What you see is what you get: activity-based probes in single-cell analysis of enzymatic activities

Abstract

Molecular imaging methods can provide spatio-temporal information about the distribution of biomolecules or biological processes, such as certain enzymatic activities, in single cells. Within a cell, it is possible to define the subcellular location of a target, its trafficking through the cell, colocalization with other biomolecules of interest and involvement in certain cell biological processes. On the other hand, single-cell imaging promises to distinguish cells that are phenotypically different from each other. The corresponding cellular diversity comprises the presence of functionally distinct cells in a population (‘phenotypic heterogeneity’), as well as dynamic cellular responses to external stimuli (‘phenotypic plasticity’), which is highly relevant, e.g. during cell differentiation, activation (of immune cells), or cell death. This review focuses on applications of a certain class of chemical probes, the so-called activity-based probes (ABPs), for visualization of enzymatic activities in the single-cell context. It discusses the structure of ABPs and other chemical probes, exemplary applications of ABPs in single-cell studies in human, mouse and bacterial systems and considerations to be made with regard to data interpretation.

Identification of fungal lignocellulose-degrading biocatalysts secreted by Phanerochaete chrysosporium via activity-based protein profiling

“…As reporters, biotin, fluorophores, or so-called two-step reporter tags such as alkyne or azide moieties are frequently used 6 . In the last years, intensive efforts have been undertaken, both to develop new ABPs targeting new enzyme families and to establish their use in, amongst others, drug discovery (target and lead discovery, target engagement) [7][8][9][10][11][12] , plant biology 13 , or microbiology [14][15][16][17][18] .…”

From Differential Stains to Next Generation Physiology: Chemical Probes to Visualize Bacterial Cell Structure and Physiology

“…The ABPs can rapidly and irreversibly bind to catalytically active target enzymes by selectively and covalently modifying active site residues of the enzyme (Figure 1C). The ABPs basically comprises of three elements: (i) reactive group (also called 'warhead'), usually an electrophilic group that covalently bind with a conserved active site nucleophile; (ii) linker region or binding group that can modulate the reactivity and specificity of the probe profile [74,75]; (iii) reporter tag for the identification, enrichment and/or visualization of labeled (as reviewed in [74,[76][77][78][79]). The modular nature of ABPs in combination with their covalent interaction with probe targets, makes ABPs one of the most versatile group of chemical probes.…”

“…The single-cell frontier and the assessment of cellular phenotypic heterogeneity is another area where chemical probes are instrumental [79]. Bacterial biofilms, antibiotic persistence and the elusive viable-but-non-culturable bacteria are examples based on the presence of functionally distinct subpopulations within pathogen populations.…”

Activity‐Based Protein Profiling in Methicillin‐Resistant Staphylococcus aureus Reveals the Broad Reactivity of a Carmofur‐Derived Probe

“…A broad variety of probes has been designed to target diverse enzyme families and the application range spans from identification of drug targets and off-targets by chemical proteomics [1] to in vivo imaging or single-cell imaging studies in diverse organisms as extensively reviewed elsewhere. [2][3][4][5][6] Some probes have been designed to target enzyme families in the broadest possible way [7] and have been applied for profiling studies in diverse organisms, whereas other probes have primarily been designed and validated for interaction with a particular target in a certain organism of interest. Repurposing these probes and validating their potential interactions with as of yet unidentified targets in other biological specimen may provide a short-cut for the discovery and functional validation of previously uncharacterized enzymes.…”

“…Activity‐based protein profiling (ABPP) uses functionalized active‐site directed small molecule probes known as activity‐based probes (ABPs) to selectively label active enzymes, which can then be detected, identified and quantified through various analytical methods. A broad variety of probes has been designed to target diverse enzyme families and the application range spans from identification of drug targets and off‐targets by chemical proteomics [1] to in vivo imaging or single‐cell imaging studies in diverse organisms as extensively reviewed elsewhere [2–6] …”