scholarly journals GnRH Antagonists Produce Differential Modulation of the Signaling Pathways Mediated by GnRH Receptors

2019 ◽  
Vol 20 (22) ◽  
pp. 5548 ◽  
Author(s):  
Samantha Sperduti ◽  
Silvia Limoncella ◽  
Clara Lazzaretti ◽  
Elia Paradiso ◽  
Laura Riccetti ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hek293 Cells ◽  
Gnrh Antagonists ◽  
Element Binding Protein ◽  
Mouse Pituitary ◽  
Responsive Element ◽  
Extracellular Regulated Kinase

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1–2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LβT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, β-catenin activation and mouse luteinizing-hormone β-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LβT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM–1 µM, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM–1 µM range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in LβT2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 µM GnRH-induced β-catenin activation, Lhb gene expression increase occurring upon LβT2 cell treatment by 1 µM GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting Lhb gene transcription.

scholarly journals G-protein-coupled receptor 30 interacts with receptor activity-modifying protein 3 and confers sex-dependent cardioprotection

2013 ◽  
Vol 51 (1) ◽  
pp. 191-202 ◽  
Author(s):  
Patricia M Lenhart ◽  
Stefan Broselid ◽  
Cordelia J Barrick ◽  
L M Fredrik Leeb-Lundberg ◽  
Kathleen M Caron
Keyword(s):  
G Protein ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cardiac Cells ◽  
Receptor Activity ◽  
Hek293 Cells ◽  
G Protein Coupled

Receptor activity-modifying protein 3 (RAMP3) is a single-pass transmembrane protein known to interact with and affect the trafficking of several G-protein-coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with GPR30, also known as G-protein-coupled estrogen receptor 1. GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Using bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3−/− mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3+/+ and Ramp3−/− mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3 and sex dependent. Our results demonstrate that GPR30–RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo and that RAMP3 is required for GPR30-mediated cardioprotection.

scholarly journals Two human menopausal gonadotrophin (hMG) preparations display different early signaling in vitro

2020 ◽  
Vol 26 (12) ◽  
pp. 894-905
Author(s):  
Livio Casarini ◽  
Laura Riccetti ◽  
Elia Paradiso ◽  
Riccardo Benevelli ◽  
Clara Lazzaretti ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Composition ◽  
Human Menopausal Gonadotrophin ◽  
Cautious Interpretation ◽  
Transfer Signal ◽  
Hcg Receptor

Abstract Commercial hMG drugs are marketed for the treatment of infertility and consist of highly purified hormones acting on receptors expressed in target gonadal cells. Menopur® and Meriofert® are combined preparation of FSH and hCG and are compared in vitro herein. To this purpose, the molecular composition of the two drugs was analyzed by immunoassay. The formation of FSH receptor and LH/hCG receptor (FSHR; LHCGR) heteromer, intracellular Ca2+ and cAMP activation, β-arrestin 2 recruitment and the synthesis of progesterone and estradiol were evaluated in transfected HEK293 and human primary granulosa lutein cells treated by drugs administered within the pg-mg/ml concentration range. Molecular characterization revealed that Meriofert® has a higher FSH:hCG ratio than Menopur® which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert® had a higher potency than Menopur® in inducing a cAMP increase. Moreover, Meriofert® revealed a higher potency than Menopur® in recruiting β-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-β-arrestin 2 complexes, as evidenced by a decrease in bioluminescence resonance energy transfer signal. This drug-specific activation of intracellular signaling pathways is consistent with the molecular composition of these preparations and impacts downstream progesterone and estradiol production, with Menopur® more potent than Meriofert® in inducing the synthesis of both the steroids. These findings are suggestive of distinct in-vivo activities of these preparations, but require cautious interpretation and further validation from clinical studies.

scholarly journals Identification of Key Receptor Residues Discriminating Human Chorionic Gonadotropin (hCG)- and Luteinizing Hormone (LH)-Specific Signaling

2020 ◽  
Vol 22 (1) ◽  
pp. 151
Author(s):  
Clara Lazzaretti ◽  
Valentina Secco ◽  
Elia Paradiso ◽  
Samantha Sperduti ◽  
Claudia Rutz ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
Chorionic Gonadotropin ◽  
Intracellular Signaling ◽  
Specific Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Hek293 Cells ◽  
Amino Acid Residues

