scholarly journals Conformational plasticity of the intracellular cavity of GPCR−G-protein complexes leads to G-protein promiscuity and selectivity

2019 ◽  
pp. 201820944 ◽  
Author(s):  
Manbir Sandhu ◽  
Anja M. Touma ◽  
Matthew Dysthe ◽  
Fredrik Sadler ◽  
Sivaraj Sivaramakrishnan ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Complexes ◽  
Dependent Manner ◽  
Secondary Messenger ◽  
C Terminus ◽  
Conformational Plasticity ◽  
Small Ensemble ◽  
Dynamics Simulations ◽  
The Impact ◽  
Dose Dependent

While the dynamics of the intracellular surface in agonist-stimulated GPCRs is well studied, the impact of GPCR dynamics on G-protein selectivity remains unclear. Here, we combine molecular dynamics simulations with live-cell FRET and secondary messenger measurements, for 21 GPCR−G-protein combinations, to advance a dynamic model of the GPCR−G-protein interface. Our data show C terminus peptides of Gαs, Gαi, and Gαq proteins assume a small ensemble of unique orientations when coupled to their cognate GPCRs, similar to the variations observed in 3D structures of GPCR−G-protein complexes. The noncognate G proteins interface with latent intracellular GPCR cavities but dissociate due to weak and unstable interactions. Three predicted mutations in β2-adrenergic receptor stabilize binding of noncognate Gαq protein in its latent cavity, allowing promiscuous signaling through both Gαs and Gαq in a dose-dependent manner. This demonstrates that latent GPCR cavities can be evolved, by design or nature, to tune G-protein selectivity, giving insights to pluridimensional GPCR signaling.

scholarly journals Gαi-derived peptide binds the µ-opioid receptor

2019 ◽  
Author(s):  
Piotr Kosson ◽  
Jolanta Dyniewicz ◽  
Piotr F. J. Lipiński ◽  
Joanna Matalinska ◽  
Aleksandra Misicka ◽  
...  
Keyword(s):  
G Protein ◽  
Opioid Receptor ◽  
Protein Complexes ◽  
Active Form ◽  
Peptide Modification ◽  
C Terminus ◽  
External Stimuli ◽  
G Protein Coupled ◽  
Dose Dependent ◽  
Peptide Interaction

G protein-coupled receptors (GPCRs) transduce external stimuli into the cell by G proteins via an allosteric mechanism. Agonist binding to the receptor stimulates GDP/GTP exchange within the heterotrimeric G protein complex. Whereas recent structures of GPCR-G protein complexes revealed that the H5, S1 and S2 domains of Gα are involved in binding the active receptor, earlier studies showed that a short peptide analogue derived from the C-terminus (H5) of the G protein transducin (Gt) is sufficient to stabilize rhodopsin in an active form. Here, we show that a Gαi-derived peptide of 12 amino acids binds the µ-opioid receptor (µOR) and acts as an allosteric modulator. The Gαi-derived peptide increases µOR affinity for its agonist morphine in a dose-dependent way. These results indicate that the GPCR-Gα peptide interaction observed so far for only rhodopsin can be extrapolated to µOR. In addition, we show that the C-terminal peptide of the Gαi subunit is sufficient to stabilize the active conformation of the receptor. Our approach opens the possibility to investigate the GPCR-G protein interface with peptide modification.

scholarly journals Inhibition of p38 Mitogen-Activated Protein Kinase Impairs Mayaro Virus Replication in Human Dermal Fibroblasts and HeLa Cells

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1156
Author(s):  
Madelaine Sugasti-Salazar ◽  
Yessica Y. Llamas-González ◽  
Dalkiria Campos ◽  
José González-Santamaría
Keyword(s):  
Protein Expression ◽  
Hela Cells ◽  
Plaque Assay ◽  
Dermal Fibroblasts ◽  
Dependent Manner ◽  
Human Dermal Fibroblasts ◽  
Mitogen Activated Protein ◽  
Mayaro Virus ◽  
The Impact ◽  
Dose Dependent

