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Hiroshi Ueda - One of the best experts on this subject based on the ideXlab platform.
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evaluation of Protein ligand Interactions using the luminescent Interaction Assay flimpia with streptavidin biotin linkage
Analytical Biochemistry, 2018Co-Authors: Takahiro Yamashita, Yuki Ohmuromatsuyama, Hideki Yamaji, Keiko Gomi, Hiroshi UedaAbstract:Abstract Post-translational modifications, such as phosphorylation, are crucial in the regulation of Protein-Protein Interactions and Protein function in cell signaling. Here, we studied the Interaction between the transactivation domain peptide of cancer suppressor Protein p53 and its negative regulator Mdm2 using a novel Protein-Protein Interaction Assay, based on the modified FlimPIA using the streptavidin-biotin Interaction to link the p53 peptide and the probe enzyme. We succeeded in detecting an attenuation in the affinity of p53 towards Mdm2 caused by the phosphorylation at Thr18. It showed that the targets, which are not easy to fuse with the FlimPIA probes, such as phosphorylated peptides can be used in this system. Also, the use of streptavidin nanobeads was found effective to get clearer signal, probably due to concentration of the detection system onto the bead surface. The system was further applied to the detection of FKBP-FRB Interaction using biotinylated FKBP domain, which suggested another potential merit of this system that allows to avoid misfolding and steric hindrance often observed for the fusion Protein approach.
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improved sensitivity of firefly luminescent intermediate based Protein Interaction Assay using ser 440 mutant with lower adenylation activity
Luminescence, 2018Co-Authors: Takahiro Yamashita, Yuki Ohmuromatsuyama, Huan Lin, Hideki Yamaji, Hiroshi UedaAbstract:Protein–Protein Interaction Assays are important in various fields including molecular biology, diagnostics, and drug screening. We recently designed a novel Protein–Protein Interaction Assay, the firefly luminescent intermediate-based Protein Interaction Assay (FlimPIA), that exploited the unique reaction mechanism of firefly luciferase (Fluc). Using two mutant Flucs, each impaired with one of the two half-reactions, namely adenylation and subsequent oxidative luminescent steps, FlimPIA detects the proximity of the two Proteins tethered to the mutant Flucs. Here, we found that introducing a mutation into a residue in the hinge region (S440) of the mutant with lowered adenylation activity (‘Acceptor’ Fluc) further improved the response of FlimPIA by lowering the residual adenylation activity. Mutants with bulkier residues showed greater inhibition, probably due to increased steric hindrance at the adenylation conformation. As a result, the FlimPIA with S440 L acceptor showed the best signal/background ratio for the detection of rapamycin-induced FKBP12–FRB Interactions.
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Improved sensitivity of firefly luminescent intermediate‐based Protein Interaction Assay using Ser 440 mutant with lower adenylation activity
Luminescence : the journal of biological and chemical luminescence, 2017Co-Authors: Yuki Ohmuro-matsuyama, Takahiro Yamashita, Huan Lin, Hideki Yamaji, Hiroshi UedaAbstract:Protein–Protein Interaction Assays are important in various fields including molecular biology, diagnostics, and drug screening. We recently designed a novel Protein–Protein Interaction Assay, the firefly luminescent intermediate-based Protein Interaction Assay (FlimPIA), that exploited the unique reaction mechanism of firefly luciferase (Fluc). Using two mutant Flucs, each impaired with one of the two half-reactions, namely adenylation and subsequent oxidative luminescent steps, FlimPIA detects the proximity of the two Proteins tethered to the mutant Flucs. Here, we found that introducing a mutation into a residue in the hinge region (S440) of the mutant with lowered adenylation activity (‘Acceptor’ Fluc) further improved the response of FlimPIA by lowering the residual adenylation activity. Mutants with bulkier residues showed greater inhibition, probably due to increased steric hindrance at the adenylation conformation. As a result, the FlimPIA with S440 L acceptor showed the best signal/background ratio for the detection of rapamycin-induced FKBP12–FRB Interactions.
