Regulator of G-protein signaling (RGS) proteins classically function as negative modulators of G-protein-coupled receptor signaling. In vitro, RGS proteins have been shown to inhibit signaling by agonists at the μ-opioid receptor, including morphine. The goal of the present study was to evaluate the contribution of endogenous RGS proteins to the antinociceptive effects of morphine and other opioid agonists. To do this, a knock-in mouse that expresses an RGS-insensitive (RGSi) mutant Gαo protein, GαoG184S (Gαo RGSi), was evaluated for morphine or methadone antinociception in response to noxious thermal stimuli. Mice expressing Gαo RGSi subunits exhibited a naltrexone-sensitive enhancement of baseline latency in both the hot-plate and warm-water tail-withdrawal tests. In the hot-plate test, a measure of supraspinal nociception, morphine antinociception was increased, and this was associated with an increased ability of opioids to inhibit presynaptic GABA neurotransmission in the periaqueductal gray. In contrast, antinociception produced by either morphine or methadone was reduced in the tail-withdrawal test, a measure of spinal nociception. In whole-brain and spinal cord homogenates from mice expressing Gαo RGSi subunits, there was a small loss of Gαo expression and an accompanying decrease in basal G-protein activity. Our results strongly support a role for RGS proteins as negative regulators of opioid supraspinal antinociception and also reveal a potential novel function of RGS proteins as positive regulators of opioid spinal antinociceptive pathways.
Regulators of G-Protein signaling (RGS) proteins are potent negative modulators of signal transduction through G-Protein coupled receptors. They function by binding to activated (GTP-bound) Gα subunits and accelerating the rate of GTP hydrolysis. Modulation of RGS activity by small molecules is an attractive mechanism to fine-tune GPCR signaling for therapeutic and research purposes. Here we describe the pharmacologic properties and mechanism of action of CCG-50014, the most potent small molecule RGS inhibitor to date. It has an IC50 for RGS4 of 30 nM and is >20-fold selective for RGS4 over other RGS proteins. CCG-50014 binds covalently to the RGS, forming an adduct on two cysteine residues located in an allosteric regulatory site. It is not a general cysteine alkylator as it does not inhibit activity of the cysteine protease papain at concentrations >3,000 fold higher than those required to inhibit RGS4 function. It is also >1,000-fold more potent as an RGS4 inhibitor than are the cysteine alkylators N-ethylmaleimide or iodoacetamide. Analysis of the cysteine reactivity of the compound shows that compound binding to Cys107 in RGS8 inhibits Gα- binding in a manner that can be reversed by cleavage of the compound-RGS disulfide bond. If the compound reacts with Cys160 in RGS8, the adduct induces RGS denaturation and activity cannot be restored by compound removal. The high potency and good selectivity of CCG-50014 make it a useful tool for studying the functional roles of RGS4.
RGS protein; Small molecule protein-protein interaction inhibitor; GPCR
Neutrophils are first responders rapidly mobilized to inflammatory sites by a tightly regulated, nonredundant hierarchy of chemoattractants. These chemoattractants engage neutrophil cell surface receptors triggering heterotrimeric G-protein Gαi subunits to exchange GDP for GTP. By limiting the duration that Gαi subunits remain GTP bound, RGS proteins modulate chemoattractant receptor signaling. Here, we show that neutrophils with a genomic knock in of a mutation that disables regulator of G-protein signaling (RGS)-Gαi2 interactions accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Intravital imaging revealed a failure of the mutant neutrophils to accumulate at and stabilize sites of sterile inflammation. Furthermore, these mice could not control a nonlethal Staphylococcus aureus infection. Neutrophil RGS proteins establish a threshold for Gαi activation, helping to coordinate desensitization mechanisms. Their loss renders neutrophils functionally incompetent.
High-throughput screening (HTS) has historically been used by the pharmaceutical industry to rapidly test hundreds of thousands of compounds to identify potential drug candidates. More recently, academic groups have used HTS to identify new chemical probes or small interfering RNA (siRNA) that can serve as experimental tools to examine the biology or physiology of novel proteins, processes, or interactions. HTS presents a significant challenge with the vast and complex nature of data generated. This report describes MScreen, a web-based, open-source cheminformatics application for chemical library and siRNA plate management, primary HTS and dose-response data handling, structure search, and administrative functions. Each project in MScreen can be secured with passwords or shared in an open information environment which enables collaborators to easily compare data from many screens, providing a useful means to identify compounds with desired selectivity. Unique features include compound, substance, mixture, and siRNA plate creation and formatting; automated dose-response fitting and quality control (QC); and user, target, and assay method administration. MScreen provides an effective means to facilitate HTS information handling and analysis in the academic setting so that users can efficiently view their screening data and evaluate results for follow-up.