Mayaro virus (MAYV) hijacks the host’s cell machinery to effectively replicate. The mitogen-activated protein kinases (MAPKs) p38, JNK, and ERK1/2 have emerged as crucial cellular factors implicated in different stages of the viral cycle. However, whether MAYV uses these MAPKs to competently replicate has not yet been determined. The aim of this study was to evaluate the impact of MAPK inhibition on MAYV replication using primary human dermal fibroblasts (HDFs) and HeLa cells. Viral yields in supernatants from MAYV-infected cells treated or untreated with inhibitors SB203580, SP600125, U0126, or Losmapimod were quantified using plaque assay. Additionally, viral protein expression was analyzed using immunoblot and immunofluorescence. Knockdown of p38⍺/p38β isoforms was performed in HDFs using the PROTACs molecule NR-7h. Our data demonstrated that HDFs are highly susceptible to MAYV infection. SB203580, a p38 inhibitor, reduced MAYV replication in a dose-dependent manner in both HDFs and HeLa cells. Additionally, SB203580 significantly decreased viral E1 protein expression. Similarly, knockdown or inhibition of p38⍺/p38β isoforms with NR-7h or Losmapimod, respectively, affected MAYV replication in a dose-dependent manner. Collectively, these findings suggest that p38 could play an important role in MAYV replication and could serve as a therapeutic target to control MAYV infection.

scholarly journals Comparison of Different Methods for Extracting the Astaxanthin from Haematococcus pluvialis: Chemical Composition and Biological Activity

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3569
Author(s):  
Yicheng Tan ◽  
Zhang Ye ◽  
Mansheng Wang ◽  
Muhammad Faisal Manzoor ◽  
Rana Muhammad Aadil ◽  
...  
Keyword(s):  
High Pressure ◽  
Biological Activity ◽  
Chemical Composition ◽  
Haematococcus Pluvialis ◽  
Activity Analysis ◽  
Cell Disruption ◽  
Dependent Manner ◽  
Dpph Activity ◽  
The Impact ◽  
Dose Dependent

In this study, the impact of different cell disruption techniques (high-pressure micro fluidization (HPMF), ionic liquids (ILs), multi-enzyme (ME), and hydrochloric acid (HCl)) on the chemical composition and biological activity of astaxanthin (AST) obtained from Haematococcus pluvialis was investigated. Results indicated that all cell disruption techniques had a significant effect on AST composition, which were confirmed by TLC and UPC2 analysis. AST recovery from HCl (HCl-AST) and ILs (ILs-AST) cell disruption techniques was dominant by free and monoesters AST, while AST recovery from HPMF (HPMF-AST) and ME (ME-AST) cell disruption techniques was composed of monoesters, diesters, and free AST. Further biological activity analysis displayed that HCl-AST showed the highest ABTS and DPPH activity, while ILs-AST showed better results against the ORAC assay. Additionally, ILs-AST exhibits a stronger anti-proliferation of HepG2 cells in a dose-dependent manner, which was ascribed to AST-induced ROS in to inhibit the proliferative of cancer cells.

scholarly journals Does Curcumin Have a Role in the Interaction between Gut Microbiota and Schistosoma mansoni in Mice?

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 767
Author(s):  
Assmaa Anter ◽  
Mohamed Abd El-Ghany ◽  
Marwa Abou El Dahab ◽  
Noha Mahana
Keyword(s):  
Schistosoma Mansoni ◽  
Intestinal Microbiota ◽  
Bacterial Species ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Pseudomonas Sp ◽  
E Coli ◽  
Predominant Species ◽  
The Impact ◽  
Dose Dependent

There is strong correlation between changes in abundance of specific bacterial species and several diseases including schistosomiasis. Several studies have described therapeutic effects of curcumin (CUR) which may arise from its regulative effects on intestinal microbiota. Thus, we examined the impact of CUR on the diversity of intestinal microbiota with/without infection by Schistosoma mansoni cercariae for 56 days. Enterobacteriaceae was dominating in a naive and S. mansoni infected mice group without CUR treatment, the most predominant species was Escherichia coli with relative density (R.D%) = 80.66% and the least one was Pseudomonas sp. (0.52%). The influence of CUR on murine microbiota composition was examined one week after oral administration of high (40) and low (20 mg/kg b.w.) CUR doses were administered three times, with two day intervals. CUR induced high variation in the Enterobacteriaceae family, characterized by a significant (p < 0.001) reduction in E. coli and asignificant (p < 0.001) increase in Pseudomonas sp. in both naïve and S. mansoni-infected mice, compared to untreated mice, in a dose-dependent manner. Additionally, our study showed the effects of high CUR doses on S. mansoni infection immunological and parasitological parameters. These data support CUR’s ability to promote Pseudomonas sp. known to produce schistosomicidal toxins and offset the sequelae of murine schistosomiasis.

