ABSTRACT
Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S2 and for positively charged residues in S1. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S2 pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.
Subject(s)
Cysteine Endopeptidases/chemistry , Cysteine Proteases/chemistry , Droseraceae/enzymology , Plant Proteins/chemistry , Animals , Caseins/chemistry , Cattle , Chromatography, Liquid , Circular Dichroism , Cloning, Molecular , Crystallography, X-Ray , Drosophila melanogaster , Glycosylation , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Kinetics , Leucine/analogs & derivatives , Leucine/chemistry , Lysine/chemistry , Models, Molecular , Papain/chemistry , Protein Folding , Protein Structure, Tertiary , Tandem Mass SpectrometryABSTRACT
OBJECTIVE: To develop enzyme-activatable Förster resonance energy transfer (FRET) substrate probes to detect matrix metalloproteinase 12 (MMP-12) and MMP-13 activities in vivo in mouse models of inflammatory arthritis. METHODS: Peptidic FRET probes activated by MMP-12 and MMP-13 were reverse designed from inhibitors selected from a phosphinic peptide inhibitor library. Selectivity of the probes was demonstrated in vitro using MMP-1, MMP-2, MMP-3, MMP-12, and MMP-13. In vivo activation of the probes was tested in the zymosan-induced mouse model of inflammation, and probe specificity was evaluated by the MMP inhibitor GM6001 and specific synthetic inhibitors of MMP-12 and MMP-13. The probes were used to monitor these enzyme activities in the collagen-induced arthritis (CIA) model in vivo. RESULTS: The MMP-12 and MMP-13 activity probes (MMP12ap and MMP13ap, respectively) discriminated between the activities of the 2 enzymes. The in vivo activation of these probes was inhibited by GM6001 and by their respective specific inhibitors. In the CIA model, MMP12ap activation peaked 5 days after disease onset and showed strong correlation with disease severity during this time (r = 0.85, P < 0.0001). MMP13ap activation increased gradually after disease onset and correlated with disease severity over a longer period of 15 days (r = 0.58, P < 0.0001). CONCLUSION: We generated two selective FRET probes that can be used to monitor MMP-12 and MMP-13 activities in live animals. MMP12ap follows the initial stage of inflammation in CIA, while MMP13ap follows the progression of the disease. The specificity of these probes is useful in monitoring the efficacy of MMP inhibitors.
Subject(s)
Arthritis, Experimental/enzymology , Matrix Metalloproteinase 12/metabolism , Matrix Metalloproteinase 13/metabolism , Synovial Membrane/enzymology , Animals , Diagnostic Imaging , Fluorescence Resonance Energy Transfer , Matrix Metalloproteinase Inhibitors/pharmacology , Mice , Synovial Membrane/drug effectsABSTRACT
This review will cover the entire hit identification process performed with biocompatible, aqueous solvated, poly[ethylene glycol] (PEG) based resins - from synthesis, through screening, to analysis. The different types of resins (including their preparation) will be discussed and compared individually. Examples of one-bead-one-compound substrate libraries will be presented, as will one-bead-two-compounds libraries used for the discovery of enzyme inhibitors. The review includes a section covering organic and bio-organic reactions performed on all-PEG resins and discusses on-bead screening of the libraries with biomolecules. Finally, analysis of compounds on single beads, either via investigation by on-bead NMR or by ladder-coding of the combinatorial compound is covered. In general, the review will focus on chemistry, libraries, synthesis, screening, and analysis, using all-PEG based resins.
