Recent advances in activity-based probes (ABPs) and affinity-based probes (A<i>f</i>BPs) for profiling of enzymes

Fang, Haixiao; Peng, Bo; Ong, Simon Yew Kuang; Wu, Qiong; Li, Lin; Yao, Shao Q.

doi:10.1039/d1sc01359a

Cited by 122 publications

(95 citation statements)

References 171 publications

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“…Whenever the catalytic mechanism of an enzyme is known, it is possible to develop chemical probes that covalently bind to the enzyme's active site and allow the detection of the enzyme via a reporter moiety grafted to the probe 266 . These compounds are named activity‐based probes (ABPs), given their dependence on the enzymatic activity rather than molecular affinity alone 267 . ABPs are generally composed by a tag, a linker and an active site binding motif incorporating a reactive group, also called ‘warhead’ 268 .…”

Section: Methods To Analyze Lysosome Morphology Positioning Motility ...mentioning

confidence: 99%

“…ABPs are generally composed by a tag, a linker and an active site binding motif incorporating a reactive group, also called ‘warhead’ 268 . For successfully monitoring the activity of lysosomal enzymes in vivo and in situ , ABPs must be able to efficiently reach lysosomes and fluoresce within the acidic environment of the lysosome lumen 267 . Fluorophores with low pKa values ( e.g ., BODIPY, DAMP, Cy5.5, etc.)…”

Section: Methods To Analyze Lysosome Morphology Positioning Motility ...mentioning

confidence: 99%

“…have optimal fluorescence in the low pH of the lysosome and have been commonly used for labelling lysosomal hydrolases. 263,267 Moreover, while fluorogenic substrates make it possible to detect the activity of lysosomal hydrolases in living cells or tissue sections, ABPs have a broader range of applications because of their ability to covalently bind the enzyme's active site. The sensitive and specific mechanism of binding of ABPs enables the visualization of endogenous enzymes in complex samples such as cell lysates and tissue homogenates, as well as in whole cells, tissue sections or even living organisms, in the case of ABPs with infrared tags detected by magnetic resonance imaging.…”

Section: Assessing Lysosomal Hydrolase Activity In Vivomentioning

confidence: 99%

“…273 In particular, probes with a tag suited for pull-down experiments such as biotin, can be employed for affinity-enrichment and identification of the labelled targets and/or interactors by proteomics, a technique termed activitybased protein profiling (ABPP). 267 A broad range of fluorogenic substrates to monitor lysosomal hydrolase activity is now commercially available, making them accessible and easy-to-use tools. Magic Red substrates, in particular, have recently been adapted for use in HCI analysis protocols to monitor lysosome function in LSD models by using Opera and Operetta systems (PerkinElmer).…”

Section: Assessing Lysosomal Hydrolase Activity In Vivomentioning

confidence: 99%

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Current methods to analyze lysosome morphology, positioning, motility and function

Barral

Staiano

Almeida

et al. 2022

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Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground‐breaking discoveries on an organelle that continues to surprise and excite cell biologists.

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Section: Methods To Analyze Lysosome Morphology Positioning Motility ...mentioning

confidence: 99%

Section: Methods To Analyze Lysosome Morphology Positioning Motility ...mentioning

confidence: 99%

Section: Assessing Lysosomal Hydrolase Activity In Vivomentioning

confidence: 99%

Section: Assessing Lysosomal Hydrolase Activity In Vivomentioning

confidence: 99%

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Current methods to analyze lysosome morphology, positioning, motility and function

Barral

Staiano

Almeida

et al. 2022

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“…ABPP revolves around the use activity-based probes (ABPs) to detect and identify specific probe-reactive enzymes within a mixture [ 25 ]. ABPs are covalent small-molecule inhibitors that contain a well-placed reactive warhead functional group, a recognition motif, and a detection handle [ 26 ]. Cyclophellitol-derived ABPs for glycoside hydrolases (GHs) use a cyclitol ring recognition motif configured to match the stereochemistry of an enzyme’s cognate glycone [ 27 , 28 ].…”

