A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase

Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening

Exploring the Temperature−-Pressure Phase Diagram of Staphylococcal Nuclease

The temperature dependence of the pressure-induced equilibrium unfolding of staphylococcal nuclease (Snase) was determined by fluorescence of the single tryptophan residue, FTIR absorption for the amide I‘ and tyrosine O−H bands, and small-angle X-ray scattering (SAXS). The results from these three techniques were similar, although the stability as measured by fluorescence was slightly lower than that measured by FTIR and SAXS. The resulting phase diagram exhibits the well-known curvature for heat and cold denaturation of proteins, due to the large decrease in heat capacity upon folding. The volume change for unfolding became less negative with increasing temperatures, consistent with a larger thermal expansivity for the unfolded state than for the folded state. Fluorescence-detected pressure-jump kinetics measurements revealed that the curvature in the phase diagram is due primarily to the rate constant for folding, indicating a loss in heat capacity for the transition state relative to the unfolded state. The similar temperature dependence of the equilibrium and activation volume changes for folding indicates that the thermal expansivities of the folded and transition states are similar. This, along with the fact that the activation volume for folding is positive over the temperature range examined, the nonlinear dependence of the folding rate constant upon temperature implicates significant dehydration in the rate-limiting step for folding of Snase

Transgenic reporter mice expressing Caspase 3/7 GloSensor.

<p>A) Schematic of the pCLEX Caspase 3/7 GloSensor transgene construct. B) Excision of the floxed EGFP-stop cassette when crossed with a Cre expressing mouse strain should result in tissue specific transcription of the reporter. C) Representative bioluminescence images of bi-transgenic (for the reporter and p48-Cre) or mono-transgenic (transgenic for the reporter in the absence of Cre) animals pre- and 30 hrs post-cerulein injection (75 ug/kg, total of 12 injections in 48 hrs). D) Quantification of BLI signal induction upon cerulein treatment. E) Representative bioluminescent and fluorescent (EGFP) ex-vivo images of pancreata from mono- or bi-transgenic animals. F) Representative bioluminescence images of bi-transgenic (right) or mono-transgic (left) animals.</p

Assessing drug sensitivity of rare and transient cell populations.

<p>A) FACS analysis of dissociated D54 cells sorted into CD133⁺ and CD133⁻ populations, P3 represents the CD133 expressing cell population. B) to E) Bioluminescence assay of CD133⁺ and CD133⁻ sorted D54 cells incubated with 200 ng/ml TRAIL (B), 50 µM MNS (C), 50 µM MK886 (D) or 12.5 µM GW7647 (E). Bioluminescence was plotted as fold induction over values obtained from vehicle treated cells. Experiments were performed in triplicates and plotted as mean ± SEM. Paired t-test was performed for all experiments and * denotes p<0.05 value at indicated time points.</p

Validation of HTS hits.

<p>A) Bioluminescence activity of cells treated with MNS was measured at 12 hours post treatment and plotted as fold induction. Experiments were performed at least in triplicates (mean ± SEM). B) Representative western blots for Luciferase, cleaved Caspase 3 and PARP or β-Actin as loading control of D54 cells treated with (25 µM) MNS for 12 hrs. C) Bioluminescence activity of cells treated with increasing concentrations of CV3988 at 12 hours post treatment. Data are plotted as fold induction over values obtained from vehicle treated cells. Experiments were performed in triplicates (mean ± SEM). D and E). Bioluminescence activity was measured at various time points using 1833 (D) or D54 (E) cells treated with CV3988 (12.5 µM), Z-VAD (20 µM) or a combination of Z-VAD plus CV3988 for 24 hrs. Data are plotted as fold induction and experiments were performed in triplicates and plotted as mean ± SEM. F) Representative western blots of Luciferase, Caspase 3, PARP or β-actin were performed on lysates obtained from D54 cells. Cells were either treated with CV3988 (12.5 µM), pre-treated with Z-VAD (20 µM) or treated with Z-VAD and CV3988 for 12 hrs.</p

High throughput screening for inducers Caspase 3/7.

<p>Glosensor expressing 1833 or D54 cells (A and B respectively) were used to screen a library of 1,280 pharmacologically active compounds (LOPAC). Fold induction of bioluminescence signal intensity over values obtained from vehicle treated cells was plotted at maximal induction (mean ± SEM).</p

Utility of Caspase 3/7 GloSensor for assessment of cell death in cells.

<p>A) Schematic of the Caspase 3/7 GloSensor reporter containing an N-terminus coding for the C-Luc domain (358–544) of luciferase and a C-terminus coding for the N-Luc domain (4–354) of luciferase and a adjoining sequence, DEVD, the Caspase 3/7 recognition sequence. B) The functional basis of the reporter, wherein Caspase 3/7 mediated cleavage at the DEVD sequence results in release of the luciferase peptides and reconstitution of the enzymatic activity and an increase in luminescence signal. C) Bioluminescence analysis of cells treated with 200 ng/ml TRAIL. Data is plotted as fold induction standardized to values obtained from vehicle treated cells. D) Western blot for Caspase 3 cleavage using D54 cells treated with TRAIL for 6 hrs. β-Actin was used to standardize loading. E) Bioluminescence analysis of D54 cells treated with varying concentrations (25–100 ng/ml) of an agonist anti-Fas antibody. Data is plotted as fold induction over values obtained from vehicle treated cells at every hour. F) Bioluminescence analysis of cells treated with a pan-Caspase inhibitor Z-VAD (20 µM), 50 µM Docetaxel or with Z-VAD and Docetaxel combined. Data is plotted as fold induction. Experiments were performed at least in triplicates and mean values were plotted ± SEM.</p