Optimization of fluorescent substrates for ADAM17 and their utility in the detection of diabetes.

ADAM17 (a disintegrin and metalloproteinase 17) is a sheddase that releases various types of membrane-associated proteins, including adhesive molecules, cytokines and their receptors, and inflammatory mediators. Evidence suggests that the enzyme is involved in the proteolytic cleavage of antiaging transmembrane protein Klotho (KL). What is more, reduced serum and urinary KL levels are observed in the early stages of chronic kidney disease. This study aimed to optimise the ADAM17 specific and selective fluorescent substrates. Then, the obtained substrate was used to detect the enzyme in urine samples of patients diagnosed with diabetes. It turned out that in all cases we were able to detect proteolytic activity, which was the opposite of the healthy samples.

Synthetic Fluorogenic Peptides Reveal Dynamic Substrate Specificity of Depalmitoylases.

Palmitoylation is a post-translational modification involving the thioesterification of cysteine residues with a 16-carbon-saturated fatty acid. Little is known about rates of depalmitoylation or the parameters that dictate these rates. Here we report a modular strategy to synthesize quenched fluorogenic substrates for the specific detection of depalmitoylase activity and for mapping the substrate specificity of individual depalmitoylases. We demonstrate that human depalmitoylases APT1 and APT2, and TgPPT1 from the parasite Toxoplasma gondii, have distinct specificities that depend on amino acid residues distal to the palmitoyl cysteine. This information informs the design of optimal and non-optimal substrates as well as isoform-selective substrates to detect the activity of a specific depalmitoylase in complex proteomes. In addition to providing tools for studying depalmitoylases, our findings identify a previously unrecognized mechanism for regulating steady-state levels of distinct palmitoylation sites by sequence-dependent control of depalmitoylation rates.

Design and application of a fluorogenic assay for monitoring inflammatory caspase activity.

Various fluorogenic assays exist for monitoring the activity of inflammatory caspases. However, there are no continuous assays that provide C-terminal substrate sequence specificity for inflammatory caspases. As a first step towards this, we have developed a continuous in vitro assay that relies on monitoring emission from tryptophan after cleavage of a quenching coumarin chromophore. The coumarin can be attached as an amino acid side chain or capping the C-terminus of the peptide. When the coumarin is a side chain, it allows for C-terminal and N-terminal sequence specificities to be explored. Using this assay, we obtained Michaelis-Menten kinetic data for four proof-of-principle peptides: WEHD-AMC (KM = 15 ± 2 µM), WEHD-MCA (KM = 93 ± 19 µM), WEHDG-MCA (KM = 21 ± 6 µM) and WEHDA-MCA (KM = 151 ± 37 µM), where AMC is 7-amino-4-methylcoumarin and MCA is ß-(7-methoxy-coumarin-4-yl)-Ala. The results indicate the viability of this new assay approach in the design of effective fluorogenic substrates for inflammatory caspases.

The capture proteasome assay (CAPA) to evaluate subtype-specific proteasome inhibitors.

We recently developed a new assay to measure proteasome activity in vitro (CAPA for capture proteasome assay) [1], based on proteasome capture on an antibody-coated plate. When used with lysates originating from cells expressing either standard proteasome, immunoproteasome or intermediate proteasomes ß5i or ß1i-ß5i, this assay allows the individual monitoring of the chymotrypsin-like, trypsin-like and caspase-like activities of the corresponding proteasome subtypes. The efficiency and specificity of four proteasome inhibitors were studied using the CAPA assay, demonstrating the potential of this assay for the development of subtype-specific proteasome inhibitors.

The capture proteasome assay: A method to measure proteasome activity in vitro.

Because of its crucial role in various cellular processes, the proteasome is the focus of intensive research for the development of proteasome inhibitors to treat cancer and autoimmune diseases. Here, we describe a new and easy assay to measure the different proteasome activities in vitro (chymotrypsin-like, caspase-like, and trypsin-like) based on proteasome capture on antibody-coated plates, namely the capture proteasome assay (CAPA). Applying the CAPA to lysates from cells expressing standard proteasome, immunoproteasome, or intermediate proteasomes ß5i or ß1i-ß5i, we can monitor the activity of the four proteasome subtypes. The CAPA provided similar results as the standard whole-cell proteasome-Glo assay without the problem of contaminating proteases requiring inhibitors. However, the profile of trypsin-like activity differed between the two assays. This could be partly explained by the presence of MgSO4 in the proteasome-Glo buffer, which inhibits the trypsin-like activity of the proteasome. The CAPA does not need MgSO4 and, therefore, provides a more precise measurement of the trypsin-like activity. The CAPA provides a quick and accurate method to measure proteasome activity in vitro in a very specific manner and should be useful for the development of proteasome inhibitors.