An expression and purification system for the biosynthesis of adenosine receptor peptides for biophysical and structural characterization.

Related Articles

An expression and purification system for the biosynthesis of adenosine receptor peptides for biophysical and structural characterization.

Protein Expr Purif. 2012 Aug;84(2):224-35

Authors: Britton ZT, Hanle EI, Robinson AS

Abstract
Biophysical and structural characterization of G protein-coupled receptors (GPCRs) has been limited due to difficulties in expression, purification, and vitro stability of the full-length receptors. "Divide and conquer" approaches aimed at the NMR characterization of peptides corresponding to specific regions of the receptor have yielded insights into the structure and dynamics of GPCR activation and signaling. Though significant progress has been made in the generation of peptides that are composed of GPCR transmembrane domains, current methods utilize fusion protein strategies that require chemical cleavage and peptide separation via chromatographic means. We have developed an expression and purification system based on fusion to ketosteroid isomerase, thrombin cleavage, and tandem affinity chromatography that enables the solubilization, cleavage, and characterization in a single detergent system relevant for biophysical and structural characterization. We have applied this expression and purification system to the production and characterization of peptides of the adenosine receptor family of GPCRs in Escherichia coli. Herein, we demonstrate using a model peptide that includes extracellular loop 3, transmembrane domain 7, and a portion of the carboxy-terminus of the adenosine A(2)a receptor that the peptide is sufficiently pure for biophysical characterization, where it adopts ?-helical structure. Furthermore, we demonstrate the utility of this system by optimizing the construct for thrombin processing and apply the system to peptides with more complex structures.

PMID: 22722102 [PubMed - in process]

NF-κB NF-kB signaling pathway NF-kB pathway

[CYLD deubiquitinase as a recurrent target in oncogenic processes].

Related Articles

[CYLD deubiquitinase as a recurrent target in oncogenic processes].

Med Sci (Paris). 2011 Jun-Jul;27(6-7):626-31

Authors: Bonnet M, Courtois G

Abstract
CYLD deubiquitinase has been originally defined as a tumor suppressor based exclusively on genetic findings. Indeed, inactivation of CYLD in humans results in familial cylindromatosis and multiple trichoepithelioma, two pathologies characterized by the development of tumors originating specifically from the skin appendages. A set of recent publications has revealed that recurrent inactivation of CYLD occurs through diverse mechanisms in several forms of cancer, unequivocally confirming its tumor suppressor function. This property is associated with the critical role played by CYLD in negatively regulating several signaling pathway, among them the NF-?B signaling pathway.

PMID: 21718647 [PubMed - indexed for MEDLINE]

find out more info

MEKK1-MKK4-JNK-AP1 pathway negatively regulates Rgs4 expression in colonic smooth muscle cells.

Related Articles

MEKK1-MKK4-JNK-AP1 pathway negatively regulates Rgs4 expression in colonic smooth muscle cells.

PLoS One. 2012;7(4):e35646

Authors: Zhang Y, Li F, Liu S, Wang H, Mahavadi S, Murthy KS, Khalili K, Hu W

Abstract
BACKGROUND: Regulator of G-protein Signaling 4 (RGS4) plays an important role in regulating smooth muscle contraction, cardiac development, neural plasticity and psychiatric disorder. However, the underlying regulatory mechanisms remain elusive. Our recent studies have shown that upregulation of Rgs4 by interleukin (IL)-1? is mediated by the activation of NF?B signaling and modulated by extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, and phosphoinositide-3 kinase. Here we investigate the effect of the c-Jun N-terminal kinase (JNK) pathway on Rgs4 expression in rabbit colonic smooth muscle cells.
METHODOLOGY/PRINCIPAL FINDINGS: Cultured cells at first passage were treated with or without IL-1? (10 ng/ml) in the presence or absence of the selective JNK inhibitor (SP600125) or JNK small hairpin RNA (shRNA). The expression levels of Rgs4 mRNA and protein were determined by real-time RT-PCR and Western blot respectively. SP600125 or JNK shRNA increased Rgs4 expression in the absence or presence of IL-1? stimulation. Overexpression of MEKK1, the key upstream kinase of JNK, inhibited Rgs4 expression, which was reversed by co-expression of JNK shRNA or dominant-negative mutants for MKK4 or JNK. Both constitutive and inducible upregulation of Rgs4 expression by SP600125 was significantly inhibited by pretreatment with the transcription inhibitor, actinomycin D. Dual reporter assay showed that pretreatment with SP600125 sensitized the promoter activity of Rgs4 in response to IL-1?. Mutation of the AP1-binding site within Rgs4 promoter increased the promoter activity. Western blot analysis confirmed that IL-1? treatment increased the phosphorylation of JNK, ATF-2 and c-Jun. Gel shift and chromatin immunoprecipitation assays validated that IL-1? increased the in vitro and ex vivo binding activities of AP1 within rabbit Rgs4 promoter.
CONCLUSION/SIGNIFICANCE: Activation of MEKK1-MKK4-JNK-AP1 signal pathway plays a tonic inhibitory role in regulating Rgs4 transcription in rabbit colonic smooth muscle cells. This negative regulation may aid in maintaining the transient level of RGS4 expression.

