origami-based version of the apoptosome, a multi-protein complex that regulates apoptosis by co-localizing multiple caspase-9 monomers. Tethering of both wildtype and inactive caspase-9 variants to a DNA origami platform demonstrates that enzymatic activity is induced by proximity-driven dimerization with half-of-sites reactivity, and additionally, reveals a multivalent activity enhancement in oligomers of three and four enzymes. Our results offer fundamental insights in caspase-9 activity regulation and demonstrate that DNA origami-based protein assembly platforms have the potential to inform the function of other multi-enzyme complexes involved in inflammation, innate immunity and cell death.

Protease-Activatable Scaffold Proteins as Versatile Molecular Hubs in Synthetic Signaling Networks.

Protease signaling and scaffold-induced control of protein-protein interactions represent two important mechanisms for intracellular signaling. Here we report a generic and modular approach to control the activity of scaffolding proteins by protease activity, creating versatile molecular platforms to construct synthetic signaling networks. Using 14-3-3 proteins as a structurally well-characterized and important class of scaffold proteins, three different architectures were explored to achieve optimal protease-mediated control of scaffold activity, fusing either one or two monovalent inhibitory ExoS peptides or a single bivalent ExoS peptide to T14-3-3 using protease-cleavable linkers. Analysis of scaffolding activity before and after protease-induced cleavage revealed optimal control of 14-3-3 activity for the system that contained monovalent ExoS peptides fused to both the N-and C-terminus, each blocking a single T14-3-3 binding site. The protease-activatable 14-3-3 scaffolds were successfully applied to construct a three-step signaling cascade in which dimerization and activation of FGG-caspase-9 on an orthogonal supramolecular platform resulted in activation of a 14-3-3 scaffold, which in turn allowed 14-3-3-templated complementation of a split-luciferase. In addition, by combining 14-3-3-templated activation of caspase-9 with a caspase-9-activatable 14-3-3 scaffold, the first example of a synthetic self-activating protease signaling network was created. Protease-activatable 14-3-3 proteins thus represent a modular platform whose properties can be rationally engineered to fit different applications, both to create artificial in vitro synthetic molecular networks and as a novel signaling hub to re-engineer intracellular signaling pathways.

Bright Bioluminescent BRET Sensor Proteins for Measuring Intracellular Caspase Activity.

FRET-based caspase activity probes have become important tools to monitor apoptotic cell signaling. However, their dependence on external illumination is incompatible with light sensitive cells and hampers applications that suffer from autofluorescence and light scattering. Here we report the development of three caspase sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) that retain the advantages of genetically encoded, ratiometric optical probes but do not require external illumination. These sensors consist of the bright and stable luciferase NanoLuc and the fluorescent protein mNeonGreen, fused together via a linker containing a recognition site for caspase-3, -8, or -9. In vitro characterization showed that each caspase sensor displayed a robust 10-fold decrease in BRET ratio upon linker cleavage, with modest caspase specificity. Importantly, whereas scattering and background fluorescence precluded FRET-based detection of intracellular caspase activity in plate-reader assays, such measurements could be easily performed using our caspase BRET sensors in a high throughput format. The brightness of the BRET sensors also enabled long-term single-cell imaging, allowing BRET-based recording of cell heterogeneity in caspase activity in a heterogenic cell population.

Small-Molecule-Induced and Cooperative Enzyme Assembly on a 14-3-3 Scaffold.

Scaffold proteins regulate cell signalling by promoting the proximity of putative interaction partners. Although they are frequently applied in cellular settings, fundamental understanding of them in terms of, amongst other factors, quantitative parameters has been lagging behind. Here we present a scaffold protein platform that is based on the native 14-3-3 dimeric protein and is controllable through the action of a small-molecule compound, thus permitting study in an in vitro setting and mathematical description. Robust small-molecule regulation of caspase-9 activity through induced dimerisation on the 14-3-3 scaffold was demonstrated. The individual parameters of this system were precisely determined and used to develop a mathematical model of the scaffolding concept. This model was used to elucidate the strong cooperativity of the enzyme activation mediated by the 14-3-3 scaffold. This work provides an entry point for the long-needed quantitative insights into scaffold protein functioning and paves the way for the optimal use of reengineered 14-3-3 proteins as chemically inducible scaffolds in synthetic systems.

Current techniques for management of transverse displaced olecranon fractures.