chemoinformatics; data analysis software; open source; high-throughput screening
Recently regulators of G protein signalling (RGS) proteins have emerged as potential therapeutic targets since they provide an alternative method of modulating the activity of GPCRs, the target of so many drugs. Inhibitors of RGS proteins must block a protein-protein interaction (RGS-Gα), but also be cell and, depending on the therapeutic target, blood brain barrier permeable. A lead compound (1a) was identified as an inhibitor of RGS4 in a screening assay and this has now been optimised for activity, selectivity and solubility. The newly developed ligands (11b, 13) display substantial selectivity over the closely related RGS8 protein, lack the off-target calcium mobilisation activity of the lead 1a and have excellent aqueous solubility. They are currently being evaluated in vivo in rodent models of depression.
RGS4; RGS protein; thiadiazolidinone; GPCR; protein-protein interaction
A method is described for the quantitative analysis of protein-protein interactions using the Flow Cytometry Protein Interaction Assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead-associated fluorescence in a flow cytometer. This method can be used to calculate protein-protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the Regulator of G-Protein Signaling protein, RGS19, in either a saturation or competition format. An adaptation of this method that is compatible for High Throughput screening is also provided.
RGS; G protein; Protein-Protein Interaction; FCPIA; High Throughput Screening; Multiplexing
Previous studies have implicated a role of Gαi proteins as co-regulators of Toll –like receptor (TLR) activation. These studies largely derived from examining the effect of Gαi protein inhibitors or genetic deletion of Gαi proteins. However the effect of increased Gαi protein function or Gαi protein expression on TLR activation has not been investigated. We hypothesized that gain of function or increased expression of Gαi proteins suppresses TLR2 and TLR4 -induced inflammatory cytokines. Novel transgenic mice with genomic “knock-in” of a Regulator of G-protein Signaling (RGS)-insensitive Gnai2 allele (Gαi2 G184S/G184S; GS/GS) were employed. These mice express essentially normal levels of Gαi2 protein, however the Gαi2 is insensitive to its negative regulator RGS thus rendering more sustained Gαi2 protein activation following ligand/receptor binding. In subsequent studies, we generated Raw 264.7 cells that stably overexpress Gαi2 protein (Raw Gαi2). Peritoneal macrophages, splenocytes and mouse embryonic fibroblasts (MEF) were isolated from WT and GS/GS mice and were stimulated with LPS, Pam3CSK4 or Poly (I:C). We also subjected WT and GS/GS mice to endotoxic shock (LPS 25mg/kg i.p.) and plasma TNFα and IL-6 production were determined. We found that in vitro LPS and Pam3CSK4 induced TNFα and IL-6 production are decreased in macrophages from GS/GS mice compared with WT mice (p<0.05). In vitro LPS and Pam3CSK4 induced IL-6 production in splenocytes and in vivo LPS induced IL-6 were suppressed in GS/GS mice. Poly (I:C) induced TNFα and IL-6 in vitro demonstrated no difference between GS/GS mice and WT mice. LPS induced IL-6 production was inhibited in MEFs from GS/GS mice similarly to macrophage and splenocytes. In parallel studies, Raw Gαi2 cells also exhibit decreased TNFα and IL-6 production in response to LPS and Pam3CSK4. These studies support our hypothesis that Gαi2 proteins are novel negative regulators of TLR activation.
Gαi protein; TLR signaling; LPS; endotoxemia; inflammatory cytokines
Intracellular signaling cascades are a series of regulated protein-protein interactions that may provide a number of targets for potential drug discovery. Here, we examine the interaction of Regulators of G protein signaling (RGS) proteins with the G protein Gαo, using a flow cytometry protein interaction assay (FCPIA). FCPIA accurately measures nanomolar binding constants of this protein-protein interaction, and has been used in high throughput screening. This report focuses on five RGS proteins (4, 6, 7, 8 and 16). In order to increase the content of screens, we assessed high throughput screening of these RGS proteins in multiplex, by establishing binding constants of each RGS with Gαo in isolation, and then in a multiplex format with five RGS proteins present. In order to use this methodology as a higher-content multiplex protein-protein interaction screen, we established Z' factor values for RGS proteins in multiplex of 0.73 to 0.92, indicating this method is suitable for screening using FCPIA. To increase throughput, we also compressed a set of 8,000 compounds by combining 4 compounds in a single assay well. Subsequent deconvolution of the compounds mixtures verified the identification of active compounds at specific RGS targets in our mixtures using the polyplexed FCPIA method.
