9 research outputs found
Ligand-Directed Chemistry of AMPA Receptors Confers Live-Cell Fluorescent Biosensors
AMPA-type glutamate
receptors (AMPARs) mediate fast excitatory
synaptic transmission in the central nervous system. Dysregulation
of AMPAR function is associated with many kinds of neurological, neurodegenerative,
and psychiatric disorders. As a result, molecules capable of controlling
AMPAR functions are potential therapeutic agents. Fluorescent semisynthetic
biosensors have attracted considerable interest for the discovery
of ligands selectively acting on target proteins. Given the large
protein complex formation of AMPARs in live cells, biosensors using
full-length AMPARs retaining original functionality are ideal for
drug screening. Here, we demonstrate that fluorophore-labeled AMPARs
prepared by ligand-directed acyl imidazole chemistry can act as turn-on
fluorescent biosensors for AMPAR ligands in living cells. These biosensors
selectively detect orthosteric ligands of AMPARs among the glutamate
receptor family. Notably, the dissociation constants of agonists and
antagonists for AMPARs were determined in live cells, which revealed
that the ligand-binding properties of AMPARs to agonists are largely
different in living cells, compared with noncellular conditions. We
also show that these sensors can be applied to detecting allosteric
modulators or subunit-selective ligands of AMPARs. Thus, our protein-based
biosensors can be useful for discovering pharmaceutical agents to
treat AMPAR-related neurological disorders
Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>
Characterizing
the protein constituents of a specific
organelle
and protein neighbors of a protein of interest (POI) is essential
for understanding the function and state of the organelle and protein
networks associated with the POI. Proximity labeling (PL) has emerged
as a promising technology for specific and efficient spatial proteomics.
Nevertheless, most enzymes adopted for PL still have limitations:
APEX requires cytotoxic H2O2 for activation
and thus is poor in biocompatibility for in vivo application,
BioID shows insufficient labeling kinetics, and TurboID suffers from
high background biotinylation. Here, we introduce a bacterial tyrosinase
(BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background
protein tagging. BmTyr is genetically encodable and enables subcellular-resolved
PL and proteomics in living cells. We further designed a strategy
of ligand-tethered BmTyr for in vivo PL, which unveiled
the surrounding proteome of a neurotransmitter receptor (Grm1 and
Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr
is one promising enzyme that can improve and expand PL-based applications
and discoveries
Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>
Characterizing
the protein constituents of a specific
organelle
and protein neighbors of a protein of interest (POI) is essential
for understanding the function and state of the organelle and protein
networks associated with the POI. Proximity labeling (PL) has emerged
as a promising technology for specific and efficient spatial proteomics.
Nevertheless, most enzymes adopted for PL still have limitations:
APEX requires cytotoxic H2O2 for activation
and thus is poor in biocompatibility for in vivo application,
BioID shows insufficient labeling kinetics, and TurboID suffers from
high background biotinylation. Here, we introduce a bacterial tyrosinase
(BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background
protein tagging. BmTyr is genetically encodable and enables subcellular-resolved
PL and proteomics in living cells. We further designed a strategy
of ligand-tethered BmTyr for in vivo PL, which unveiled
the surrounding proteome of a neurotransmitter receptor (Grm1 and
Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr
is one promising enzyme that can improve and expand PL-based applications
and discoveries
Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>
Characterizing
the protein constituents of a specific
organelle
and protein neighbors of a protein of interest (POI) is essential
for understanding the function and state of the organelle and protein
networks associated with the POI. Proximity labeling (PL) has emerged
as a promising technology for specific and efficient spatial proteomics.
Nevertheless, most enzymes adopted for PL still have limitations:
APEX requires cytotoxic H2O2 for activation
and thus is poor in biocompatibility for in vivo application,
BioID shows insufficient labeling kinetics, and TurboID suffers from
high background biotinylation. Here, we introduce a bacterial tyrosinase
(BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background
protein tagging. BmTyr is genetically encodable and enables subcellular-resolved
PL and proteomics in living cells. We further designed a strategy
of ligand-tethered BmTyr for in vivo PL, which unveiled
the surrounding proteome of a neurotransmitter receptor (Grm1 and
Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr
is one promising enzyme that can improve and expand PL-based applications
and discoveries
Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>
Characterizing
the protein constituents of a specific
organelle
and protein neighbors of a protein of interest (POI) is essential
for understanding the function and state of the organelle and protein
networks associated with the POI. Proximity labeling (PL) has emerged
as a promising technology for specific and efficient spatial proteomics.