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Ultrasensitive Firefly Luminescent Intermediate-Based Protein-Protein Interaction Assay (FlimPIA) Based on the Functional Complementation of Mutant Firefly Luciferases.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Yuki Ohmuro-matsuyama, Hiroshi UedaAbstract:We recently developed a Protein-Protein Interaction Assay, FlimPIA (Firefly luminescent intermediate-based Protein-Protein Interaction Assay) based on the catalytic mechanism of firefly luciferase (Fluc) that can be divided into two half-reactions: the adenylation step and the oxidative luminescent steps. We engineered two mutant Fluc enzymes named "Donor" and "Acceptor" where the oxidative luminescent activity of the Donor is almost eliminated and the adenylation activity of the Acceptor is suppressed. When the Donor and the Acceptor are each fused to one of two interacting partners, and put together to interact, the Donor and the Acceptor come sufficiently close such that the Acceptor can react with the luciferyl-adenylate intermediate (LH2-AMP) produced by the Donor, and thus emit luminescence.FlimPIA can be used in vitro and in cultured cells. Owing to recent improvements, it has several advantages in terms of signal/background ratio, detectable size of interacting partner, and sensitivity over conventional Protein-Protein Interaction Assays based on Forster/fluorescence resonance energy transfer and Protein-fragment complementation performed in vitro. Here, we describe a protocol to make use of the latest version of FlimPIA which shows even lower background and higher signal than previously described ones.
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A Protein-Protein Interaction Assay FlimPIA Based on the Functional Complementation of Mutant Firefly Luciferases.
Methods of Molecular Biology, 2016Co-Authors: Yuki Ohmuro-matsuyama, Hiroshi UedaAbstract:: There is a significant focus on detecting and Assaying Protein-Protein Interactions (PPIs) in biology and biotechnology. Protein-fragment complementation Assay (PCA) is one of the most widely used methods to detect PPI by splitting the enzyme-coding or fluorescent Protein-coding polypeptide, as well as Forster resonance energy transfer (FRET). Here, we describe a novel PPI Assay FlimPIA (firefly luminescent intermediate-based Protein-Protein Interaction Assay) by a unique approach of splitting the two major catalytic steps (half reactions) of firefly luciferase (FLuc).
Yuki Ohmuro-matsuyama - One of the best experts on this subject based on the ideXlab platform.
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Improved sensitivity of firefly luminescent intermediate‐based Protein Interaction Assay using Ser 440 mutant with lower adenylation activity
Luminescence : the journal of biological and chemical luminescence, 2017Co-Authors: Yuki Ohmuro-matsuyama, Takahiro Yamashita, Huan Lin, Hideki Yamaji, Hiroshi UedaAbstract:Protein–Protein Interaction Assays are important in various fields including molecular biology, diagnostics, and drug screening. We recently designed a novel Protein–Protein Interaction Assay, the firefly luminescent intermediate-based Protein Interaction Assay (FlimPIA), that exploited the unique reaction mechanism of firefly luciferase (Fluc). Using two mutant Flucs, each impaired with one of the two half-reactions, namely adenylation and subsequent oxidative luminescent steps, FlimPIA detects the proximity of the two Proteins tethered to the mutant Flucs. Here, we found that introducing a mutation into a residue in the hinge region (S440) of the mutant with lowered adenylation activity (‘Acceptor’ Fluc) further improved the response of FlimPIA by lowering the residual adenylation activity. Mutants with bulkier residues showed greater inhibition, probably due to increased steric hindrance at the adenylation conformation. As a result, the FlimPIA with S440 L acceptor showed the best signal/background ratio for the detection of rapamycin-induced FKBP12–FRB Interactions.
