In Vivo Delivery and Activation of Masked Fluorogenic Hydrolase Substrates by Endogenous Hydrolases in C. elegans

Abstract

Protein expression and localization are often studied in vivo by tagging molecules with green fluorescent protein (GFP), yet subtle changes in protein levels are not easily detected. To develop a sensitive in vivo method to amplify fluorescence signals and allow cell-specific quantification of protein abundance changes, we sought to apply an enzyme-activated cellular fluorescence system in vivo by delivering ester-masked fluorophores to Caenorhabditis elegans neurons expressing porcine liver esterase (PLE). To aid uptake into sensory neuron membranes, we synthesized two novel fluorogenic hydrolase substrates with long hydrocarbon tails. Recombinant PLE activated these fluorophores in vitro. In vivo activation occurred in sensory neurons, along with potent activation in intestinal lysosomes quantifiable by imaging and microplate and partially attributable to gut esterase 1 (GES-1) activity. These data demonstrate the promise of biorthogonal hydrolases and their fluorogenic substrates as in vivo neuronal imaging tools and for characterizing endogenous C. elegans hydrolase substrate specificities.

Fluorogenic structure activity library pinpoints molecular variations in substrate specificity of structurally homologous esterases

“…reactivity (19,20,23,75), fragment-based inhibitor design analogous to serine protease fluorogenic SAS libraries (37,39,40), and in vivo imaging of cell type-specific hydrolase activity (24,33).…”

“…To increase hydrolytic stability and to distance the cleavable moiety from the fluorescent reporter, stable moieties have been inserted between the hydrolytic bond and the fluorophore (16, 18, 24, 30 -32). Among these stable moieties, the acyloxymethyl ether class of fluorogenic substrates has found utility in orthogonal cell labeling, substrate specificity screening, and in vivo enzyme characterization (16,24,(33)(34)(35)(36).…”

“…We previously developed a small, general library of fluorogenic ester substrates based on acyloxymethyl ether fluorescein (24,31,41,42). We then applied this library to broadly characterize the structural factors controlling the substrate specificity of esterases, to propose biological functions for uncharacterized esterases, and to identify unusual biocatalytic reactions (31,33,36,(41)(42)(43). This preliminary fluorogenic library provided sensitive detection of even weak binding substrates within a high-throughput and straightforward assay design (35,(41)(42)(43).…”

Comparative analysis of the human serine hydrolase OVCA2 to the model serine hydrolase homolog FSH1 from S. cerevisiae

“…[34] These fluorogenic substrates provide low background hydrolysis, sensitive kinetic measurements, and a broad screen of ester substrates across subclasses of metabolic serine hydrolases. [35][36][37][38] In addition to these substrates, chromogenic pnitrophenyl ester substrates with varying alkyl chain lengths from 2-to 14-carbons were used to subclassify OVCA2 based on substrate preference for more lipophilic substrates. [36,39] These p-nitrophenyl ester substrates have greater solubility and only one ester group for chromogenic protection, but have a higher background hydrolysis rate and lower sensitivity than the fluorogenic substrates.…”

Recent Advances in Design of Fluorescence-Based Assays for High-Throughput Screening

“…A voltage-sensitive dye was designed to be the substrate of porcine liver esterase (PLE), and its fluorescence increased after being cleaved by PLE. PLE can be selectively expressed in cells of interest (e.g., neurons), so only these cells can have PLE on their surface to catalyze the dye and show fluorescence . Zhao et al described an exquisite label-free fluorescent and colorimetric dual-readout assay of tyrosinase activity, showing a good example of employing enzymes to convert the nonfluorescent substrates into fluorescent products.…”