188 research outputs found
Ligand-receptor-G-protein molecular assemblies on beads for mechanistic studies and screening by flow cytometry.
ABSTRACT G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Gi␣2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein. Streptavidin-coated polystyrene beads used biotinylated anti-FLAG antibodies to bind FLAG-tagged G proteins for ternary complex assembly. Validation was achieved by showing time and concentration dependence of ternary complex formation. Affinity measurements of ligand for receptor on particles, of the ligand-receptor complex for G protein on the particles, and receptor-Gi␣2 fusion protein for G␥, were consistent with comparable assemblies in detergent suspension. Performance was assessed in applications representing the potential of these assemblies for ternary complex mechanisms. We showed the relationship for a family of ligands between LR and LRG affinity and characterized the affinity of both the wild-type and GFP fusion receptors with G protein. We also showed the potential of kinetic measurements to allow observation of individual steps of GTP-induced ternary complex disassembly and discriminated a fast step caused by RG disassembly compared with the slower step of G␣␥ disassembly. GPCRs interact with extracellular stimuli, such as photons, hormones, neurotransmitters, and odorants The formyl peptide receptor (FPR) responds to the presence of N-formyl methionine-containing peptides resulting from bacterial and mitochondrial protein synthesis, as well as other hydrophobic peptide
The Role of Cadmium and Nickel in Estrogen Receptor Signaling and Breast Cancer: Metalloestrogens or Not?
During the last half-century, incidences of breast cancer have increased globally. Various factors —genetic and environmental— have been implicated in the initiation and progression of this disease. One potential environmental risk factor that has not received a lot of attention is the exposure to heavy metals. While several mechanisms have been put forth describing how high concentrations of heavy metals play a role in carcinogenesis, it is unclear whether chronic, lowlevel exposure to certain heavy metals (i.e. cadmium and nickel), can directly result in the development and progression of cancer. Cadmium and nickel have been hypothesized to play a role in breast cancer development by acting as metalloestrogens— metals that bind to estrogen receptors and mimic the actions of estrogen. Since the lifetime exposure to estrogen is a wellestablished risk factor for breast cancer, anything that mimics its activity will likely contribute to the etiology of the disease. However, heavy metals, depending on their concentration, are capable of binding to a variety of proteins and may exert their toxicities by disrupting multiple cellular functions, complicating the analysis of whether heavy metal-induced carcinogenesis is mediated by the estrogen receptor. The purpose of this review is to discuss the various epidemiological, in vivo, and in vitro studies that show a link between the heavy metals, cadmium and nickel, and breast cancer development. We will particularly focus on the studies that test whether or not these two metals act as metalloestrogens in order to assess the strength of the data supporting this hypothesis
Functional Characterization of EngAMS, a P-Loop GTPase of Mycobacterium smegmatis
Bacterial P-loop GTPases belong to a family of proteins that selectively hydrolyze a small molecule guanosine tri-phosphate (GTP) to guanosine di-phosphate (GDP) and inorganic phosphate, and regulate several essential cellular activities such as cell division, chromosomal segregation and ribosomal assembly. A comparative genome sequence analysis of different mycobacterial species indicates the presence of multiple P-loop GTPases that exhibit highly conserved motifs. However, an exact function of most of these GTPases in mycobacteria remains elusive. In the present study we characterized the function of a P-loop GTPase in mycobacteria by employing an EngA homologue from Mycobacterium smegmatis, encoded by an open reading frame, designated as MSMEG_3738. Amino acid sequence alignment and phylogenetic analysis suggest that MSMEG_3738 (termed as EngAMS) is highly conserved in mycobacteria. Homology modeling of EngAMS reveals a cloverleaf structure comprising of α/β fold typical to EngA family of GTPases. Recombinant EngAMS purified from E. coli exhibits a GTP hydrolysis activity which is inhibited by the presence of GDP. Interestingly, the EngAMS protein is co-eluted with 16S and 23S ribosomal RNA during purification and exhibits association with 30S, 50S and 70S ribosomal subunits. Further studies demonstrate that GTP is essential for interaction of EngAMS with 50S subunit of ribosome and specifically C-terminal domains of EngAMS are required to facilitate this interaction. Moreover, EngAMS devoid of N-terminal region interacts well with 50S even in the absence of GTP, indicating a regulatory role of the N-terminal domain in EngAMS-50S interaction
