Ratiometric assay of CARM1 activity using a FRET-based fluorescent probe

One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.     

A high-throughput FRET-based assay for determination of Atg4 activity

Atg4 is required for cleaving Atg8, allowing it to be conjugated to phosphatidylethanolamine on phagophore membranes, a key step in autophagosome biogenesis. Deconjugation of Atg8 from autophagosomal membranes could be also a regulatory step in controlling autophagy. Therefore, the activity of Atg4 is important for autophagy and could be a target for therapeutic intervention. In this study, a sensitive and specific method to measure the activity of two Atg4 homologs in mammalian cells, Atg4A and Atg4B, was developed using a fluorescence resonance energy transfer (FRET)-based approach. Thus LC3B and GATE-16, two substrates that could be differentially cleaved by Atg4A and Atg4B, were fused with CFP and YFP at the N- and C-terminus, respectively, allowing FRET to occur. The FRET signals decreased in proportion to the Atg4-mediated cleavage, which separated the two fluorescent proteins. This method is highly efficient for measuring the enzymatic activity and kinetics of Atg4A and Atg4B under in vitro conditions. Applications of the assay indicated that the activity of Atg4B was dependent on its catalytic cysteine and expression level, but showed little changes under several common autophagy conditions. In addition, the assays displayed excellent performance in high throughput format and are suitable for screening and analysis of potential modulators. In summary, the FRET-based assay is simple and easy to use, is sensitive and specific, and is suitable for both routine measurement of Atg4 activity and high-throughput screening.     

A high-throughput FRET-based assay for determination of Atg4 activity

Atg4 is required for cleaving Atg8, allowing it to be conjugated to phosphatidylethanolamine on phagophore membranes, a key step in autophagosome biogenesis. Deconjugation of Atg8 from autophagosomal membranes could be also a regulatory step in controlling autophagy. Therefore, the activity of Atg4 is important for autophagy and could be a target for therapeutic intervention. In this study, a sensitive and specific method to measure the activity of two Atg4 homologs in mammalian cells, Atg4A and Atg4B, was developed using a fluorescence resonance energy transfer (FRET)-based approach. Thus LC3B and GATE-16, two substrates that could be differentially cleaved by Atg4A and Atg4B, were fused with CFP and YFP at the N- and C-terminus, respectively, allowing FRET to occur. The FRET signals decreased in proportion to the Atg4-mediated cleavage, which separated the two fluorescent proteins. This method is highly efficient for measuring the enzymatic activity and kinetics of Atg4A and Atg4B under in vitro conditions. Applications of the assay indicated that the activity of Atg4B was dependent on its catalytic cysteine and expression level, but showed little changes under several common autophagy conditions. In addition, the assays displayed excellent performance in high throughput format and are suitable for screening and analysis of potential modulators. In summary, the FRET-based assay is simple and easy to use, is sensitive and specific, and is suitable for both routine measurement of Atg4 activity and high-throughput screening.     

Novel hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants using a modified CUPRAC method

Reactive oxygen species (ROS) such as superoxide anion, hydroxyl ((*)OH), peroxyl, and alkoxyl radicals may attack biological macromolecules giving rise to oxidative stress-originated diseases. Since (*)OH is very short-lived, secondary products resulting from (*)OH attack to various probes are measured. Although the measurement of aromatic hydroxylation with HPLC/electrochemical detection is more specific than the low-yield TBARS test, it requires sophisticated instrumentation. As a more convenient and less costly alternative, we used p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37 degrees C-incubated solutions for 2h. The CUPRAC (i.e., our original method for total antioxidant capacity assay) absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of (*)OH scavengers, the difference being proportional to the scavenging ability of the tested compound. A rate constant for the reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. The second-order rate constants of the scavengers were determined with competition kinetics by means of a linear plot of A(0)/A as a function of C(scavenger)/C(probe), where A(0) and A are the CUPRAC absorbances of the system in the absence and presence of scavenger, respectively, and C is the molar concentration of relevant species. The 2,4- and 3,5-dimethoxybenzoates were the best probes in terms of linearity and sensitivity. Iodide, metabisulfite, hexacyanoferrate(II), thiourea, formate, and dimethyl sulfoxide were shown by the modified CUPRAC assay to be more effective scavengers than mannitol, glucose, lysine, and simple alcohols, as in the TBARS assay. The developed method is less lengthy, more specific, and of a higher yield than the classical TBARS assay. The hydroxyl radical scavenging rate constants of ascorbic acid, formate, and hexacyanoferrate(II) that caused interference in other assays could be easily found with the proposed procedure.