Evidence that estrogen receptor β enhances MMP-13 promoter activity in HIG-82 cells and that this enhancement can be influenced by ligands and involves specific promoter sites

2007 ◽  
Vol 85 (3) ◽  
pp. 326-336 ◽  
Author(s):  
Ting Lu ◽  
Yamini Achari ◽  
Jerome B. Rattner ◽  
David A. Hart
Keyword(s):  
Estrogen Receptor ◽  
Promoter Activity ◽  
Estrogen Receptor Β ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Connective Tissues ◽  
Matrix Metalloproteinase 13 ◽  
Promoter Construct ◽  
The Impact ◽  
Dose Dependent

Degradation of articular cartilage is characteristic of osteoarthritis, and matrix metalloproteinase-13 (MMP-13) has been implicated in this condition. Estrogen receptors (ERs) are present in connective tissues, indicating these tissues' potential responsiveness to estrogen. We based this study on the hypothesis that estrogen receptor β (ERβ) can modulate MMP-13 promoter activity. Transfection of cells with ERβ constructs led to the induction of the endogenous MMP-13 gene, as evidenced by increased mRNA levels. The results also indicated that MMP-13 promoter construct activity in the HIG-82 cell line significantly increased when ERβ was present, and that estrogen downregulated this response in a dose-dependent manner. ERβ was shown to enhance MMP-13 expression somewhat more strongly than ERα, and the impact of a number of selective ER modulators (tamoxifen, raloxifene, and ICI 182,780) on ERβ enhancement of promoter activity was found to be significantly less than that of estrogen. Furthermore, transcription regulatory sites in the MMP-13 promoter, specifically AP-1 and PEA-3, were shown to act in conjunction to mediate ERβ effects. Thus, ERβ likely influences MMP-13 promoter expression in normal and disease processes.

Characterization of the role of CaMKI-like kinase (CKLiK) in human granulocyte function

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 1076-1083 ◽  
Author(s):  
Sandra Verploegen ◽  
Laurien Ulfman ◽  
Hanneke W. M. van Deutekom ◽  
Corneli van Aalst ◽  
Henk Honing ◽  
...  
Keyword(s):  
Respiratory Burst ◽  
Critical Role ◽  
Neutrophil Migration ◽  
Platelet Activating Factor ◽  
Dependent Manner ◽  
Functional Responses ◽  
Effector Functions ◽  
C Terminus ◽  
Human Granulocytes ◽  
Dose Dependent

AbstractActivation of granulocyte effector functions, such as induction of the respiratory burst and migration, are regulated by a variety of relatively ill-defined signaling pathways. Recently, we identified a novel Ca2+/calmodulin-dependent kinase I-like kinase, CKLiK, which exhibits restricted mRNA expression to human granulocytes. Using a novel antibody generated against the C-terminus of CKLiK, CKLiK was detected in CD34+-derived neutrophils and eosinophils, as well as in mature peripheral blood granulocytes. Activation of human granulocytes by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF), but not the phorbol ester PMA (phorbol 12-myristate-13-acetate), resulted in induction of CKLiK activity, in parallel with a rise of intracellular Ca2+ [Ca2+]i. To study the functionality of CKLiK in human granulocytes, a cell-permeable CKLiK peptide inhibitor (CKLiK297-321) was generated which was able to inhibit kinase activity in a dose-dependent manner. The effect of this peptide was studied on specific granulocyte effector functions such as phagocytosis, respiratory burst, migration, and adhesion. Phagocytosis of Aspergillus fumigatus particles was reduced in the presence of CKLiK297-321 and fMLP-induced reactive oxygen species (ROS) production was potently inhibited by CKLiK297-321 in a dose-dependent manner. Furthermore, fMLP-induced neutrophil migration on albumin-coated surfaces was perturbed, as well as β2-integrin-mediated adhesion. These findings suggest a critical role for CKLiK in modulating chemoattractant-induced functional responses in human granulocytes.