Section: Introductionmentioning

confidence: 99%

Activity-based protein profiling reveals dynamic substrate-specific cellulase secretion by saprotrophic basidiomycetes

McGregor

Boer

Santos

et al. 2022

Biotechnol Biofuels

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Background Fungal saccharification of lignocellulosic biomass occurs concurrently with the secretion of a diverse collection of proteins, together functioning as a catalytic system to liberate soluble sugars from insoluble composite biomaterials. How different fungi respond to different substrates is of fundamental interest to the developing biomass saccharification industry. Among the cornerstones of fungal enzyme systems are the highly expressed cellulases (endo-β-glucanases and cellobiohydrolases). Recently, a cyclophellitol-derived activity-based probe (ABP-Cel) was shown to be a highly sensitive tool for the detection and identification of cellulases. Results Here we show that ABP-Cel enables endo-β-glucanase profiling in diverse fungal secretomes. In combination with established ABPs for β-xylanases and β-d-glucosidases, we collected multiplexed in-gel fluorescence activity-based protein profiles of 240 secretomes collected over ten days from biological replicates of ten different basidiomycete fungi grown on maltose, wheat straw, or aspen pulp. Our results reveal the remarkable dynamics and unique enzyme fingerprints associated with each species substrate combination. Chemical proteomic analysis identifies significant arsenals of cellulases secreted by each fungal species during growth on lignocellulosic biomass. Recombinant production and characterization of a collection of probe-reactive enzymes from GH5, GH10, and GH12 confirm that ABP-Cel shows broad selectivity towards enzymes with endo-β-glucanase activity. Conclusion Using small-volume samples with minimal sample preparation, the results presented here demonstrate the ready accessibility of sensitive direct evidence for fungal enzyme secretion during early stages of growth on complex lignocellulosic substrates.

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Reactivity‐Tunable Fluorescent Platform for Selective and Biocompatible Modification of Cysteine or Lysine

Ren,

Li,

Peng

et al. 2024

Advanced Science

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Chemoselective modification of specific residues within a given protein poses a significant challenge, as the microenvironment of amino acid residues in proteins is variable. Developing a universal molecular platform with tunable chemical warheads can provide powerful tools for precisely labeling specific amino acids in proteins. Cysteine and lysine are hot targets for chemoselective modification, but current cysteine/lysine‐selective warheads face challenges due to cross‐reactivity and unstable reaction products. In this study, a versatile fluorescent platform is developed for highly selective modification of cysteine/lysine under biocompatible conditions. Chloro‐ or phenoxy‐substituted NBSe derivatives effectively labeled cysteine residues in the cellular proteome with high specificity. This finding also led to the development of phenoxy‐NBSe phototheragnostic for the diagnosis and activatable photodynamic therapy of GSH‐overexpressed cancer cells. Conversely, alkoxy‐NBSe derivatives are engineered to selectively react with lysine residues in the cellular environment, exhibiting excellent anti‐interfering ability against thiols. Leveraging a proximity‐driven approach, alkoxy‐NBSe probes are successfully designed to demonstrate their utility in bioimaging of lysine deacetylase activity. This study also achieves integrating a small photosensitizer into lysine residues of proteins in a regioselective manner, achieving photoablation of cancer cells activated by overexpressed proteins.

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Recent advances in activity-based probes (ABPs) and affinity-based probes (AfBPs) for profiling of enzymes

Abstract: This review highlights the recent advances in the use of activity-based probes (ABPs) and affinity-based probes (AfBPs), and summarizes their design strategies (based on inhibitors and substrates) and detection approaches.

Cited by 122 publications

References 171 publications

Current methods to analyze lysosome morphology, positioning, motility and function

Current methods to analyze lysosome morphology, positioning, motility and function

Activity-based protein profiling reveals dynamic substrate-specific cellulase secretion by saprotrophic basidiomycetes

Reactivity‐Tunable Fluorescent Platform for Selective and Biocompatible Modification of Cysteine or Lysine

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