G protein; RGS; Flow Cytometry; FCPIA; High throughput screening
Guanine nucleotide-exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytokskeletal changes, motility, growth, survival, and gene transcription. The RhoGEF Leukemia-Associated RhoGEF (LARG) is a member of the Regulators of G-protein Signaling Homology Domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide binding assay utilizing BODIPY-Texas Red-GTPγS (BODIPY-TR-GTPγS), we performed a ten-thousand compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a non-fluorescent radioactive guanine nucleotide binding assay measuring LARG-stimulated [35S] GTPγS binding to RhoA, thus ruling out non-specific fluorescent effects. All five compounds selectively inhibited LARG-stimulated RhoA [35S] GTPγS binding, but had little to no effect upon RhoA or Gαo [35S] GTPγS binding. Therefore, these five compounds should serve as promising starting points for the development of small molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.
high-throughput screening; fluorescence polarization; RhoGEF; RhoA; LARG; Drug Discovery
Arrhythmia; Automaticity; Mouse mutants; Potassium channels; G proteins
Regulator of G protein signalling (RGS) proteins act as molecular ‘off switches’ that terminate G protein signalling by catalyzing the hydrolysis of Gα-bound GTP to GDP. Many different Gαi-coupled receptors have been implicated in the cardioprotective effects of ischaemic preconditioning. However, the role of RGS proteins in modulating cardioprotection has not been previously investigated. We used mice that were homozygous (GS/GS) or heterozygous (GS/+) for a mutation in Gαi2 rendering it RGS-insensitive (G184S) to determine whether interactions between endogenous RGS proteins and Gαi2 modulate Gαi-mediated protection from ischaemic injury.
Methods and results
Langendorff-perfused mouse hearts were subjected to 30 min global ischaemia and 2 h reperfusion. Infarcts in GS/GS (14.5% of area at risk) and GS/+ (22.6% of AAR) hearts were significantly smaller than those of +/+ hearts (37.2% of AAR) and recovery of contractile function was significantly enhanced in GS/GS and GS/+ hearts compared with +/+ hearts. The cardioprotective phenotype was not reversed by wortmannin or U0126 but was reversed by 5-hydroxydecanoic acid and HMR 1098, indicating that RGS-insensitive Gαi2 protects the heart through a mechanism that requires functional ATP-dependent potassium channels but does not require acute activation of extracellular-regulated kinase or Akt signalling pathways.
This is the first study to demonstrate that Gαi2-mediated cardioprotection is suppressed by RGS proteins. These data suggest that RGS proteins may provide novel therapeutic targets to protect the heart from ischaemic injury.
Ischaemic preconditioning; Ischaemia reperfusion injury; Regulator of G protein signalling (RGS); Gi2
Proper development of the mammalian brain requires that neural progenitor cells balance self-renewal and differentiation under precise temporal and spatial regulation, but the underlying mechanisms are not well understood. In this study, we identify Gα subunit as a positive regulator of mammalian neurogenesis, working with the RGS-mediated ephrin-B signaling pathway as two opposing forces to maintain a balance between self-renewal and differentiation in the developing mouse cerebral cortex. Multiple Gαi subunits are expressed by cortical neural progenitor cells during the course of cortical neurogenesis. Activation of Gαi signaling, through in utero electroporation mediated expression of wild-type and constitutively active Gαi subunits, counteracts the function of ephrin-B in cortical neural progenitors to induce differentiation. Genetic knock-in of an RGS-insensitive G184SGαi2 causes early cell cycle exit and a reduction of cortical neural progenitor cells and leads to a defect in the production of late born cortical neurons, similar to what is observed in mutant mice with deficiency in ephrin-B reverse signaling pathway. This study reveals a role of Gα subunit in mammalian neurogenesis and uncovers a developmental mechanism, coordinated by the Gα and ephrin-B signaling pathways, for control of the balance between self-renewal and differentiation in neural progenitor cells.
Gα subunit; Ephrin-B/RGS signaling; neural progenitor cells; cortical neurogenesis; self-renewal and differentiation
Recent evidence suggests that G protein coupled receptors, especially those linked to Gαi, contribute to the mechanisms of anesthetic action. Regulator of G protein signaling (RGS) proteins bind to activated Gαi and inhibit its signal transduction. Genomic knock-in mice with an RGS-insensitive Gαi2 G184S (Gαi2 GS) allele exhibit enhanced Gαi2 signaling and provide a novel approach for investigating the role of Gαi2 signaling and RGS proteins in general anesthesia.