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Ultrasensitive Firefly Luminescent Intermediate-Based Protein-Protein Interaction Assay (FlimPIA) Based on the Functional Complementation of Mutant Firefly Luciferases.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Yuki Ohmuro-matsuyama, Hiroshi UedaAbstract:We recently developed a Protein-Protein Interaction Assay, FlimPIA (Firefly luminescent intermediate-based Protein-Protein Interaction Assay) based on the catalytic mechanism of firefly luciferase (Fluc) that can be divided into two half-reactions: the adenylation step and the oxidative luminescent steps. We engineered two mutant Fluc enzymes named "Donor" and "Acceptor" where the oxidative luminescent activity of the Donor is almost eliminated and the adenylation activity of the Acceptor is suppressed. When the Donor and the Acceptor are each fused to one of two interacting partners, and put together to interact, the Donor and the Acceptor come sufficiently close such that the Acceptor can react with the luciferyl-adenylate intermediate (LH2-AMP) produced by the Donor, and thus emit luminescence.FlimPIA can be used in vitro and in cultured cells. Owing to recent improvements, it has several advantages in terms of signal/background ratio, detectable size of interacting partner, and sensitivity over conventional Protein-Protein Interaction Assays based on Forster/fluorescence resonance energy transfer and Protein-fragment complementation performed in vitro. Here, we describe a protocol to make use of the latest version of FlimPIA which shows even lower background and higher signal than previously described ones.
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A Protein-Protein Interaction Assay FlimPIA Based on the Functional Complementation of Mutant Firefly Luciferases.
Methods of Molecular Biology, 2016Co-Authors: Yuki Ohmuro-matsuyama, Hiroshi UedaAbstract:: There is a significant focus on detecting and Assaying Protein-Protein Interactions (PPIs) in biology and biotechnology. Protein-fragment complementation Assay (PCA) is one of the most widely used methods to detect PPI by splitting the enzyme-coding or fluorescent Protein-coding polypeptide, as well as Forster resonance energy transfer (FRET). Here, we describe a novel PPI Assay FlimPIA (firefly luminescent intermediate-based Protein-Protein Interaction Assay) by a unique approach of splitting the two major catalytic steps (half reactions) of firefly luciferase (FLuc).
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Ultra sensitive firefly luciferase‐based Protein‐Protein Interaction Assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions
Biotechnology journal, 2015Co-Authors: Makoto Kurihara, Yuki Ohmuro-matsuyama, Takahiro Yamashita, Hideki Yamaji, Keiichi Ayabe, Hiroshi UedaAbstract:Detecting and Assaying Protein-Protein Interactions are significant research procedures in biology and biotechnology. We recently reported a novel Assay to detect Protein-Protein Interaction, i.e. firefly luminescent intermediate-based Protein-Protein Interaction Assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This Assay detects neighboring of two mutant Flucs, namely, a "Donor" that catalyzes the adenylation of firefly luciferin to produce a luciferyl-adenylate intermediate, and an "Acceptor" that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the Assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl-adenylate formed during the catalysis and an increased ATP-based Km value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to ∼40 was attained, which promises wider use of the Assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena.
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Improved Protein–Protein Interaction Assay FlimPIA by the Entrapment of Luciferase Conformation
Analytical chemistry, 2013Co-Authors: Yuki Ohmuro-matsuyama, Yuko Hara, Hiroshi UedaAbstract:Recently we reported a novel Protein–Protein Interaction Assay FlimPIA (firefly luminescent intermediate-based Protein–Protein Interaction Assay) based on the functional complementation of two mutant firefly luciferases (Fluc), each defective in its one of two half reactions. The Assay detects approximation of two mutant Flucs, namely, a “Donor” that catalyzes ATP-driven luciferin adenylation to produce a luciferyl-adenylate intermediate, and an “Acceptor” that mainly catalyzes subsequent oxidative luminescent reaction. However, there was a problem in FlimPIA that the remaining adenylation activity of the Acceptor constituted its background signal and hampered its wider use. In this study, we aimed at reducing the background signal by trapping the Acceptor to the “oxidation” conformation, either chemically or by disulfide bonding. The results showed higher sensitivity and detection over the longer distance of the developed Assay compared to conventional FlimPIA, Fluc-based Protein-fragment complementation...