PMID: 22545125 [PubMed - indexed for MEDLINE]

G-protein Receptors gpcr pathway NF-κB

Ginsenoside Rg1 protection against ?-amyloid peptide-induced neuronal apoptosis via estrogen receptor ? and glucocorticoid receptor-dependent anti-protein nitration pathway.

Related Articles

Ginsenoside Rg1 protection against ?-amyloid peptide-induced neuronal apoptosis via estrogen receptor ? and glucocorticoid receptor-dependent anti-protein nitration pathway.

Neuropharmacology. 2012 Sep;63(3):349-61

Authors: Wu J, Pan Z, Wang Z, Zhu W, Shen Y, Cui R, Lin J, Yu H, Wang Q, Qian J, Yu Y, Zhu D, Lou Y

Abstract
Ginsenoside Rg1 (Rg1) acts as a neuroprotective agent against various insults, however, the underlying mechanism has not been fully elucidated yet. Here, we report that Rg1 protects primary rat cerebrocortical neurons against ?-amyloid peptide????? (A??????) injury via estrogen receptor ? (ER?) and glucocorticoid receptor (GR)-dependent anti-protein nitration pathway. In primary rat cerebrocortical neuron cultures under basal conditions, Rg1 leads to nuclear translocation of ER? and GR, induces related responsive gene PR, pS? and MKP-1, SGK transcription. Meantime, Rg1 also increases the basal level of ERK1/2 phosphorylation. In the presence of toxic level of A??????, Rg1 maintains ERK1/2 phosphorylation, attenuates iNOS expression, NO production, and inhibits NF-?B nuclear translocation, protein nitration and cell death. The antiapoptotic effects of Rg1 via both ER? and GR were abolished by small interfering RNAs (siRNA). ERK1/2 phosphorylation inhibitor U0126 can block downstream iNOS expression and NO generation. Interestingly, the anti-protein nitration effect of Rg1 is well matched with ER? and GR activation, although its anti-ROS production effect is in an ER?- and GR-independent manner. These results suggest that Rg1 ameliorates A??????-induced neuronal apoptosis at least in part by two complementary ER?- and GR-dependent downstream pathways: (1) upregulation of ERK1/2 phosphorylation followed by inhibiting iNOS expression, NO generation and protein tyrosine nitration. (2) reduction NF-?B nuclear translocation. These data provide new understanding into the mechanisms of Rg1 anti-apoptotic functions after A?????? exposure, suggesting that ER? and GR-dependent anti-protein tyrosine nitration pathway might take an important role in the neuroprotective effect of Rg1.

PMID: 22534050 [PubMed - indexed for MEDLINE]

G-protein Receptors gpcr pathway NF-κB

The noncanonical NF-?B pathway.

Related Articles

The noncanonical NF-?B pathway.

Immunol Rev. 2012 Mar;246(1):125-40

Authors: Sun SC

Abstract
The noncanonical nuclear factor-?B (NF-?B) signaling pathway mediates activation of the p52/RelB NF-?B complex and, thereby, regulates specific immunological processes. This NF-?B pathway relies on the inducible processing of NF-?B2 precursor protein, p100, as opposed to the degradation of I?B? in the canonical NF-?B pathway. A central signaling component of the noncanonical NF-?B pathway is NF-?B-inducing kinase (NIK), which functions together with a downstream kinase, IKK? (inhibitor of NF-?B kinase ?), to induce phosphorylation-dependent ubiquitination and processing of p100. Under normal conditions, NIK is targeted for continuous degradation by a tumor necrosis factor (TNF) receptor-associated factor-3 (TRAF3)-dependent E3 ubiquitin ligase. In response to signals mediated by a subset of TNF receptor superfamily members, NIK becomes stabilized as a result of TRAF3 degradation, leading to the activation of noncanonical NF-?B. This review discusses both the historical perspectives and the recent progress in the regulation and biological function of the noncanonical NF-?B pathway.