BACKGROUND: displaced transverse fractures of the olecranon are the most common fractures occurring in the elbow in adults that requires operative intervention. METHODS: a literature search was performed on PubMed, Web of Science, Science Direct/Scopus, Google Scholar and Google using the keywords 'olecranon', 'fracture', 'internal fixation' and 'tension band wiring', with no limit for time or restrictions to language. RESULTS: thirty-one clinical articles were selected: 20 retrospective studies, 9 prospective cohort studies, and 2 randomized control trials. The CMS ranged from 18 to 66 (mean 41.68): overall, the quality of the studies was poor, and no moderate or good quality studies were found. The mean follow-up was 46.7 months (range 1 to 350 months). Several complications occurred after surgery: prominent hardware, skin breakdown, wire migration and infections occurred frequently. Removal of the hardware was required in 472 patients, usually after complaints, but also removal was routinely undertaken. CONCLUSIONS: tension band wiring is still the most widely applied method to operatively manage olecranon fractures, with the transcortical method of using K-wires the most satisfactory. Plate fixation is a good alternative as complications are minimal. Other techniques using absorbable sutures are less investigated, but are promising, especially in children.

Improved Immunoassay Sensitivity in Serum as a Result of Polymer-Entrapped Quantum Dots: 'Papaya Particles'.

Fluorescent labels are widely employed in biomarker quantification and diagnostics, however they possess narrow Stokes shifts and can photobleach, limiting multiplexed detection applications and compromising sensitivity. In contrast, quantum dots do not photobleach and have much wider Stokes shifts, but a paucity of robust surface attachment chemistries for bioconjugation has limited their uptake in biomedical diagnostics. We report a novel class of biofunctional fluorescent labels based on trapping of ∼10(4) quantum dots within a core nanoparticle. The doped particles act as scaffolds for generation of a multilayered shell consisting of a functionalized hydrophilic polymer with covalently attached receptors for analyte capture. These constructs, which conceptually resemble a papaya fruit, are chemically stable, remain monodispersed for >6 months in buffer, and show utility in immunoassay applications. Using monoclonal antibody fragments against nonstructural protein dengue NS1, an early biomarker for dengue fever, antibody immobilization capacity was 75-fold higher compared with traditional carbodiimide protein coupling. In the model dengue immunoassay, we observed a 15-fold lower limit of detection and 4-fold higher fluorescence intensity with the "papaya particles" compared to current "best-in-class" commercial reagents. Direct deployment in human serum allowed sensitive detection of different NS1 serotypes with lower limits of detection within the clinically relevant range (1-10 ng/mL), and sufficient specificity for identification of the dengue serotype was achieved for concentrations >10 ng/mL (DV1-3) and >50 ng/mL (DV4). The combination of chemical and physical stability and high binding capacity combined with the intrinsic advantages of quantum dots may enable more simple, robust diagnostic assays in the future.

Biomechanical studies on transverse olecranon and patellar fractures: a systematic review with the development of a new scoring method.

INTRODUCTION: Several methods of transverse patellar and olecranon fixation have been described. This article compares biomechanical studies of various fixation methods using a newly developed scoring method. SOURCE OF DATA: The databases PubMed, Web of Science, Science Direct, Google Scholar and Google were searched for relevant studies. AREAS OF AGREEMENT: Fixation hardware failure remains a problem. Various materials and fixation techniques have been tested to provide an improved fixation of transverse olecranon and patellar fractures. AREAS OF CONTROVERSY: The difference in biomechanical testing setup between the studies makes it hard to compare different fixation techniques. GROWING POINTS: The newly developed grading method was proved to be unbiased and reliable; however, extra specifications need to be added at some criteria when adopting the scoring method. AREAS TIMELY FOR DEVELOPING RESEARCH: Non-metallic constructs may provide an improvement to the currently used metallic tension band wiring technique; however, clinical research is required.

Treatment of transverse patellar fractures: a comparison between metallic and non-metallic implants.

INTRODUCTION: Several methods of transverse patellar fixation have been described. This study compares the clinical outcome and the occurrence of complications of various fixation methods. SOURCES OF DATA: The databases PubMed, Web of Science, Science Direct, Google Scholar and Google were searched. AREAS OF AGREEMENT: A direct comparison between fixation techniques using mixed or non-metallic implants and metallic K-wire and tension band fixation shows no significant difference in clinical outcome between both groups. Additionally, studies reporting novel operation techniques show good clinical results. AREAS OF CONTROVERSY: Studies describing the treatment of patients using non-metallic or mixed implants are fewer compared with those using metallic fixation. GROWING POINTS: A large variety of clinical scoring systems were used for assessing the results of treatment, which makes direct comparison difficult. AREAS TIMELY FOR DEVELOPING RESEARCH: More data of fracture treatment using non-metallic or mixed implants is needed to achieve a more balanced comparison.