Fluorescent-conjugated polymer superquenching facilitates highly sensitive detection of proteases

Kumaraswamy, Sriram; Bergstedt, Troy S.; Shi, Xuefei; Rininsland, Frauke; Kushon, Stuart A.; Xia, Wensheng; Ley, Kevin D.; Achyuthan, Komandoor E.; McBranch, D.; Whitten, David G.

doi:10.1073/pnas.0402367101

Cited by 207 publications

(162 citation statements)

References 23 publications

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“…Approximately 10 g of cellular extract were mixed to ␥-secretase substrate (GVVIATVK, flanked by biotin and a fluorescence quencher, 20 M) in 20 l (final volume) of assay buffer (50 mM Tris-HCl,, pH 7, 2 mM EDTA, 0.05% bovine serum albumin, 2 mM reduced glutathione). After 2 h of incubation in the dark, 40 l of QTL Sensor TM (41) was added, and the fluorescence was measured in a microplate reader with excitation/emission wavelengths of 430/595 nm, respectively. Results are expressed as ␤-and ␥-secretase activities/mg of cellular protein.…”

Section: Methodsmentioning

confidence: 99%

Lithium Chloride Increases the Production of Amyloid-β Peptide Independently from Its Inhibition of Glycogen Synthase Kinase 3

Feyt

Kienlen‐Campard

Leroy

et al. 2005

Journal of Biological Chemistry

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Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-␤ peptide (A␤) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of A␤. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of ␤-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of A␤ by increasing the ␤-cleavage of amyloid precursor protein, generating more substrate for an unmodified ␥-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of ␤-catenin and slightly decreased A␤ production. It is concluded that the LiCl-mediated increase in A␤ production is not related to GSK3 inhibition.Alzheimer disease, the most frequent cause of dementia, is characterized by the presence of typical microscopic lesions in the brain of affected patients. The coexistence of intraneuronal neurofibrillary tangles and extracellular senile plaques allows confirmation of the clinical diagnosis of the disease (1).Neurofibrillary tangles are made of paired helical filaments (PHFs) 2 containing the microtubule-associated protein, tau (2-4). In Alzheimer disease, tau is hyperphosphorylated, and many serine and threonine residues (5, 6) that are found to be phosphorylated in PHF tau can be phosphorylated by GSK3 in both in vitro and transfected cells (7-9). Senile plaques contain an amyloid core that is mainly constituted of amyloid-␤ peptide (A␤) (10), which is derived from the amyloid precursor protein (APP) (11,12). The APP gene encodes 10 different APP isoforms (13) with an amino acid content varying from 365 to 770 amino acids. The neuronal APP is a single pass type I transmembrane protein containing 695 amino acids (11) that is processed by amyloidogenic and nonamyloidogenic catabolic pathways. The ␤-cleavage of APP, catalyzed by the well characterized aspartyl protease ␤-site APP-cleaving enzyme 1 (BACE1) (14), produces a C-terminal fragment of APP (␤CTF), which is further cleaved by ␥-secretase to generate A␤. The ␥-secretase activity is found as a multiprotein complex containing at least four different proteins: Aph-1, nicastrin, presenilin, and Pen-2 (15, 16). APP can also be cleaved within the A␤ sequence by an ␣-secretase. The ␣-cleavage of APP generates a soluble N-terminal fragment (s␣APP) and a 83-membrane-anchored C-terminal fragment (C83). Experimental evidence indicates that the ␣-cleavage of APP695 could be performed by members of the desintegrin and metalloprotease family, ADAM10 and ADAM17 (17). The short intracellular C-terminal domain of APP can be phosphorylated in vitro and in...