Yuki Ohmuromatsuyama - One of the best experts on this subject based on the ideXlab platform.
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evaluation of Protein ligand Interactions using the luminescent Interaction Assay flimpia with streptavidin biotin linkage
Analytical Biochemistry, 2018Co-Authors: Takahiro Yamashita, Yuki Ohmuromatsuyama, Hideki Yamaji, Keiko Gomi, Hiroshi UedaAbstract:Abstract Post-translational modifications, such as phosphorylation, are crucial in the regulation of Protein-Protein Interactions and Protein function in cell signaling. Here, we studied the Interaction between the transactivation domain peptide of cancer suppressor Protein p53 and its negative regulator Mdm2 using a novel Protein-Protein Interaction Assay, based on the modified FlimPIA using the streptavidin-biotin Interaction to link the p53 peptide and the probe enzyme. We succeeded in detecting an attenuation in the affinity of p53 towards Mdm2 caused by the phosphorylation at Thr18. It showed that the targets, which are not easy to fuse with the FlimPIA probes, such as phosphorylated peptides can be used in this system. Also, the use of streptavidin nanobeads was found effective to get clearer signal, probably due to concentration of the detection system onto the bead surface. The system was further applied to the detection of FKBP-FRB Interaction using biotinylated FKBP domain, which suggested another potential merit of this system that allows to avoid misfolding and steric hindrance often observed for the fusion Protein approach.
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improved sensitivity of firefly luminescent intermediate based Protein Interaction Assay using ser 440 mutant with lower adenylation activity
Luminescence, 2018Co-Authors: Takahiro Yamashita, Yuki Ohmuromatsuyama, Huan Lin, Hideki Yamaji, Hiroshi UedaAbstract:Protein–Protein Interaction Assays are important in various fields including molecular biology, diagnostics, and drug screening. We recently designed a novel Protein–Protein Interaction Assay, the firefly luminescent intermediate-based Protein Interaction Assay (FlimPIA), that exploited the unique reaction mechanism of firefly luciferase (Fluc). Using two mutant Flucs, each impaired with one of the two half-reactions, namely adenylation and subsequent oxidative luminescent steps, FlimPIA detects the proximity of the two Proteins tethered to the mutant Flucs. Here, we found that introducing a mutation into a residue in the hinge region (S440) of the mutant with lowered adenylation activity (‘Acceptor’ Fluc) further improved the response of FlimPIA by lowering the residual adenylation activity. Mutants with bulkier residues showed greater inhibition, probably due to increased steric hindrance at the adenylation conformation. As a result, the FlimPIA with S440 L acceptor showed the best signal/background ratio for the detection of rapamycin-induced FKBP12–FRB Interactions.
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ultra sensitive firefly luciferase based Protein Protein Interaction Assay flimpia attained by hinge region engineering and optimized reaction conditions
Biotechnology Journal, 2016Co-Authors: Makoto Kurihara, Hiroshi Ueda, Yuki Ohmuromatsuyama, Takahiro Yamashita, Hideki Yamaji, Keiichi AyabeAbstract:: Detecting and Assaying Protein-Protein Interactions are significant research procedures in biology and biotechnology. We recently reported a novel Assay to detect Protein-Protein Interaction, i.e. firefly luminescent intermediate-based Protein-Protein Interaction Assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This Assay detects neighboring of two mutant Flucs, namely, a "Donor" that catalyzes the adenylation of firefly luciferin to produce a luciferyl-adenylate intermediate, and an "Acceptor" that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the Assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl-adenylate formed during the catalysis and an increased ATP-based Km value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to ∼40 was attained, which promises wider use of the Assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena.
