Expression of opioid and anti-opioid receptors in Chinese hamster ovary cells after exposure to dimethyl sulfoxide

Talmont, Franck; Mollereau, Catherine; Zajac, Jean‐Marie

doi:10.1016/j.ab.2011.09.001

Cited by 4 publications

(2 citation statements)

References 8 publications

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“…We have had reasonable success with different cell systems which include the methylotrophic yeast Pichia pastoris, the Chinese hamster ovary (CHO) cells and the human neuronal SH-SY5Y cells. 29,[34][35][36][37][38] In 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 the course of these studies and using the Pichia pastoris system we had also conducted the first and up to date single proteomic study of hMOR by MALDI-TOF-MS on tryptic digests of the protein obtaining a 27% of sequence coverage. 26 To reproduce these early experiments and further optimize the proteomic methodologies for hMOR we have produced a new batch of pure receptor protein using the Pichia pastoris system and applied proteomic and MS methodologies for sequencing the receptor by following the flow chart of Scheme 1.…”

Section: Resultsmentioning

confidence: 99%

Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human μ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines

Rosa

Bech-Serra²,

Canals³

et al. 2015

J. Proteome Res.

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Human μ-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR.

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Section: Resultsmentioning

confidence: 99%

Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human μ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines

Rosa

Bech-Serra²,

Canals³

et al. 2015

J. Proteome Res.

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“…For instance, its administration may increase functional expression of several receptors e.g. NMUR, bradykinin or opioid in cell cultures [67][68][69]. Moreover, DMSO is considered to affect the motor activity and to modulate the sleep architecture in rats [70][71].…”

Section: Introductionmentioning

confidence: 99%

Escitalopram alters local expression of noncanonical stress-related neuropeptides in the rat brain via NPS receptor signaling

et al. 2022

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Background: Neuropeptide S (NPS) is a multifunctional regulatory factor that exhibits a potent anxiolytic activity in animal models. However, there are no reports dealing with the potential molecular relationships between the anxiolytic activity of selective serotonin reuptake inhibitors (SSRIs) and NPS signaling, especially in the context of novel stress-related neuropeptides action. The present work therefore focused on gene expression of novel stress neuropeptides in the rat brain after acute treatment with escitalopram and in combination with neuropeptide S receptor (NPSR) blockade. Methods: Studies were carried out on adult, male Sprague-Dawley rats that were divided into 5 groups: animals injected with saline (control) and experimental rats treated with escitalopram (at single dose 10mg/kg daily), escitalopram and SHA-68, a selective NPSR antagonist (at single dose 40mg/kg), SHA-68 alone and corresponding vehicle (solvent SHA-68) control. To measure anxiety-like behavior and locomotor activity the open field test (OFT) was performed. All individuals were sacrificed under anaesthesia and the whole brain excised. Total mRNA was isolated from homogenized samples of amygdala, hippocampus, hypothalamus, thalamus, cerebellum and brainstem.Real time-PCR was used for estimation of related NPS, NPSR, neuromedin U (NMU), NMU receptor 2 (NMUR2) and nesfatin-1 precursor nucleobindin-2 (NUCB2) gene expression.Results: Acute escitalopram administration affects the local expression of the examined neuropeptides mRNA in a varied manner depending on brain location. An increase of NPSR and NUCB2 mRNA expression in the hypothalamus and brainstem was abolished by SHA-68 coadministration, while NMU mRNA expression was upregulated after NPSR blockade in the hippocampus and cerebellum. Conclusions: Pharmacological effects of escitalopram may be connected with local NPSR-related alterations in NPS/NMU/NMUR2 and nesfatin-1 gene expression at the level of selected rat brain regions. A novel alternative mode of SSRI action can be therefore cautiously proposed.

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Biological properties and perspective applications of “Bio-neuter” chemicals?

Ghanemi

2014

Saudi Pharmaceutical Journal

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Expression of opioid and anti-opioid receptors in Chinese hamster ovary cells after exposure to dimethyl sulfoxide

Cited by 4 publications

References 8 publications

Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human μ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines

Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human μ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines

Escitalopram alters local expression of noncanonical stress-related neuropeptides in the rat brain via NPS receptor signaling

Biological properties and perspective applications of “Bio-neuter” chemicals?

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