Characterization, application and potential uses of biotin-tagged inhibitors for lymphocyte serine proteases (granzymes)

Winkler, Ulrike; Allison, Nigel; Woodard, Susan L.; Gault, Ruth A.; Ewoldt, Gerald R.; Kam, Chih-Min; Abuelyaman, Ahmed S.; Powers, James C.; Hudig, Dorothy

doi:10.1016/0161-5890(96)00025-9

Cited by 15 publications

(9 citation statements)

References 34 publications

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“…However, this inhibitor displayed remarkable specificity for NTE in these experiments and thus appeared too selective to be useful as a general probe for serine hydrolases. Powers and colleagues had generated isocoumarin inhibitors coupled to biotin as serine hydrolase inhibitors (19,20). Although these isocoumarins reacted with a greater range of serine hydrolases than the aforementioned saligenin phosphoramidate, the requirement that these compounds alkylate a second functional group in the enzyme active site to achieve stable irreversible inhibition suggested that a significant number of serine hydrolases might show poor sensitivity to such reagents.…”

Section: Resultsmentioning

confidence: 99%

Activity-based protein profiling: The serine hydrolases

Liu

Patricelli

Cravatt

1999

Proc. Natl. Acad. Sci. U.S.A.

1,034

935

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With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FPbiotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.

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Section: Resultsmentioning

confidence: 99%

Activity-based protein profiling: The serine hydrolases

Liu

Patricelli

Cravatt

1999

Proc. Natl. Acad. Sci. U.S.A.

1,034

935

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“…Isocoumarin derivatives and DCI have been used as molecular probes to characterize the biological role of lymphocyte granule proteases in cytotoxic lymphocytes (CTL) cells. ,,, DCI inhibits all five granzymes and inactivates cytolysis. , The lysis is restored when the inhibited granzymes are reactivated by hydroxylamine, which indicates that serine proteases are essential for hydrolysis. Biotinylated isocoumarins were successfully used in the detection of granzymes in CTLs. ,, …”

Section: Isocoumarinsmentioning

confidence: 99%

Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases

et al. 2002

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“…To date, only a few groups have tried to make chemical functional ABPs for Grs, but the major issue was poor selectivity of these probes for individual enzymes. 12 The first ABP for GrB and GrA designed using combinatorial chemistry methods was reported by the Craik group; however, the kinetic inhibition constant rates of the designed ABPs were quite low (k obs /I = 460 M −1 s −1 for GrB and 2000 M −1 s −1 for GrA), making them problematic for use in biological assays. 13 T h i s c o n t e n t i s Here, we describe a novel ABP that will allow us to distinguish GrA from other Grs and allow for GrA detection in biological samples.…”

Section: ■ Introductionmentioning

confidence: 99%

Detection of Active Granzyme A in NK92 Cells with Fluorescent Activity-Based Probe

et al. 2020

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Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.

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Characterization, application and potential uses of biotin-tagged inhibitors for lymphocyte serine proteases (granzymes)

Cited by 15 publications

References 34 publications

Activity-based protein profiling: The serine hydrolases

Activity-based protein profiling: The serine hydrolases

Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases

Detection of Active Granzyme A in NK92 Cells with Fluorescent Activity-Based Probe

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