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Section: Methodsmentioning

confidence: 99%

Lithium Chloride Increases the Production of Amyloid-β Peptide Independently from Its Inhibition of Glycogen Synthase Kinase 3

Feyt

Kienlen‐Campard

Leroy

et al. 2005

Journal of Biological Chemistry

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“…These assays require specialized equipment and͞or suffer from low fluorescence intensity change as a function of enzyme activity and generally cannot be used to detect phosphorylation of natural, chemically unmodified protein substrates. The use of native substrates is attractive because inhibitor screens may yield novel inhibitors that affect the enzyme docking site, which can be at a site distant from the active site.We sought to enhance sensitivity in the measurement of enzymatic activity by amplifying the fluorescence signal using superquenching (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). This phenomenon has been described in several reports and is based on the finding that photoluminescence of conjugated polymers and related polymeric ensembles can be quenched by means of energy and͞or electron transfer to small molecule quenchers (15)(16)(17)22).…”

mentioning

confidence: 99%

Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities

Rininsland¹,

Xia²,

Wittenburg³

et al. 2004

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

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138

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An assay technology for high-throughput screening of kinase and phosphatase activities is introduced. The format is based upon superquenching of fluorescent-conjugated polymers by dyelabeled kinase͞phosphatase peptide substrates. The sensor platform is composed of highly fluorescent-conjugated polyelectrolytes colocated with the phosphate coordinating metal ion gallium on microspheres. Phosphorylated peptide substrates containing a quencher bind specifically to the metal ions by means of phosphate groups, resulting in quench of polymer fluorescence. The modulation of fluorescence signal is proportional to kinase or phosphatase activity and is monitored as a turn-off or turn-on signal, respectively. The assay is homogeneous and simple and can be run either as an endpoint measurement or in a kinetic mode. The assay meets the sensitivity required for high-throughput screening of kinase or phosphatase inhibitors and is a valuable tool for drug discovery. A modified version of the assay allows for the detection of protein phosphorylation. P hosphorylation and dephosphorylation of proteins by kinase and phosphatase enzymes mediate the regulation of cellular metabolism, growth, differentiation, and proliferation (1-3). Aberrations in kinase and phosphatase activities can lead to inflammation and diseases such as cancer (4, 5). More than 500 kinases and phosphatases are thought to be involved in the regulation of cellular activity and are possible targets for drug therapy (6). Of the kinases, Ϸ90% phosphorylate serine residues, 10% threonine, and 0.1% tyrosine residues (7). Although it has become possible to develop anti-phospho-tyrosine antibodies (8), those against phospho-serine and threonine residues are of low affinity and are often specific to only one kinase (9). Currently, non-antibody-based high-throughput screening (HTS) assays are based on methods such as time-resolved fluorescence (TRF) (10), fluorescence polarization (FP) (11-13), or fluorescence resonance energy transfer (FRET) (14). These assays require specialized equipment and͞or suffer from low fluorescence intensity change as a function of enzyme activity and generally cannot be used to detect phosphorylation of natural, chemically unmodified protein substrates. The use of native substrates is attractive because inhibitor screens may yield novel inhibitors that affect the enzyme docking site, which can be at a site distant from the active site.We sought to enhance sensitivity in the measurement of enzymatic activity by amplifying the fluorescence signal using superquenching (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). This phenomenon has been described in several reports and is based on the finding that photoluminescence of conjugated polymers and related polymeric ensembles can be quenched by means of energy and͞or electron transfer to small molecule quenchers (15)(16)(17)22). In previous studies, it was found that one quencher molecule can quench the photoluminescence of up to several hundred polymer repeat units (25)(26)(27).Our sensor platform compri...

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“…Another option may be the use of fluorescent amplifying conjugated polymers, which are composed of a large number of connected fluorophores that do not self-quench. These polymers can be many times brighter than single organic fluorophores and yet still be efficiently quenched by a single quenching moiety 33,34 ; however, additional work in this area is still needed. It should be noted that intermittent imaging, with a frame rate >1 frame per sec, cannot be performed to image a single RNA transcript over extended periods of time because it is difficult to ensure that it is the same RNA transcript being tracked from frame-to-frame.…”

Section: Discussionmentioning

confidence: 99%

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Song

Zhang

Huang

et al. 2014

JoVE

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The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3'-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts. Video LinkThe video component of this article can be found at

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Fluorescent-conjugated polymer superquenching facilitates highly sensitive detection of proteases

Cited by 207 publications

References 23 publications

Lithium Chloride Increases the Production of Amyloid-β Peptide Independently from Its Inhibition of Glycogen Synthase Kinase 3

Lithium Chloride Increases the Production of Amyloid-β Peptide Independently from Its Inhibition of Glycogen Synthase Kinase 3

Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

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