PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-7 (7)
 

Clipboard (0)
None

Select a Filter Below

Journals
Authors
more »
Year of Publication
Document Types
author:("perron, Julie")
1.  Analysis of RXR/THR and RXR/PPARG2 Heterodimerization by Bioluminescence Resonance Energy Transfer (BRET) 
PLoS ONE  2013;8(12):e84569.
Background
Nuclear receptors (NR) regulate transcription of genes involved in many biological processes such as development, cell proliferation, differentiation and cell death. Amongst them, PPARG2 and THR control tissue glucose and lipid homeostasis which are deregulated in severe pathophysiological conditions such as metabolic syndromes.
Methodology/Principal Findings
Here, we describe a real time BRET approach to monitor heterodimerization between RXR and PPARG2 or THR in vitro or in living cells. The presence of a specific DNA target was required to induce in vitro a BRET shift reflecting heterodimerization of RXR/PPARG2 or RXR/THR. As in electrophoretic mobility shift assay (EMSA), the stringency and specificity of the BRET shift assay depended upon assay condition optimization including MgCl2 concentration. For the nuclear receptors, we found by mutagenesis analysis that each heterodimer partner must harbor an intact DNA binding domain to induce BRET and heterodimerization on a DNA target. Moreover the interaction between the PPARG2 ligand binding domain and the RXR DNA binding domain stabilized the heterodimer on its DNA target. BRET microscopy in living cells highlighted the heterodimerization of RXR/PPARG2 within the nucleus clustered in discrete foci that may represent active target gene transcription regulation regions. BRET imaging also suggested that heterodimerization between RXR and PPARG2 required the DNA binding of PPARG2.
Conclusions/Significance
The BRET approach described here allowed us to study the dynamic interactions which exist between NR in vitro or in living cells and can provide important information on heterodimerization modes, affinity with a given RE and subcellular localization of the heterodimers. This method could be used to study real time changes of NR heterodimers occurring on DNA depending upon cell activation, chromatin state and help to define the mechanisms of ligands or drug action designed to target NRs.
doi:10.1371/journal.pone.0084569
PMCID: PMC3877338  PMID: 24391967
2.  A genetically encoded IL-1β BRET sensor to monitor inflammasome activity 
Inflammation is fundamental for protecting the organism against infection and injury. However, a failure to control immune response results in chronic inflammation and several associated disorders such as pain and loss of function. Initiation of inflammation is orchestrated by cytokines, among which interleukin (IL)-1β is particularly important. IL-1β is synthesized as an inactive protein that has to be processed by the inflammasome to generate the mature bioactive form. Conventional techniques cannot monitor IL-1β activation with high spatial and temporal resolution. Here, we present a ratiometric biosensor that allows monitoring IL-1β processing in real-time, with a temporal resolution of seconds and with a single cell spatial resolution. Using this sensor, we describe for the first time the kinetic of the inflammasome activity in living macrophages. With this new probe we also demonstrated that the pro-IL-1β processing occurs all over the cytoplasm.
doi:10.4049/jimmunol.1201349
PMCID: PMC3437522  PMID: 22815289
3.  Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability 
The Journal of Cell Biology  2012;198(2):251-263.
Synaptic activity–dependent remodeling of the glutamate receptor scaffold complex generates a negative feedback loop that limits further NMDA receptor activation.
Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor–mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.
doi:10.1083/jcb.201110101
PMCID: PMC3410417  PMID: 22801779
4.  REV, A BRET-Based Sensor of ERK Activity 
Networks of signaling molecules are activated in response to environmental changes. How are these signaling networks dynamically integrated in space and time to process particular information? To tackle this issue, biosensors of single signaling pathways have been engineered. Bioluminescence resonance energy transfer (BRET)-based biosensors have proven to be particularly efficient in that matter due to the high sensitivity of this technology to monitor protein–protein interactions or conformational changes in living cells. Extracellular signal-regulated kinases (ERK) are ubiquitously expressed and involved in many diverse cellular functions that might be encoded by the strength and spatio-temporal pattern of ERK activation. We developed a BRET-based sensor of ERK activity, called Rluc8-ERKsubstrate-Venus (REV). As expected, BRET changes of REV were correlated with ERK phosphorylation, which is required for its kinase activity. In neurons, the nature of the stimuli determines the strength, the location, or the moment of ERK activation, thus highlighting how acute modulation of ERK may encode the nature of initial stimulus to specify the consequences of this activation. This study provides evidence for suitability of REV as a new biosensor to address biological questions.