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improved Protein Protein Interaction Assay flimpia by the entrapment of luciferase conformation
Analytical Chemistry, 2014Co-Authors: Yuki Ohmuromatsuyama, Yuko Hara, Hiroshi UedaAbstract:Recently we reported a novel Protein–Protein Interaction Assay FlimPIA (firefly luminescent intermediate-based Protein–Protein Interaction Assay) based on the functional complementation of two mutant firefly luciferases (Fluc), each defective in its one of two half reactions. The Assay detects approximation of two mutant Flucs, namely, a “Donor” that catalyzes ATP-driven luciferin adenylation to produce a luciferyl-adenylate intermediate, and an “Acceptor” that mainly catalyzes subsequent oxidative luminescent reaction. However, there was a problem in FlimPIA that the remaining adenylation activity of the Acceptor constituted its background signal and hampered its wider use. In this study, we aimed at reducing the background signal by trapping the Acceptor to the “oxidation” conformation, either chemically or by disulfide bonding. The results showed higher sensitivity and detection over the longer distance of the developed Assay compared to conventional FlimPIA, Fluc-based Protein-fragment complementation...
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a Protein Protein Interaction Assay based on the functional complementation of mutant firefly luciferases
Analytical Chemistry, 2013Co-Authors: Yuki Ohmuromatsuyama, Keiichi Ayabe, Kota Nakano, Aoi Kimura, Masaki Ihara, Takeshi Wada, Hiroshi UedaAbstract:We report a novel bioluminescent Protein–Protein Interaction (PPI) Assay, which is based on the functional complementation of two mutant firefly luciferases (Fluc). The chemical reaction catalyzed by Fluc is divided into two half reactions of ATP-driven luciferin adenylation and subsequent oxidative reactions. In the former adenylation half-reaction, a luciferyl-adenylate (LH2-AMP) intermediate is produced from LH2 and ATP. With this intermediate, the latter oxidative reactions produce oxyluciferin via proton abstraction at the C4 carbon of LH2-AMP. We created and optimized two Fluc mutants; one is named “Donor”, which virtually lacks oxidative activity, while the other, named “Acceptor”, is almost defective in the adenylation activity. Then, the two mutants are fused to interacting partners, and prepared as pure Proteins. When the Interaction between the partners is induced, higher efficiency of LH2-AMP transfer between the Donor and Acceptor enzymes resulted in increased luminescence. The Assay was foun...
Keiichi Ayabe - One of the best experts on this subject based on the ideXlab platform.
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ultra sensitive firefly luciferase based Protein Protein Interaction Assay flimpia attained by hinge region engineering and optimized reaction conditions
Biotechnology Journal, 2016Co-Authors: Makoto Kurihara, Hiroshi Ueda, Yuki Ohmuromatsuyama, Takahiro Yamashita, Hideki Yamaji, Keiichi AyabeAbstract:: Detecting and Assaying Protein-Protein Interactions are significant research procedures in biology and biotechnology. We recently reported a novel Assay to detect Protein-Protein Interaction, i.e. firefly luminescent intermediate-based Protein-Protein Interaction Assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This Assay detects neighboring of two mutant Flucs, namely, a "Donor" that catalyzes the adenylation of firefly luciferin to produce a luciferyl-adenylate intermediate, and an "Acceptor" that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the Assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl-adenylate formed during the catalysis and an increased ATP-based Km value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to ∼40 was attained, which promises wider use of the Assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena.
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Ultra sensitive firefly luciferase‐based Protein‐Protein Interaction Assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions
Biotechnology journal, 2015Co-Authors: Makoto Kurihara, Yuki Ohmuro-matsuyama, Takahiro Yamashita, Hideki Yamaji, Keiichi Ayabe, Hiroshi UedaAbstract:Detecting and Assaying Protein-Protein Interactions are significant research procedures in biology and biotechnology. We recently reported a novel Assay to detect Protein-Protein Interaction, i.e. firefly luminescent intermediate-based Protein-Protein Interaction Assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This Assay detects neighboring of two mutant Flucs, namely, a "Donor" that catalyzes the adenylation of firefly luciferin to produce a luciferyl-adenylate intermediate, and an "Acceptor" that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the Assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl-adenylate formed during the catalysis and an increased ATP-based Km value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to ∼40 was attained, which promises wider use of the Assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena.