(1) The human luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptor (LHCGR) discriminates its two hormone ligands and differs from the murine receptor (Lhr) in amino acid residues potentially involved in qualitative discerning of LH and hCG. The latter gonadotropin is absent in rodents. The aim of the study is to identify LHCGR residues involved in hCG/LH discrimination. (2) Eight LHCGR cDNAs were developed, carrying “murinizing” mutations on aminoacidic residues assumed to interact specifically with LH, hCG, or both. HEK293 cells expressing a mutant or the wild type receptor were treated with LH or hCG and the kinetics of cyclic adenosine monophosphate (cAMP) and phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) activation was analyzed by bioluminescence resonance energy transfer (BRET). (3) Mutations falling within the receptor leucine reach repeat 9 and 10 (LRR9 and LRR10; K225S +T226I and R247T), of the large extracellular binding domain, are linked to loss of hormone-specific induced cAMP increase, as well as hCG-specific pERK1/2 activation, leading to a Lhr-like modulation of the LHCGR-mediated intracellular signaling pattern. These results support the hypothesis that LHCGR LRR domain is the interaction site of the hormone β-L2 loop, which differs between LH and hCG, and might be fundamental for inducing gonadotropin-specific signals. (4) Taken together, these data identify LHCGR key residues likely evolved in the human to discriminate LH/hCG specific binding.

scholarly journals The Molecular Chaperone DNAJB6, but Not DNAJB1, Suppresses the Seeded Aggregation of Alpha-Synuclein in Cells

2019 ◽  
Vol 20 (18) ◽  
pp. 4495 ◽  
Author(s):  
Natasja Deshayes ◽  
Sertan Arkan ◽  
Christian Hansen
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Lewy Bodies ◽  
Resonance Energy ◽  
Alpha Synuclein ◽  
Hek293 Cells ◽  
Induced Aggregation ◽  
In Cells

Alpha-synuclein (α-Syn) can misfold and aggregate, causing the degeneration of dopaminergic neurons, as seen in Parkinson’s disease (PD). We recently demonstrated that DNAJB6, a co-chaperone found in Lewy bodies (LB), suppresses the aggregation of α-Syn in cells and in vitro. In this study, we compared the capacities of DNAJB1 and DNAJB6 to suppress the seeded α-Syn aggregation in HEK293 cells expressing α-Syn tagged with cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). The aggregation of α-Syn was seeded by the transfection of the cells with recombinant α-Syn pre-formed fibrils (PFFs), following the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated knockout (KO) of these two genes, respectively. We quantified the α-Syn aggregation by fluorescence microscopy and fluorescence resonance energy transfer (FRET) analysis. We detected significantly more aggregates in the DNAJB6 KO cells compared with the parental cells, whereas the DNAJB1 KO had no effect on the α-Syn aggregation. This is the first evidence that DNAJB6 can suppress α-Syn aggregation, induced by exogenous α-Syn seeds, in cells. Next, we explored whether this mechanism could be dependent on protein degradation pathways. We observed that the increase in the α-Syn PFF-induced aggregation in the DNAJB6 KO cells compared with the parental cells was strongly diminished upon the incubation of the cells with the proteasomal inhibitor MG132. These results consolidate that DNAJB6 is a suppressor of α-Syn aggregation, and suggest that DNAJB6 may target misfolded and/or aggregated α-Syn for proteasomal degradation.

Quantitative Ratiometric pH Imaging Using a 1,2-Dioxetane Chemiluminescence Resonance Energy Transfer Sensor

2020 ◽  
Author(s):  
Lucas S. Ryan ◽  
Jeni Gerberich ◽  
Uroob Haris ◽  
ralph mason ◽  
Alexander Lippert
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Whole Body ◽  
Photon Flux ◽  
Ph Homeostasis ◽  
Ph Imaging ◽  
Confounding Variables ◽  
Significant Difference