PMID: 22435551 [PubMed - indexed for MEDLINE]

NF-kB pathway NF-kB signaling NF-kappaB signaling pathway

Adhesion G Protein-Coupled Receptors: Signaling, Pharmacology & Mechanisms of Activation.

Related Articles

Adhesion G Protein-Coupled Receptors: Signaling, Pharmacology & Mechanisms of Activation.

Mol Pharmacol. 2012 Jul 20;

Authors: Paavola KJ, Hall RA

Abstract
The adhesion G protein-coupled receptors (GPCRs) are a distinct family of more than 30 receptors in vertebrate genomes. These receptors have been shown to play pivotal roles in a diverse range of biological functions and are characterized by extremely large N-termini featuring various adhesion domains capable of mediating cell-cell and cell-matrix interactions. The adhesion GPCR N-termini also contain GPCR proteolytic site (GPS) motifs that undergo autocatalytic cleavage during receptor processing to create mature GPCRs existing as non-covalently attached complexes between the N-terminus and transmembrane regions. There is mounting evidence that adhesion GPCRs can couple to G proteins to activate a variety of different downstream signaling pathways. Furthermore, recent studies have demonstrated that adhesion GPCR N-termini can bind to multiple ligands, which may differentially activate receptor signaling and/or mediate cell adhesion. Additionally, studies on several distinct adhesion GPCRs have revealed that truncations of the N-termini result in constitutively-active receptors, suggesting a model of receptor activation in which removal of the N-terminus may be a key event in stimulating receptor signaling. Since mutations to certain adhesion GPCRs cause human disease, and since many members of this receptor family exhibit highly discrete distribution patterns in different tissues, the adhesion GPCRs represent a class of potentially important drug targets that have not yet been exploited. For this reason, understanding the mechanisms of activation for these receptors and elucidating their downstream signaling pathways can provide insights with the potential to lead to novel therapeutics.

PMID: 22821233 [PubMed - as supplied by publisher]

NF-kappaB signaling pathway NF-kB signaling pathway NF-kB pathway

The extreme C-terminal region of G?s differentially couples to the luteinizing hormone and beta2-adrenergic receptors.

Related Articles

The extreme C-terminal region of G?s differentially couples to the luteinizing hormone and beta2-adrenergic receptors.

Mol Endocrinol. 2011 Aug;25(8):1416-30

Authors: DeMars G, Fanelli F, Puett D

Abstract
The mechanisms of G protein coupling to G protein-coupled receptors (GPCR) share general characteristics but may exhibit specific interactions unique for each GPCR/G protein partnership. The extreme C terminus (CT) of G protein ?-subunits has been shown to be important for association with GPCR. Hypothesizing that the extreme CT of G?(s) is an essential component of the molecular landscape of the GPCR, human LH receptor (LHR), and ?(2)-adrenergic receptor (?(2)-AR), a model cell system was created for the expression and manipulation of G?(s) subunits in LHR(+) s49 ck cells that lack endogenous G?(s). On the basis of studies involving truncations, mutations, and chain extensions of G?(s), the CT was found to be necessary for LHR and ?(2)-AR signaling. Some general similarities were found for the responses of the two receptors, but significant differences were also noted. Computational modeling was performed with a combination of comparative modeling, molecular dynamics simulations, and rigid body docking. The resulting models, focused on the G?(s) CT, are supported by the experimental observations and are characterized by the interaction of the four extreme CT amino acid residues of G?(s) with residues in LHR and ?(2)-AR helix 3, (including R of the DRY motif), helix 6, and intracellular loop 2. This portion of G?(s) recognizes the same regions of the two GPCR, although with differences in the details of selected interactions. The predicted longer cytosolic extensions of helices 5 and 6 of ?(2)-AR are expected to contribute significantly to differences in G?(s) recognition by the two receptors.

PMID: 21622536 [PubMed - indexed for MEDLINE]

NF-kB pathway NF-kB signaling NF-kappaB signaling pathway