G-quadruplex-based fluorometric biosensor for label-free and homogenous detection of protein acetylation-related enzymes activities.

Reversible protein acetylation, one of the key types of post-translational modifications, is composed of histone acetylation and deacetylation, which is typically catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) respectively. Herein, a label-free fluorescent method has been established for the homogeneous bioassay of HAT/HDAC activity and respective inhibitors. The proposed approach is primarily based on the electrostatic interaction between G-quadruplexes (G4s) and acetylation-related peptides, which results in marked change of fluorescent intensity of G4/Thioflavin T (ThT) complexes. This HAT (p300) activity assay is exceedingly sensitive and selective, with a linear range from 0.1 to 120nM and a detection limit of 0.05nM. Moreover, this biosensor is feasible to detect the HDAC (Sirt1) activity with a linear range from 1 to 450nM and a detection limit of 1nM. The potency of this assay is further demonstrated by detecting HAT/HDAC activity in cell lysates and evaluating HAT and HDAC-targeted inhibitors, C464 and EX 527, respectively. The proposed assay is convenient, label-free and cost-efficient, which is promising for HAT/HDAC-targeted epigenetic research and pharmaceutical development.

A poly(ADP-ribose) polymerase-1 activity assay based on the FRET between a cationic conjugated polymer and supercharged green fluorescent protein.

A label-free and convenient strategy for PARP-1 activity assay and inhibitors assessment has been developed based on the fluorescence resonance energy transfer (FRET) between a cationic conjugated polymer (CCP) and supercharged green fluorescent protein (scGFP).

Resurfaced fluorescent protein as a sensing platform for label-free detection of copper(II) ion and acetylcholinesterase activity.

Protein engineering by resurfacing is an efficient approach to provide new molecular toolkits for biotechnology and bioanalytical chemistry. H39GFP is a new variant of green fluorescent protein (GFP) containing 39 histidine residues in the primary sequence that was developed by protein resurfacing. Herein, taking H39GFP as the signal reporter, a label-free fluorometric sensor for Cu(2+) sensing was developed based on the unique multivalent metal ion-binding property of H39GFP and fluorescence quenching effect of Cu(2+) by electron transfer. The high affinity of H39GFP with Cu(2+) (Kd, 16.2 nM) leads to rapid detection of Cu(2+) in 5 min with a low detection limit (50 nM). Using acetylthiocholine (ATCh) as the substrate, this H39GFP/Cu(2+) complex-based sensor was further applied for the turn-on fluorescence detection of acetylcholinesterase (AChE) activity. The assay was based on the reaction between Cu(2+) and thiocholine, the hydrolysis product of ATCh by AChE. The proposed sensor is highly sensitive (limit of detection (LOD) = 0.015 mU mL(-1)) and is feasible for screening inhibitors of AChE. Furthermore, the practicability of this method was demonstrated by the detection of pesticide residue (carbaryl) in real food samples. Hence, the successful applications of H39GFP in the detection of metal ion and enzyme activity present the prospect of resurfaced proteins as versatile biosensing platforms.

Cyclic-AMP-dependent protein kinase (PKA) activity assay based on FRET between cationic conjugated polymer and chromophore-labeled peptide.

A sensitive fluorescence turn-on biosensing platform for protein kinase activity assay has been developed based on fluorescence resonance energy transfer (FRET) between a fluorophore labeled peptide and a water soluble cationic conjugated polymer (CCP). The CCP-based assay is based on the electrostatic interaction between the peptide and the CCP. The FRET efficiency will change with the changing charges around the peptide after phosphorylation. The feasibility of this method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.3 mU µL(-1)). Based on its simple mechanism, this assay is also sensitive and robust enough to be applied to the evaluation of PKA inhibitor H-89. The IC50 value, the half maximal inhibitory concentration, was 40 nM. Furthermore, our method has excellent selectivity. CCP-based assay is sensitive, versatile, cost-effective and easy to operate, so, this method is a promising candidate for kinase activity assay and inhibitor screening.

Immuno-stimulating properties of diosgenyl saponins isolated from Paris polyphylla.

The effects of three diosgenyl saponins isolated from Paris polyphylla on the immuno-stimulating activity in relation to phagocytosis, respiratory burst, and nitric oxide production in mouse macrophage cells RAW 264.7 have been investigated. Our results showed that all three diosgenyl saponins significantly enhanced phagocytic activity that increased with the concentration of saponins to reach a maximum, and then tended to decrease with higher concentrations. Saponins with sugar moiety directly induced respiratory burst response in RAW 264.7 cells that increased with the concentrations and reached a maximum, then decreased with higher concentrations after 2-h incubations, however, diosgenin had no PMA-triggered respiratory burst response. Treatment of RAW 264.7 cells with saponins with sugar moiety for 24-h caused a significant increase in the production of nitric oxide, while diosgenin had no effect at all. Consequently, relationship between molecular structures of three diosgenyl saponins and their immunomodulatory activities was discussed, and a possible mechanism of immuno-stimulating function of diosgenyl saponins was accordingly explored.