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A Protein-Protein Interaction Assay based on the functional complementation of mutant firefly luciferases.
Analytical chemistry, 2013Co-Authors: Yuki Ohmuro-matsuyama, Keiichi Ayabe, Kota Nakano, Aoi Kimura, Masaki Ihara, Takeshi Wada, Hiroshi UedaAbstract:We report a novel bioluminescent Protein-Protein Interaction (PPI) Assay, which is based on the functional complementation of two mutant firefly luciferases (Fluc). The chemical reaction catalyzed by Fluc is divided into two half reactions of ATP-driven luciferin adenylation and subsequent oxidative reactions. In the former adenylation half-reaction, a luciferyl-adenylate (LH2-AMP) intermediate is produced from LH2 and ATP. With this intermediate, the latter oxidative reactions produce oxyluciferin via proton abstraction at the C4 carbon of LH2-AMP. We created and optimized two Fluc mutants; one is named "Donor", which virtually lacks oxidative activity, while the other, named "Acceptor", is almost defective in the adenylation activity. Then, the two mutants are fused to interacting partners, and prepared as pure Proteins. When the Interaction between the partners is induced, higher efficiency of LH2-AMP transfer between the Donor and Acceptor enzymes resulted in increased luminescence. The Assay was found to work both in vitro and in cultured cells with strong signals. This would be the first example of reconstituting two divided reactions of one enzyme to detect PPI, which will not only be utilized as a robust PPI Assay, but also open a way to control the activity of similar enzymes in acyl/adenylate-forming enzyme superfamily.
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A Protein–Protein Interaction Assay Based on the Functional Complementation of Mutant Firefly Luciferases
Analytical Chemistry, 2013Co-Authors: Yuki Ohmuro-matsuyama, Keiichi Ayabe, Kota Nakano, Aoi Kimura, Masaki Ihara, Takeshi Wada, Hiroshi UedaAbstract:We report a novel bioluminescent Protein–Protein Interaction (PPI) Assay, which is based on the functional complementation of two mutant firefly luciferases (Fluc). The chemical reaction catalyzed by Fluc is divided into two half reactions of ATP-driven luciferin adenylation and subsequent oxidative reactions. In the former adenylation half-reaction, a luciferyl-adenylate (LH2-AMP) intermediate is produced from LH2 and ATP. With this intermediate, the latter oxidative reactions produce oxyluciferin via proton abstraction at the C4 carbon of LH2-AMP. We created and optimized two Fluc mutants; one is named “Donor”, which virtually lacks oxidative activity, while the other, named “Acceptor”, is almost defective in the adenylation activity. Then, the two mutants are fused to interacting partners, and prepared as pure Proteins. When the Interaction between the partners is induced, higher efficiency of LH2-AMP transfer between the Donor and Acceptor enzymes resulted in increased luminescence. The Assay was foun...