Nevertheless, most enzymes adopted for PL still have limitations:
APEX requires cytotoxic H2O2 for activation
and thus is poor in biocompatibility for in vivo application,
BioID shows insufficient labeling kinetics, and TurboID suffers from
high background biotinylation. Here, we introduce a bacterial tyrosinase
(BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background
protein tagging. BmTyr is genetically encodable and enables subcellular-resolved
PL and proteomics in living cells. We further designed a strategy
of ligand-tethered BmTyr for in vivo PL, which unveiled
the surrounding proteome of a neurotransmitter receptor (Grm1 and
Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr
is one promising enzyme that can improve and expand PL-based applications
and discoveries
Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>
Characterizing
the protein constituents of a specific
organelle
and protein neighbors of a protein of interest (POI) is essential
for understanding the function and state of the organelle and protein
networks associated with the POI. Proximity labeling (PL) has emerged
as a promising technology for specific and efficient spatial proteomics.
Nevertheless, most enzymes adopted for PL still have limitations:
APEX requires cytotoxic H2O2 for activation
and thus is poor in biocompatibility for in vivo application,
BioID shows insufficient labeling kinetics, and TurboID suffers from
high background biotinylation. Here, we introduce a bacterial tyrosinase
(BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background
protein tagging. BmTyr is genetically encodable and enables subcellular-resolved
PL and proteomics in living cells. We further designed a strategy
of ligand-tethered BmTyr for in vivo PL, which unveiled
the surrounding proteome of a neurotransmitter receptor (Grm1 and
Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr
is one promising enzyme that can improve and expand PL-based applications
and discoveries
Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>
Characterizing
the protein constituents of a specific
organelle
and protein neighbors of a protein of interest (POI) is essential
for understanding the function and state of the organelle and protein
networks associated with the POI. Proximity labeling (PL) has emerged
as a promising technology for specific and efficient spatial proteomics.
Nevertheless, most enzymes adopted for PL still have limitations:
APEX requires cytotoxic H2O2 for activation
and thus is poor in biocompatibility for in vivo application,
BioID shows insufficient labeling kinetics, and TurboID suffers from
high background biotinylation. Here, we introduce a bacterial tyrosinase
(BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background
protein tagging. BmTyr is genetically encodable and enables subcellular-resolved
PL and proteomics in living cells. We further designed a strategy
of ligand-tethered BmTyr for in vivo PL, which unveiled
the surrounding proteome of a neurotransmitter receptor (Grm1 and
Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr
is one promising enzyme that can improve and expand PL-based applications
and discoveries
Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>
Characterizing
the protein constituents of a specific
organelle
and protein neighbors of a protein of interest (POI) is essential
for understanding the function and state of the organelle and protein
networks associated with the POI. Proximity labeling (PL) has emerged
as a promising technology for specific and efficient spatial proteomics.
Nevertheless, most enzymes adopted for PL still have limitations:
APEX requires cytotoxic H2O2 for activation
and thus is poor in biocompatibility for in vivo application,
BioID shows insufficient labeling kinetics, and TurboID suffers from
high background biotinylation. Here, we introduce a bacterial tyrosinase
(BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background
protein tagging. BmTyr is genetically encodable and enables subcellular-resolved
PL and proteomics in living cells. We further designed a strategy
of ligand-tethered BmTyr for in vivo PL, which unveiled
the surrounding proteome of a neurotransmitter receptor (Grm1 and
Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr
is one promising enzyme that can improve and expand PL-based applications
and discoveries
Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>
Characterizing
the protein constituents of a specific
organelle
and protein neighbors of a protein of interest (POI) is essential
for understanding the function and state of the organelle and protein
networks associated with the POI. Proximity labeling (PL) has emerged
as a promising technology for specific and efficient spatial proteomics.
Nevertheless, most enzymes adopted for PL still have limitations:
APEX requires cytotoxic H2O2 for activation
and thus is poor in biocompatibility for in vivo application,
BioID shows insufficient labeling kinetics, and TurboID suffers from
high background biotinylation. Here, we introduce a bacterial tyrosinase
(BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background
protein tagging. BmTyr is genetically encodable and enables subcellular-resolved
PL and proteomics in living cells. We further designed a strategy
of ligand-tethered BmTyr for in vivo PL, which unveiled
the surrounding proteome of a neurotransmitter receptor (Grm1 and
Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr
is one promising enzyme that can improve and expand PL-based applications
and discoveries