Beneficial Effects of Estrogen in a Mouse Model of Cerebrovascular Insufficiency
BACKGROUND: The M(5) muscarinic acetylcholine receptor is known to play a crucial role in mediating acetylcholine dependent dilation of cerebral blood vessels. Previously, we reported that male M(5) muscarinic acetylcholine knockout mice (M5R(-/-) mice) suffer from a constitutive constriction of cerebral arteries, reduced cerebral blood flow, dendritic atrophy, and short-term memory loss, without necrosis and/or inflammation in the brain. METHODOLOGY/PRINCIPAL FINDINGS: We employed the Magnetic Resonance Angiography to study the area of the basilar artery in male and female M5R(-/-) mice. Here we show that female M5R(-/-) mice did not show the reduction in vascular area observed in male M5R(-/-) mice. However, ovariectomized female M5R(-/-) mice displayed phenotypic changes similar to male M5R(-/-) mice, strongly suggesting that estrogen plays a key role in the observed gender differences. We found that 17beta-estradiol (E2) induced nitric oxide release and ERK activation in a conditional immortalized mouse brain cerebrovascular endothelial cell line. Agonists of ERalpha, ERbeta, and GPR30 promoted ERK activation in this cell line. Moreover, in vivo magnetic resonance imaging studies showed that the cross section of the basilar artery was restored to normal in male M5R(-/-) mice treated with E2. Treatment with E2 also improved the performance of male M5R(-/-) mice in a cognitive test and reduced the atrophy of neural dendrites in the cerebral cortex and hippocampus. M5R(-/-) mice also showed astrocyte swelling in cortex and hippocampus using the three-dimensional reconstruction of electron microscope images. This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and the number of synapses. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that M5R(-/-) mice represent an excellent novel model system to study the beneficial effects of estrogen on cerebrovascular function and cognition. E2 may offer new therapeutic perspectives for the treatment of cerebrovascular insufficiency related memory dysfunction
Impaired leukocyte influx in cervix of postterm women not responding to prostaglandin priming
<p>Abstract</p> <p>Background</p> <p>Prolonged pregnancies are associated with increased rate of maternal and fetal complications. Post term women could be divided into at least two subgroups, one where parturition is possible to induce by prostaglandins and one where it is not. Our aim was to study parameters in cervical biopsies in women with spontaneous delivery at term (controls) and compare to those that are successfully induced post term (responders), and those that are not induced (non-responders), by local prostaglandin treatment.</p> <p>Methods</p> <p>Stromal parameters examined in this study were the accumulation of leukocytes (CD45, CD68), mRNAs and/or proteins for the extracellular matrix degrading enzymes (matrix metalloproteinase (MMP)-2, MMP-8 and MMP-9), their inhibitors (tissue inhibitor of MMP (TIMP)-1 and TIMP-2), interleukin-8 (IL-8), the platelet activating factor-receptor (PAF-R), syndecan-1 and estrogen binding receptors (estrogen receptor (ER)α, ERβ and G-coupled protein receptor (GPR) 30) as well as the proliferation marker Ki-67.</p> <p>Results</p> <p>The influx of leukocytes as assessed by CD45 was strongest in the responders, thereafter in the controls and significantly lower in the non-responders. IL-8, PAF-R and MMP-9, all predominantly expressed in leukocytes, showed significantly reduced immunostaining in the group of non-responders, while ERα and GPR30 were more abundant in the non-responders, as compared to the controls.</p> <p>Conclusion</p> <p>The impaired leukocyte influx, as reflected by the reduced number of CD45 positive cells as well as decreased immunostaining of IL-8, PAF-R and MMP-9 in the non-responders, could be one explanation of the failed ripening of the cervix in post term women. If the decreased leukocyte influx is a primary explanation to absent ripening or secondary, as a result of other factors, is yet to be established.</p
Human umbilical endothelial cells (HUVECs) have a sex: characterisation of the phenotype of male and female cells