<p>Regulation of physiological pH is integral for proper whole-body and cellular function, and disruptions in pH homeostasis can be both a cause and effect of disease. In light of this, many methods have been developed to monitor pH in cells and animals. In this study, we report a chemiluminescence resonance energy transfer (CRET) probe Ratio-pHCL-1, comprised of an acrylamide 1,2-dioxetane chemiluminescent scaffold with an appended pH-sensitive carbofluorescein fluorophore. The probe provides an accurate measurement of pH between 6.8-8.4, making it viable tool for measuring pH in biological systems. Further, its ratiometric output is independent of confounding variables. Quantification of pH can be accomplished both using common fluorimetry and advanced optical imaging methods. Using an IVIS Spectrum, pH can be quantified through tissue with Ratio-pHCL-1, which has been shown in vitro and precisely calibrated in sacrificed mouse models. Initial studies showed that intraperitoneal injections of Ratio-pHCL-1 into sacrificed mice produce a photon flux of more than 10^10 photons per second, and showed a significant difference in ratio of emission intensities between pH 6.0, 7.0, and 8.0.</p> <b></b><i></i><u></u><sub></sub><sup></sup><br>

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

scholarly journals Biological nanoscale fluorescent probes: From structure and performance to bioimaging

2020 ◽  
Vol 39 (1) ◽  
pp. 209-221
Author(s):  
Jiafeng Wan ◽  
Xiaoyuan Zhang ◽  
Kai Zhang ◽  
Zhiqiang Su
Keyword(s):  
Fluorescent Probes ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Biological Imaging ◽  
Fluorescent Materials ◽  
And Performance

Abstract In recent years, nanomaterials have attracted lots of attention from researchers due to their unique properties. Nanometer fluorescent materials, such as organic dyes, semiconductor quantum dots (QDs), metal nano-clusters (MNCs), carbon dots (CDs), etc., are widely used in biological imaging due to their high sensitivity, short response time, and excellent accuracy. Nanometer fluorescent probes can not only perform in vitro imaging of organisms but also achieve in vivo imaging. This provides medical staff with great convenience in cancer treatment. Combined with contemporary medical methods, faster and more effective treatment of cancer is achievable. This article explains the response mechanism of three-nanometer fluorescent probes: the principle of induced electron transfer (PET), the principle of fluorescence resonance energy transfer (FRET), and the principle of intramolecular charge transfer (ICT), showing the semiconductor QDs, precious MNCs, and CDs. The excellent performance of the three kinds of nano fluorescent materials in biological imaging is highlighted, and the application of these three kinds of nano fluorescent probes in targeted biological imaging is also introduced. Nanometer fluorescent materials will show their significance in the field of biomedicine.

scholarly journals Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels

2018 ◽  
Vol 115 (47) ◽  
pp. 12051-12056 ◽  
Author(s):  
Akil A. Puckerin ◽  
Donald D. Chang ◽  
Zunaira Shuja ◽  
Papiya Choudhury ◽  
Joachim Scholz ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hek293 Cells ◽  
Channel Blockers ◽  
Wild Type ◽  
Voltage Dependent ◽  
Somatosensory Neurons ◽  
Potential Applications ◽  
Voltage Dependent Calcium Channels ◽  
Potential Therapeutics

Genetically encoded inhibitors for voltage-dependent Ca2+ (CaV) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca2+ (CaV1/CaV2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary CaVβ-subunits. To investigate latent CaVβ-independent components of inhibition, we coexpressed each RGK individually with CaV1 (CaV1.2/CaV1.3) or CaV2 (CaV2.1/CaV2.2) channels reconstituted in HEK293 cells with either wild-type (WT) β2a or a mutant version (β2a,TM) that does not bind RGKs. All four RGKs strongly inhibited CaV1/CaV2 channels reconstituted with WT β2a. By contrast, when channels were reconstituted with β2a,TM, Rem inhibited only CaV1.2, Rad selectively inhibited CaV1.2 and CaV2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT CaVβ, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted CaV1.2 while Rad[R208A/L235A] inhibited CaV1.2/CaV2.2 but not CaV1.3/CaV2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous CaV1.2 channels in ventricular cardiomyocytes and selectively blocked 25 and 62%, respectively, of HVA currents in somatosensory neurons of the dorsal root ganglion, corresponding to their distinctive selectivity for CaV1.2 and CaV1.2/CaV2.2 channels. Thus, we have exploited latent β-binding–independent Rem and Rad inhibition of specific CaV1/CaV2 channels to develop selective GECCIs with properties unmatched by current small-molecule CaV channel blockers.

scholarly journals Rational design of protein-specific folding modifiers

2020 ◽  
Author(s):  
Anirban Das ◽  
Anju Yadav ◽  
Mona Gupta ◽  
R Purushotham ◽  
Vishram L. Terse ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rational Design ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Measurements ◽  
Folding Nucleus ◽  
Dynamics Simulations ◽  
General Method

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

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