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a Protein Protein Interaction Assay based on the functional complementation of mutant firefly luciferases
Analytical Chemistry, 2013Co-Authors: Yuki Ohmuromatsuyama, Keiichi Ayabe, Kota Nakano, Aoi Kimura, Masaki Ihara, Takeshi Wada, Hiroshi UedaAbstract:We report a novel bioluminescent Protein–Protein Interaction (PPI) Assay, which is based on the functional complementation of two mutant firefly luciferases (Fluc). The chemical reaction catalyzed by Fluc is divided into two half reactions of ATP-driven luciferin adenylation and subsequent oxidative reactions. In the former adenylation half-reaction, a luciferyl-adenylate (LH2-AMP) intermediate is produced from LH2 and ATP. With this intermediate, the latter oxidative reactions produce oxyluciferin via proton abstraction at the C4 carbon of LH2-AMP. We created and optimized two Fluc mutants; one is named “Donor”, which virtually lacks oxidative activity, while the other, named “Acceptor”, is almost defective in the adenylation activity. Then, the two mutants are fused to interacting partners, and prepared as pure Proteins. When the Interaction between the partners is induced, higher efficiency of LH2-AMP transfer between the Donor and Acceptor enzymes resulted in increased luminescence. The Assay was foun...
F. Letourneur - One of the best experts on this subject based on the ideXlab platform.
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Acidic clusters target transmembrane Proteins to the contractile vacuole in Dictyostelium cells.
Journal of cell science, 2006Co-Authors: V. Mercanti, C. Blanc, Y. Lefkir, P. Cosson, F. LetourneurAbstract:The mechanisms responsible for the targeting of transmembrane integral Proteins to the contractile vacuole (CV) network in Dictyostelium discoideum are unknown. Here we show that the transfer of the cytoplasmic domain of a CV-resident Protein (Rh50) to a reporter transmembrane Protein (CsA) is sufficient to address the chimera (CsA-Rh50) to the CV. We identified two clusters of acidic residues responsible for this targeting, and these motifs interacted with the gamma-adaptin AP-1 subunit in a yeast Protein-Protein Interaction Assay. For the first time we report the existence of an indirect transport pathway from the plasma membrane to the CV via endosomes. Upon internalization, the small fraction of CsA-Rh50 present at the cell surface was first concentrated in endosomes distinct from early and late p80-positive endosomes and then slowly transported to the CV. Together our results suggest the existence of an AP-1-dependent selective transport to the contractile vacuole in Dictyostelium.
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Acidic clusters target transmembrane Proteins to the contractile vacuole in Dictyostelium cells.
Journal of Cell Science, 2006Co-Authors: V. Mercanti, C. Blanc, Y. Lefkir, P. Cosson, F. LetourneurAbstract:The mechanisms responsible for the targeting of transmembrane integral Proteins to the contractile vacuole (CV) network in Dictyostelium discoideum are unknown. Here we show that the transfer of the cytoplasmic domain of a CV-resident Protein (Rh50) to a reporter transmembrane Protein (CsA) is sufficient to address the chimera (CsA-Rh50) to the CV. We identified two clusters of acidic residues responsible for this targeting, and these motifs interacted with the gamma-adaptin AP-1 subunit in a yeast Protein-Protein Interaction Assay. For the first time we report the existence of an indirect transport pathway from the plasma membrane to the CV via endosomes. Upon internalization, the small fraction of CsA-Rh50 present at the cell surface was first concentrated in endosomes distinct from early and late p80-positive endosomes and then slowly transported to the CV. Together our results suggest the existence of an AP-1-dependent selective transport to the contractile vacuole in Dictyostelium.The mechanisms responsible for the targeting of transmembrane integral Proteins to the contractile vacuole (CV) network in Dictyostelium discoideum are unknown. Here we show that the transfer of the cytoplasmic domain of a CV-resident Protein (Rh50) to a reporter transmembrane Protein (CsA) is sufficient to address the chimera (CsA-Rh50) to the CV. We identified two clusters of acidic residues responsible for this targeting, and these motifs interacted with the gamma-adaptin AP-1 subunit in a yeast Protein-Protein Interaction Assay. For the first time we report the existence of an indirect transport pathway from the plasma membrane to the CV via endosomes. Upon internalization, the small fraction of CsA-Rh50 present at the cell surface was first concentrated in endosomes distinct from early and late p80-positive endosomes and then slowly transported to the CV. Together our results suggest the existence of an AP-1-dependent selective transport to the contractile vacuole in Dictyostelium.