scholarly journals The Extreme C-Terminal Region of Gαs Differentially Couples to the Luteinizing Hormone and β2-Adrenergic Receptors

2011 ◽  
Vol 25 (8) ◽  
pp. 1416-1430 ◽  
Author(s):  
Geneva DeMars ◽  
Francesca Fanelli ◽  
David Puett
Keyword(s):  
G Protein ◽  
Comparative Modeling ◽  
Cell System ◽  
Amino Acid Residues ◽  
Intracellular Loop ◽  
C Terminus ◽  
Model Cell ◽  
Rigid Body Docking ◽  
Molecular Landscape ◽  
Dynamics Simulations

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 α-subunits has been shown to be important for association with GPCR. Hypothesizing that the extreme CT of Gαs is an essential component of the molecular landscape of the GPCR, human LH receptor (LHR), and β2-adrenergic receptor (β2-AR), a model cell system was created for the expression and manipulation of Gαs subunits in LHR+ s49 ck cells that lack endogenous Gαs. On the basis of studies involving truncations, mutations, and chain extensions of Gαs, the CT was found to be necessary for LHR and β2-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 Gαs CT, are supported by the experimental observations and are characterized by the interaction of the four extreme CT amino acid residues of Gαs with residues in LHR and β2-AR helix 3, (including R of the DRY motif), helix 6, and intracellular loop 2. This portion of Gαs 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 β2-AR are expected to contribute significantly to differences in Gαs recognition by the two receptors.

scholarly journals Thrombin stimulates wortmannin-inhibitable phosphoinositide 3-kinase and membrane blebbing in CHRF-288 cells

1996 ◽  
Vol 314 (3) ◽  
pp. 805-810 ◽  
Author(s):  
Geeta S. VEMURI ◽  
Jin ZHANG ◽  
Rusong HUANG ◽  
James H. KEEN ◽  
Susan E. RITTENHOUSE
Keyword(s):  
G Protein ◽  
Morphological Changes ◽  
Shape Change ◽  
Specific Inhibitor ◽  
Thrombin Receptor ◽  
Dependent Manner ◽  
Permeabilized Cells ◽  
Phosphoinositide 3 Kinase ◽  
Change Response ◽  
Dose Dependent

We have investigated thrombin-stimulated morphological changes and the activation of phosphoinositide 3-kinase (PI 3-K), as manifested by the accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (labelled with 32P or myo-[3H]inositol), in CHRF-288 cells, a leukaemic cell line derived from a platelet progenitor cell. We report that these cells, when exposed to thrombin or SFLLRN (the peptide Ser-Phe-Leu-Leu-Arg-Asn, a thrombin-receptor ligand) rapidly change shape, forming membrane ‘blebs’, detectable by differential interference contrast or confocal microscopy, as well as labelled 3-phosphorylated phosphoinositides. The ‘blebs’ are distinguishable from ‘ruffles’ or lamellae, since they do not contain phalloidin-detectable actin. Studies with permeabilized cells indicate that PI 3-K is activated synergistically by thrombin+guanosine 5´-[γ-thio]triphosphate. Two forms of PI 3-K, i.e. PI 3-K(γ) and p85/PI 3-K, regulated by Gβγ subunits of heterotrimeric G-protein and the small G-protein Rho, respectively, are present in these cells, as is true for platelets. Wortmannin, a known potent and specific inhibitor of PI 3-K activities, inhibits thrombin-stimulated accumulation of 3-phosphorylated phosphoinositides in a dose-dependent manner (IC50 ~ 10nM), without affecting phospholipase C activation. Pretreatment of CHRF-288 cells with either wortmannin (100 nM) or an unrelated synthetic PI 3-K inhibitor, LY294002 (50 μM), abolishes thrombin-receptor-stimulated blebbing. These results suggest that thrombin-stimulated accumulation of 3-phosphorylated phosphoinositide(s) is required for the shape-change response in CHRF-288 cells.

scholarly journals Quercetin mediated inhibition of Staphylococcus aureus biofilms and the impact of the isolate phenotype and genotype