Attenuation of Salt-Induced Cardiac Remodeling and Diastolic Dysfunction by the GPER Agonist G-1 in Female mRen2.Lewis Rats
The G protein-coupled estrogen receptor (GPER) is expressed in various tissues including the heart. Since the mRen2.Lewis strain exhibits salt-dependent hypertension and early diastolic dysfunction, we assessed the effects of the GPER agonist (G-1, 40 nmol/kg/hr for 14 days) or vehicle (VEH, DMSO/EtOH) on cardiac function and structure.Intact female mRen2.Lewis rats were fed a normal salt (0.5% sodium; NS) diet or a high salt (4% sodium; HS) diet for 10 weeks beginning at 5 weeks of age.Prolonged intake of HS in mRen2.Lewis females resulted in significantly increased blood pressure, mildly reduced systolic function, and left ventricular (LV) diastolic compliance (as signified by a reduced E deceleration time and E deceleration slope), increased relative wall thickness, myocyte size, and mid-myocardial interstitial and perivascular fibrosis. G-1 administration attenuated wall thickness and myocyte hypertrophy, with nominal effects on blood pressure, LV systolic function, LV compliance and cardiac fibrosis in the HS group. G-1 treatment significantly increased LV lusitropy [early mitral annular descent (e')] independent of prevailing salt, and improved the e'/a' ratio in HS versus NS rats (P<0.05) as determined by tissue Doppler.Activation of GPER improved myocardial relaxation in the hypertensive female mRen2.Lewis rat and reduced cardiac myocyte hypertrophy and wall thickness in those rats fed a high salt diet. Moreover, these advantageous effects of the GPER agonist on ventricular lusitropy and remodeling do not appear to be associated with overt changes in blood pressure
Genomics of Signaling Crosstalk of Estrogen Receptor α in Breast Cancer Cells
BACKGROUND: The estrogen receptor alpha (ERalpha) is a ligand-regulated transcription factor. However, a wide variety of other extracellular signals can activate ERalpha in the absence of estrogen. The impact of these alternate modes of activation on gene expression profiles has not been characterized. METHODOLOGY/PRINCIPAL FINDINGS: We show that estrogen, growth factors and cAMP elicit surprisingly distinct ERalpha-dependent transcriptional responses in human MCF7 breast cancer cells. In response to growth factors and cAMP, ERalpha primarily activates and represses genes, respectively. The combined treatments with the anti-estrogen tamoxifen and cAMP or growth factors regulate yet other sets of genes. In many cases, tamoxifen is perverted to an agonist, potentially mimicking what is happening in certain tamoxifen-resistant breast tumors and emphasizing the importance of the cellular signaling environment. Using a computational analysis, we predicted that a Hox protein might be involved in mediating such combinatorial effects, and then confirmed it experimentally. Although both tamoxifen and cAMP block the proliferation of MCF7 cells, their combined application stimulates it, and this can be blocked with a dominant-negative Hox mutant. CONCLUSIONS/SIGNIFICANCE: The activating signal dictates both target gene selection and regulation by ERalpha, and this has consequences on global gene expression patterns that may be relevant to understanding the progression of ERalpha-dependent carcinomas
Tamoxifen mechanically deactivates hepatic stellate cells via the G protein-coupled estrogen receptor
Tamoxifen has been used for many years to target estrogen receptor signalling in breast cancer cells. Tamoxifen is also an agonist of the G protein-coupled estrogen receptor (GPER), a GPCR ubiquitously expressed in tissues that mediates the acute response to estrogens. Here we report that tamoxifen promotes mechanical quiescence in hepatic stellate cells (HSCs), stromal fibroblast-like cells whose activation triggers and perpetuates liver fibrosis in hepatocellular carcinomas. This mechanical deactivation is mediated by the GPER/RhoA/myosin axis and induces YAP deactivation. We report that tamoxifen decreases the levels of hypoxia-inducible factor-1 alpha (HIF-1α) and the synthesis of extracellular matrix proteins through a mechanical mechanism that involves actomyosin-dependent contractility and mechanosensing of tissue stiffness. Our results implicate GPER-mediated estrogen signalling in the mechanosensory-driven activation of HSCs and put forward estrogenic signalling as an option for mechanical reprogramming of myofibroblast-like cells in the tumour microenvironment. Tamoxifen, with half a century of safe clinical use, might lead this strategy of drug repositioning.Peer reviewe
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