Evaluation of Enzymatic Activities in Living Systems with Small-molecular Fluorescent Substrate Probes

Komatsu, Toshimitsu; Urano, Yasuteru

doi:10.2116/analsci.31.257

Cited by 44 publications

(46 citation statements)

References 77 publications

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“…β -Galactosidase ( β -gal), an enzyme that functions to remove galactose residues from gangliosides, glycoproteins, sphingolipids, and keratin sulfate; has been widely used as a marker enzyme to identify cell types, study transcriptional regulation, and investigate gene expression[1]. Thus, developing probes to detect the activity of this important enzyme has been a crucial research topic.…”

Section: Introductionmentioning

confidence: 99%

A novel near-infrared fluorescent probe for sensitive detection of β-galactosidase in living cells

Zhang

Liu

Dutta

et al. 2017

Analytica Chimica Acta

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A novel near-infrared fluorescent probe for β-galactosidase has been developed based on a hemicyanine skeleton, which is conjugated with a D-galactose residue via a glycosidic bond. The probe serves as a substrate of β-galactosidase and displays rapid and sensitive turn-on fluorescent responses to β-galactosidase in aqueous solution. A 12.8-fold enhancement of fluorescence intensity at 703 nm was observed after incubation of 10 nM of β-galactosidase with 5 μM probe for 10 min. The probe can sensitively detect as little as 0.1 nM of β-galactosidase and shows linear responses to the enzyme concentration below 1.4 nM. The kinetic study showed that the probe has high binding affinity to β-galactosidase with Km = 3.6 μM. The probe was used to detect β-galactosidase in living cells by employing the premature cell senescence model. The probe exhibited strong fluorescent signals in senescent cells but not in normal cells, which demonstrats that the probe is able to detect the endogenous senescence-associated β-galactosidase in living cells.

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Section: Introductionmentioning

confidence: 99%

A novel near-infrared fluorescent probe for sensitive detection of β-galactosidase in living cells

Zhang

Liu

Dutta

et al. 2017

Analytica Chimica Acta

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“…[3] Only af ew fluorescent substrates have been reported for methyltransferases,partly because the structural change caused by methyl-group transfer is relatively minor compared to that caused by hydrolysis or oxidation/reduction, and is difficult to use as atrigger of fluorescence change.However,there is increasing interest in understanding the cellular roles of methyl-transfer reactions,s ince members of the large enzyme families,s uch as DNAm ethyltransferases and histone methyltransferases,have important physiological functions and are considered to be potential targets for drug development to treat tumors and metabolic diseases. High-throughput screening of our chemical library with this assays ystem identified PIMT inhibitors that may be useful as leads in the design of chemical probes for controlling PIMT activity.…”

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confidence: 99%

“…[2] Many fluorescent substrates have been reported, but most of them target hydrolases and oxidoreductases. [3] Only af ew fluorescent substrates have been reported for methyltransferases,partly because the structural change caused by methyl-group transfer is relatively minor compared to that caused by hydrolysis or oxidation/reduction, and is difficult to use as atrigger of fluorescence change.…”

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Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity

et al. 2016

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We have established a coupled assay system targeting protein l-isoaspartyl methyltransferase (PIMT), a key enzyme in the metabolism of isoaspartyl peptides and proteins. The system utilizes a fluorogenic peptide probe containing an isoaspartyl residue at the P1' position of the caspase-3 recognition sequence. Following PIMT-catalyzed methyl transfer reaction, the methylated probe is specifically cleaved by caspase-3 to give fluorescence activation. High-throughput screening of our chemical library with this assay system identified PIMT inhibitors that may be useful as leads in the design of chemical probes for controlling PIMT activity.

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“…Various types of omics studies (e.g., proteomics, metabolomics) have been used to find biomarkers, but altered enzymatic activity is not always observable with widely used omics platforms. 1,2) For example, changes in protein expression level are not always reflected in functional activity, since protein functions are regulated dynamically by multiple factors, including posttranslational modifications, protein-protein interactions, and intrinsic inhibitors. 1) Since enzymatic activities in bio-fluids are often measured for diagnostic purposes (e.g., alkaline phosphatases [3][4][5] and gamma-glutamyl transferases [6][7][8] ), a better strategy might be to directly screen for altered enzymatic activities in those bio-samples.…”

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confidence: 99%

Identification of Lung Inflammation-Related Elevation of Acylamino Acid Releasing Enzyme (APEH) Activity Using an Enzymomics Approach

Komatsu

Yoshioka

Hanaoka

et al. 2016

CHEMICAL & PHARMACEUTICAL BULLETIN

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Disease-associated alterations of enzymatic functions are potentially useful as disease biomarkers, and here we show that an enzymomics (omics of active enzymes) approach, in which enzymatic activities are screened with panels of substrates, can be an effective way to identify such alterations. In the present study, we used a panel of fluorogenic substrates to search for altered enzyme activities in bronchoalveolar lavage fluid (BALF) from a mouse model of lung inflammation. We found that acylamino acid releasing enzyme (APEH) activity was highly elevated, apparently reflecting the increased population of immune cells in the inflamed lung.Key words chemical biology; fluorescent probe; enzymomics; biomarker; lung inflammation Cellular functions are mediated by various enzymes, and altered enzymatic functions are often observed during disease onset and progression. Such changes can be helpful for clinical diagnosis and monitoring, and therefore there is great interest in the discovery of novel biomarker candidates. Various types of omics studies (e.g., proteomics, metabolomics) have been used to find biomarkers, but altered enzymatic activity is not always observable with widely used omics platforms.1,2) For example, changes in protein expression level are not always reflected in functional activity, since protein functions are regulated dynamically by multiple factors, including posttranslational modifications, protein-protein interactions, and intrinsic inhibitors.1) Since enzymatic activities in bio-fluids are often measured for diagnostic purposes (e.g., alkaline phosphatases [3][4][5] and gamma-glutamyl transferases [6][7][8] ), a better strategy might be to directly screen for altered enzymatic activities in those bio-samples. This approach can be termed enzymomics (omics of active enzymes).For this purpose, two key technologies are required: firstly, a method to simultaneously monitor multiple enzymatic activities towards a range of substrates, and secondly, a method to identify target proteins exhibiting activities of interest. In this study, we developed a panel of fluorogenic substrates to read out the enzymatic activities of various peptidases, and used it to search for altered enzymatic activities in bronchoalveolar lavage fluid (BALF) from a mouse model of lung inflammation induced by lipopolysaccharide (LPS) and formyl peptide formyl-methionyl-leucyl-phenylalanine (fMLF) (Fig. 1). We found that one of the substrates showed a strongly enhanced fluorescence signal in inflammatory BALF. We then identified the enzyme mediating this activity by means of our recently developed diced electrophoresis gel (DEG) assay. In DEG assay, enzymatic activities are detected after two-dimensional native electrophoresis-based separation of the proteome, and the target protein exhibiting the activity is identified by protein identifying technologies like LC-MS/MS. 9-11)BALF is already used in diagnosis of airway diseases, and elastase is well established as one of the biomarkers of lung inflammation.12,13) Increased elas...

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Evaluation of Enzymatic Activities in Living Systems with Small-molecular Fluorescent Substrate Probes

Cited by 44 publications

References 77 publications

A novel near-infrared fluorescent probe for sensitive detection of β-galactosidase in living cells

A novel near-infrared fluorescent probe for sensitive detection of β-galactosidase in living cells

Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity

Identification of Lung Inflammation-Related Elevation of Acylamino Acid Releasing Enzyme (APEH) Activity Using an Enzymomics Approach

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