2021 ◽  
Vol 42 (3) ◽  
pp. 615-624
Author(s):  
E.H. Eldrehmy ◽  
◽  
S.M. Abdel-Hafez ◽  
Y.S. Alghamdi ◽  
M.M. Soliman ◽  
...  
Keyword(s):  
Microtiter Plate ◽  
Antibiofilm Activity ◽  
Dependent Manner ◽  
Inhibition Rate ◽  
Pcr Assay ◽  
Gram Staining ◽  
Biofilm Production ◽  
Pcr Assays ◽  
The Impact ◽  
Dose Dependent

Aim: This study was designed to assess the antibiofilm activity of quercetin on characterized S. aureus isolates. Methodology: This study evaluated 36 S. aureus isolates, each of which was identified using Gram staining, culture, biochemical, and PCR assays. Isolates were cultured and their biofilm production was evaluated using Congo red agar (CRA) plates, microtiter plate tests and PCR, and the effects of quercetin were examined. Results: The CRA results revealed that eight (22.3%) S. aureus isolates were strongly positive for biofilm production and an additional 18 isolates (50%) showed moderate biofilm capacity. The remaining 10 isolates were negative (27.7%) for biofilm production. S. aureus isolates were divided into strong positive, intermediate, and negative groups, 27.8%, 44.5%, and 27.7%, respectively. Scanning electron microscopy showed that the biofilm-producing isolates appeared as aggregates of cells within a heavy matrix. In addition, PCR assay identified IcaA and IcaD (66.6% for both) biofilm production genes in most isolates and IcaC (61.1%), IcaB, FnbB (33.3% for both), and Fib (22.2%) in several other strains. Quercetin significantly inhibited biofilm activity in biofilm producing S. aureus isolates in a dose-dependent manner, with an inhibition rate of 29.6-87.7%. Interpretation: Biofilm production is dependent on Ica gene phenotype and strains with an IcaABCD or IcaABD phenotype produce more biofilm than strains with IcaAD phenotype. Quercetin significantly inhibited S. aureus biofilm production, irrespective of Ica phenotype.

scholarly journals Pilot investigation on the dose-dependent impact of irradiation on primary human alveolar osteoblasts in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna-Klara Amler ◽  
Domenic Schlauch ◽  
Selin Tüzüner ◽  
Alexander Thomas ◽  
Norbert Neckel ◽  
...  
Keyword(s):  
Bone Cells ◽  
Marker Gene ◽  
Marker Genes ◽  
Dependent Manner ◽  
Current Standard ◽  
Functional Changes ◽  
Standard Procedures ◽  
The Impact ◽  
Dose Dependent

AbstractRadiotherapy of head and neck squamous cell carcinoma can lead to long-term complications like osteoradionecrosis, resulting in severe impairment of the jawbone. Current standard procedures require a 6-month wait after irradiation before dental reconstruction can begin. A comprehensive characterization of the irradiation-induced molecular and functional changes in bone cells could allow the development of novel strategies for an earlier successful dental reconstruction in patients treated by radiotherapy. The impact of ionizing radiation on the bone-forming alveolar osteoblasts remains however elusive, as previous studies have relied on animal-based models and fetal or animal-derived cell lines. This study presents the first in vitro data obtained from primary human alveolar osteoblasts. Primary human alveolar osteoblasts were isolated from healthy donors and expanded. After X-ray irradiation with 2, 6 and 10 Gy, cells were cultivated under osteogenic conditions and analyzed regarding their proliferation, mineralization, and expression of marker genes and proteins. Proliferation of osteoblasts decreased in a dose-dependent manner. While cells recovered from irradiation with 2 Gy, application of 6 and 10 Gy doses not only led to a permanent impairment of proliferation, but also resulted in altered cell morphology and a disturbed structure of the extracellular matrix as demonstrated by immunostaining of collagen I and fibronectin. Following irradiation with any of the examined doses, a decrease of marker gene expression levels was observed for most of the investigated genes, revealing interindividual differences. Primary human alveolar osteoblasts presented a considerably changed phenotype after irradiation, depending on the dose administered. Mechanisms for these findings need to be further investigated. This could facilitate improved patient care by re-evaluating current standard procedures and investigating faster and safer reconstruction concepts, thus improving quality of life and social integrity.

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