Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation
Graphical abstract
Section snippets
Labeling proteases with ABPs
For the ABP dose curve experiment, recombinant proteases were diluted in 20 μL of phosphate-buffered saline: trypsin (100 ng), neutrophil elastase (1 μg), proteinase-3 (1 μg). The indicated ABP was added from 100x DMSO stocks to yield final concentrations of 0, 0.1, 0.5, and 1 μM with 1% DMSO. Samples were incubated for 30 min at 37 °C and then solubilized with 4× sample buffer (40% glycerol, 200 mM Tris-Cl [pH 6.8], 8% SDS, 0.04% bromophenol blue, 5% beta-mercaptoethanol). Proteins were resolved on a
Immunoprecipitation
Probe-labeled lysates were divided into input and immunoprecipitation (IP) samples (100 μg each). The pulldown sample was diluted in 500 μL IP buffer (PBS [pH 7.4], 1 mM EDTA, 0.5% NP-40) along with 5 μL sheep anti-mouse neutrophil elastase/ELA2 antibody (R&D). Protein A/G beads (40 μl slurry) were washed with IP buffer and then added to the sample. Tubes were rocked overnight at 4 °C. Beads were washed four times with IP buffer and once with 0.9% sodium chloride. After the last wash, all liquid was
Acute pancreatitis model
All animal experiments were approved by the Animal Ethics Committee of Monash University. Eight-week old male C57BL/6 J mice were given 12 hourly injections of caerulein (50 μg/kg in PBS) or PBS.12 After the ninth injection, the indicated ABP or vehicle control was administered intravenously (20 nmol dissolved in 100 μl 20% DMSO/PBS). Five hours later, pancreata were removed and imaged for Cy5 fluorescence using an IVIS Spectrum (Perkin Elmer). Radiance efficiency was determined using Living Image
Acute colitis model
Eight-week old male C57BL/6 J mice were administered 3% dextran sodium sulfate in the drinking water for 7 days. Colons were excised and flushed with 1 mL cold PBS. Supernatants were cleared by centrifugation. Total protein (50 μg) was labeled with Cy5-PK-DPP or Cy5-V-DPP and analyzed by fluorescent SDS-PAGE as above. Alternatively, elastase activity was measured with the fluorogenic substrate probe MeOSucc-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin (Bachem, AAPV-AMC). Protein (50 μg) was incubated in
Cy5-V-DPP stability test
Recombinant neutrophil elastase (rNE; 1 or 10 μg as indicated) was aliquoted alone or spiked into lysates prepared from normal or inflamed pancreata or luminal fluids (100 μg total protein) followed by immediate addition of Cy5-V-DPP for 30 min at 37 °C or addition after 10 min. All samples were then analyzed using fluorescent SDS-PAGE as above and then analyzed for total elastase levels by western blot (R&D AF4517; 1:1000) and donkey anti-sheep-IR800 (LiCOR; 1:10,000). Blots were scanned on an
Statistics
All experiments were performed in triplicate using at least 3 biological replicates. Data are reported as means ± SEM. Statistical significance was determined by comparing 2 groups using the Student’s t test, and p values of less than 0.05 were considered significant.
Acknowledgements
LEM is supported by an Early Career Fellowship from the National Health and Medical Research Council (NHMRC, Australia, GNT1091636). BG is the recipient of an Australian Research Council (ARC) Future Fellowship (FT130100838). NB is supported by NHMRC and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology. Work in the authors’ laboratory is funded in part by Takeda Pharmaceuticals Inc.
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2022, Journal of Biological ChemistryCitation Excerpt :A smaller subset of serine hydrolases can be more specifically interrogated by probes with a diphenylphosphonate-containing warhead, which reacts more uniquely with serine proteases. Irreversible serine protease inhibitors featuring this functional group were pioneered by Powers et al. (16, 45) and subsequently adapted as fluorescent probes (17, 26) or attached to biotin for affinity isolation and identification of serine proteases (19, 25). Optimization of peptide amino acid sequence has led to inhibitors of enhanced selectivity toward individual serine proteases, such as granzymes A and K (18).
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2020, Trends in Pharmacological SciencesCitation Excerpt :Fluorescent ABPs additionally allow for the detection of ex vivo protease activity by in-gel fluorescence, allowing the quantification and identification of probe-labeled proteases [39,44]. Alternatively, fluorescent APBs could be applied to image active proteases in tissue sections by confocal microscopy of tissue sections, affording a detailed characterization of their localization and cellular source [44] (Figure 2). While ABP studies have mainly been limited to murine models and ex vivo human studies, there is potential in the future to apply probes to detect protease activity in patients, either by in situ positron emission tomography (PET) imaging using radio-labeled ABPs [46] or by fluorescent imaging of topically applied probes during endoscopy [47].
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