doi:10.3389/fendo.2013.00095
PMCID: PMC3727045  PMID: 23908646
biosensor; bioluminescence resonance energy transfer; BRET imaging; fluorescence lifetime imaging microscopy; extracellular signal-regulated kinases; spatio-temporal signaling; Rluc8-ERKsubstrate-Venus
5.  A Genetically Encoded IL-1β Bioluminescence Resonance Energy Transfer Sensor To Monitor Inflammasome Activity 
Inflammation is fundamental for protecting the organism against infection and injury. However, a failure to control immune response results in chronic inflammation and several associated disorders such as pain and loss of function. Initiation of inflammation is orchestrated by cytokines, among which IL-1β is particularly important. IL-1β is synthesized as an inactive protein that has to be processed by the inflammasome to generate the mature bioactive form. Conventional techniques cannot monitor IL-1β activation with high spatial and temporal resolution. In this study, we present a ratiometric biosensor that allows monitoring IL-1β processing in real time, with a temporal resolution of seconds and with a single-cell spatial resolution. Using this sensor, to our knowledge, we describe for the first time the kinetic of the inflammasome activity in living macrophages. With this new probe, we also demonstrated that the pro–IL-1β processing occurs all over the cytoplasm.
doi:10.4049/jimmunol.1201349
PMCID: PMC3437522  PMID: 22815289
6.  Scaffold remodeling in space and time controls synaptic transmission 
Bioarchitecture  2012;2(2):29-32.
Scaffolding proteins that are associated with glutamate receptors in dendritic spines govern the location and function of receptors to control synaptic transmission. Unraveling the spatio-temporal dynamics of protein-protein interactions within components of the scaffolding complex will bring to light the function of these interactions. Combining bioluminescence resonance energy transfer (BRET) imaging to electrophysiological recordings, we have recently shown that GKAP, a core protein of the scaffolding complex, interacts with DLC2, a protein associated with molecular motors. Synaptic activity-induced GKAP-DLC2 interaction in spines stabilizes the scaffolding complex and enhances the NMDA currents. Interestingly, this work placed emphasis on the bioarchitectural dependence of protein-protein interaction dynamics. Depending on physiological conditions, the modulation in space and time of protein-protein interaction is acutely regulated, engendering a subtle control of synaptic transmission in the state of the individual synapse.
PMCID: PMC3383718  PMID: 22754626
bioluminescence resonance energy transfer (BRET); dendritic spine; dynein light chain 2 (DLC2); glutamate receptors; guanylate kinase-associated protein (GKAP); protein-protein interaction; scaffolding proteins; synaptic transmission
7.  A single subunit (GB2) is required for G-protein activation by the heterodimeric GABAb receptor 
The Journal of Biological Chemistry  2001;277(5):3236-3241.
Although G-protein coupled receptors (GPCRs) have been shown to assemble into functional homo or heteromers, the role of each protomer in G-protein activation is not known. Among the GPCRs, the γ-aminobutyric acid (GABA) type B receptor (GABABR) is the only one known so far that needs two subunits, GB1 and GB2, to function. The GB1 subunit contains the GABA binding site but is unable to activate G-proteins alone. In contrast the GB2 subunit which does not bind GABA, has an heptahelical domain able to activate G-proteins when assembled into dimers (Galvez et al., EMBO J. 20, 2001, 2152–2159). In the present study, we examined the role of each subunit within the GB1-GB2 heteromer, in G-protein coupling. To that aim, point mutations in the highly conserved third intracellular loop known to prevent G-protein activation of the related Ca-sensing or metabotropic glutamate receptors were introduced into GB1 and GB2. One mutation, L686P introduced in GB2 prevents the formation of a functional receptor, even though the heteromer reaches the cell surface, and even though the mutated subunit still associates with GB1 and increases GABA affinity on GB1. This was observed either in HEK293 cells where the activation of the G-protein was assessed by measurement of IP accumulation, or in cultured neurons where the inhibition of the Ca-channel current was measured. In contrast, the same mutation when introduced into GB1 does not modify the G-protein coupling properties of the heteromeric GABAB receptor either in HEK293 cells or in neurons. These data show that a single subunit in a dimeric GPCR is critical for coupling to G-proteins.
doi:10.1074/jbc.M108900200
PMCID: PMC2566549  PMID: 11711539
Animals; Benzoates; pharmacokinetics; Binding; Competitive; Cell Line; Cells; Cultured; Cerebellum; cytology; GABA Antagonists; pharmacokinetics; GTP-Binding Proteins; chemistry; metabolism; Humans; Inositol Phosphates; metabolism; Kidney; Kinetics; Ligands; Mice; Mutagenesis; Site-Directed; Neurons; cytology; Organophosphorus Compounds; pharmacokinetics; Protein Binding; Protein Subunits; Receptors; GABA-B; drug effects; genetics; physiology; Recombinant Proteins; chemistry; metabolism; Transfection; gamma-Aminobutyric Acid; pharmacology

Results 1-7 (7)