Homozygous Gαi2 GS/GS and wild type (WT) mice were anesthetized with isoflurane and time (s) to loss and resumption of righting response was quantified. During recovery from isoflurane anesthesia breathing was quantified in a plethysmography chamber for both lines of mice.
Gαi2 GS/GS mice required significantly less time for loss of righting and significantly more time for resumption of righting than WT mice. During recovery from isoflurane anesthesia, Gαi2 GS/GS mice exhibited significantly greater respiratory depression. Poincaré analyses show that GS/GS mice have diminished respiratory variability compared to WT mice.
Modulation of Gαi2 signaling by RGS proteins alters loss and resumption of wakefulness, and state-dependent changes in breathing.
Summary: Rapid expansion of available data about G Protein Coupled Receptor (GPCR) dimers/oligomers over the past few years requires an effective system to organize this information electronically. Based on an ontology derived from a community dialog involving colleagues using experimental and computational methodologies, we developed the GPCR-Oligomerization Knowledge Base (GPCR-OKB). GPCR-OKB is a system that supports browsing and searching for GPCR oligomer data. Such data were manually derived from the literature. While focused on GPCR oligomers, GPCR-OKB is seamlessly connected to GPCRDB, facilitating the correlation of information about GPCR protomers and oligomers.
Availability and Implementation: The GPCR-OKB web application is freely available at http://www.gpcr-okb.org
Supplementary information: Supplementary data are available at Bioinformatics online.
Regulators of G protein signaling (RGS) proteins act as GTPase accelerating proteins (GAPs) to negatively modulate G protein signaling and are defined by a conserved RGS domain with considerable amino acid diversity. To determine the effects of specific, purified RGS proteins on mu-opioid signaling, C6 cells stably expressing a mu-opioid receptor were rendered permeable to proteins by treatment with digitonin. Mu-opioid inhibition of forskolin-stimulated adenylyl cyclase (AC) by DAMGO, a mu-specific opioid peptide, remained fully intact in permeabilized cells. Purified RGS domain of RGS4 added to permeabilized cells resulted in a two-fold loss in DAMGO potency but had no effect in cells expressing RGS-insensitive G proteins. The inhibitory effect of DAMGO was reduced to the same extent by purified RGS4 and RGS8. In contrast, the RGS domain of RGS7 had no effect and inhibited the action of RGS8 due to weak physical association with Gαi2 and minimal GAP activity in C6 cell membranes. These data suggest that differences in conserved RGS domains of specific RGS proteins contribute to differential regulation of opioid signaling to AC and that a permeabilized cell model is useful for studying the effects of specific RGS proteins on aspects of G protein-coupled receptor signaling.
RGS proteins; mu-opioid; adenylyl cyclase; permeabilization; Gα proteins
We recently identified bis(amide) CCG-1423 (1) as a novel inhibitor of RhoA/C-mediated gene transcription that is capable of inhibiting invasion of PC-3 prostate cancer cells in a Matrigel model of metastasis. An initial structure-activity relationship study focusing on bioisosteric replacement of the amides and conformational restriction identified two compounds, 4g and 8, with improved selectivity for inhibition of RhoA/C-mediated gene transcription and attenuated cytotoxcity relative to 1. Both compounds were also capable of inhibiting cell invasion with equal efficacy to 1 but with less attendant cytotoxicity.
This paper introduces the concept of phase-locking analysis of oscillatory cellular signaling systems to elucidate biochemical circuit architecture. Phase-locking is a physical phenomenon that refers to a response mode in which system output is synchronized to a periodic stimulus; in some instances, the number of responses can be fewer than the number of inputs, indicative of skipped beats. While the observation of phase-locking alone is largely independent of detailed mechanism, we find that the properties of phase-locking are useful for discriminating circuit architectures because they reflect not only the activation but also the recovery characteristics of biochemical circuits. Here, this principle is demonstrated for analysis of a G-protein coupled receptor system, the M3 muscarinic receptor-calcium signaling pathway, using microfluidic-mediated periodic chemical stimulation of the M3 receptor with carbachol and real-time imaging of resulting calcium transients. Using this approach we uncovered the potential importance of basal IP3 production, a finding that has important implications on calcium response fidelity to periodic stimulation. Based upon our analysis, we also negated the notion that the Gq-PLC interaction is switch-like, which has a strong influence upon how extracellular signals are filtered and interpreted downstream. Phase-locking analysis is a new and useful tool for model revision and mechanism elucidation; the method complements conventional genetic and chemical tools for analysis of cellular signaling circuitry and should be broadly applicable to other oscillatory pathways.
