Detecting protein-protein interactions in living cells: development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction

Marie Gottschalk; Anders Bach; Jakob Lerche Hansen; Povl Krogsgaard-Larsen; Anders Skov Kristensen; Kristian Strømgaard

doi:10.1007/s11064-009-9998-4

Detecting protein-protein interactions in living cells: development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction

Marie Gottschalk, Anders Bach, Jakob Lerche Hansen, Povl Krogsgaard-Larsen, Anders Skov Kristensen, Kristian Strømgaard

Institut for Neurovidenskab

7 Citationer (Scopus)

Abstract

The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat-conjugated peptide for its ability to disrupt the PSD-95/NMDA receptor interaction in living cells.

Originalsprog	Engelsk
Tidsskrift	Neurochemical Research
Vol/bind	34
Udgave nummer	10
Sider (fra-til)	1729-1737
ISSN	0364-3190
DOI	https://doi.org/10.1007/s11064-009-9998-4
Status	Udgivet - 2009

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Detecting protein-protein interactions in living cells : development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction. / Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche et al.

I: Neurochemical Research, Bind 34, Nr. 10, 2009, s. 1729-1737.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

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title = "Detecting protein-protein interactions in living cells: development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction",

abstract = "The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat-conjugated peptide for its ability to disrupt the PSD-95/NMDA receptor interaction in living cells.",

keywords = "Faculty of Health and Medical Sciences",

author = "Marie Gottschalk and Anders Bach and Hansen, {Jakob Lerche} and Povl Krogsgaard-Larsen and Kristensen, {Anders Skov} and Kristian Str{\o}mgaard",

note = "Keywords: BRET assay; BRET; NMDA receptor; Protein-protein interactions; PSD-95; PDZ domain",

year = "2009",

doi = "10.1007/s11064-009-9998-4",

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T2 - development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction

AU - Gottschalk, Marie

AU - Bach, Anders

AU - Hansen, Jakob Lerche

AU - Krogsgaard-Larsen, Povl

AU - Kristensen, Anders Skov

AU - Strømgaard, Kristian

N1 - Keywords: BRET assay; BRET; NMDA receptor; Protein-protein interactions; PSD-95; PDZ domain

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N2 - The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat-conjugated peptide for its ability to disrupt the PSD-95/NMDA receptor interaction in living cells.

AB - The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat-conjugated peptide for its ability to disrupt the PSD-95/NMDA receptor interaction in living cells.

KW - Faculty of Health and Medical Sciences

U2 - 10.1007/s11064-009-9998-4

DO - 10.1007/s11064-009-9998-4

M3 - Journal article

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SN - 0364-3190

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SP - 1729

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JO - Neurochemical Research

JF - Neurochemical Research

IS - 10

ER -

Detecting protein-protein interactions in living cells: development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction

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