Abstract
Neutrophils, migrating to the site of infection, are able to release serine proteases after being activated. These serine proteases comprise cathepsin G (CatG), neutrophil elastase protease 3 (PR3), and neutrophil serine protease 4 (NSP4). A disadvantage of the uncontrolled proteolytic activity of proteases is the outcome of various human diseases, including cardiovascular diseases, thrombosis, and autoimmune diseases. Activity-based probes (ABPs) are used to determine the proteolytic activity of proteases, containing a set of three essential elements: Warhead, recognition sequence, and the reporter tag for detection of the covalent enzyme activity–based probe complex. Here, we summarize the latest findings of ABP-mediated detection of proteases in both locations intracellularly and on the cell surface of cells, thereby focusing on CatG. Particularly, application of ABPs in regular flow cytometry, imaging flow cytometry, and mass cytometry by time-of-flight (CyTOF) approaches is advantageous when distinguishing between immune cell subsets. ABPs can be included in a vast panel of markers to detect proteolytic activity and determine whether proteases are properly regulated during medication. The use of ABPs as a detection tool opens the possibility to interfere with uncontrolled proteolytic activity of proteases by employing protease inhibitors.
Highlights
TO ACTIVITY-BASED PROBES (ABPS)Proteases represent a diverse class of enzymes that cleave proteins into peptides and to amino acids, thereby controlling the proteome to a steady-state level in the organism
activity-based probe (ABP) contain three essential elements: a warhead, which is often an electrophilic group that reacts with the active site amino acid residue of the protease and results in an irreversible covalent binding; a recognition sequence, a peptide linker that matches the substrate specificity of the protease; and a reporter tag for detection of the proteaseABP complex (Oleksyszyn and Powers, 1989, Oleksyszyn and Power, 1991; Abuelyaman et al, 1994; Blum et al, 2009; Sienczyk and Oleksyszyn, 2009; Chakrabarty et al, 2019; Breidenbach et al, 2020; Kahler et al, 2020)
Other ABPs are generated for targeting the serine proteases matriptase and matriptase-2; these components contain the diphenyl phosphonate warhead, two guanidinophenyl moieties as arginine mimetics, and coumarin as the fluorophore that can be detected by direct in-gel or HPLC
Summary
Proteases represent a diverse class of enzymes that cleave proteins into peptides and to amino acids, thereby controlling the proteome to a steady-state level in the organism. Other ABPs are generated for targeting the serine proteases matriptase and matriptase-2; these components contain the diphenyl phosphonate warhead, two guanidinophenyl moieties as arginine mimetics, and coumarin (aromatic lactone) as the fluorophore that can be detected by direct in-gel or HPLC fluorescence scanning (Haussler et al, 2016; Haussler et al., 2017; Breidenbach et al, 2020). Another example of an ABP is the acyloxymethyl ketone (AOMK) component, a cysteine reactive electrophile, including. The addition of ABPs to noninvasive optical and PET/ CT imaging leads to an improvement of drug discovery, tumor categorization (Gaikwad et al, 2018), and investigation of disease progressions as well as resolution of the respective mechanism (Withana et al, 2016b)
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