Key to robust discernment of cell circuit architecture is to have as many distinct response features as possible for comparison and evaluation. One under-appreciated characteristic of oscillatory circuits is that under periodic stimulation, these systems will exhibit responses synchronized to this stimulatory input, a phenomenon termed phase-locking. We demonstrate that phase-locked response characteristics vary noticeably depending on circuit activation and recovery properties; these response characteristics thereby provide a unique set of criteria for oscillatory circuit architecture analysis. The concept is validated through experiments on an oscillatory calcium pathway in mammalian cells; the experimental setup allowed us to explore, for the first time, the properties of chemically induced phase-locking of intracellular signals. Observations of this phenomenon were then used to test the predictions of several existing mathematical models of calcium signaling. Most of the models we evaluated were unable to match all our experimental observations, suggesting that current models are missing mechanistic elements in the context of calcium signaling for the cell type and receptor/stimulant tested. The observations of phase-locking further led us to identify one simple mechanistic modification that would account for all the experimental observations. The techniques and methodology presented should be broadly applicable to a variety of biological oscillators.
Leukemia associated Rho guanine nucleotide exchange factor (LARG) activates RhoA in response to signals received by specific classes of cell surface receptors. The catalytic core of LARG is a Dbl homology (DH) domain whose activity is modulated by an adjacent pleckstrin homology (PH) domain. In this study, we used a transcriptional assay and confocal microscopy to examine the roles of several novel structural features of the LARG DH/PH domains, including a conserved and exposed hydrophobic patch on the PH domain that mediates protein-protein interactions in crystal structures of LARG and its close homolog PDZ-RhoGEF. Mutation of the hydrophobic patch has no effect on nucleotide exchange activity in vitro, but abolished the ability of LARG to activate RhoA and to induce stress fiber formation in cultured cells. The activity of these mutants could be rescued by fusion with exogenous membrane targeting domains. However, because membrane recruitment by activated Gα13 subunits was not sufficient to rescue activity of a hydrophobic patch mutant, the LARG PH domain cannot solely contribute to membrane targeting. Instead, it seems likely the domain is involved in regulatory interactions with other proteins near the membrane surface. We also show that the hydrophobic patch of the PH domain is likely important for the activity of all Lbc family RhoGEFs.
Gα13; RhoGEF; Lbc; Pleckstrin homology domain; Actin stress fibers; Membrane recruitment
Regulators of G Protein Signaling (RGS) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors. The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We previously reported the design, mechanistic evaluation and structure-activity relationships (SAR) of a disulfide-containing cyclic peptide inhibitor of RGS4, YJ34 (Ac-Val-Lys-c[Cys-Thr-Gly-Ile-Cys]-Glu-NH2, S-S) (Roof, et al. Chem Biol Drug Des 2006; 67:266-274). Using a focused one-bead, one-compound (OBOC) peptide library that contains features known to be necessary for the activity of YJ34, we now identify peptides that bind to RGS4. Six peptides showed confirmed binding to RGS4 by flow cytometry. Two analogs of peptide 2, (Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp-NH2, S-S with a free or acetylated N-terminus) inhibited RGS4-stimulated Gαo GTPase activity at 25–50 μM. They selectively inhibit RGS4 but not RGS7, RGS16 and RGS19. Their inhibition of RGS4 does not depend on cysteine-modification of RGS4, as they do not lose activity when all cysteines are removed from RGS4. Peptide 2 has been modeled to fit in the same binding pocket predicted for YJ34 but in the reverse orientation.
One-Bead; One-Compound Library (OBOC); Focused library; Regulators of G-Protein Signaling (RGS); Protein-protein interaction (PPI) inhibitors; Structure-activity relationship (SAR)
Noonan syndrome (NS OMIM 163950) is a relatively common autosomal dominant developmental disorder characterized by short stature, specific facial features, and congenital cardiac anomalies. Approximately 50–66% of cases have defined mutations in the K-ras/Raf/MEK/ERK pathway that lead to constitutive signaling, but a significant number remain unexplained. We hypothesize that enhanced signaling through Gαi2 (from the GNAI2 gene) may also produce a NS-like phenotype. This is based on a recently described mouse model in which RGS-mediated inhibition of Gαi2 is prevented by a knock-in mutation (G184S) that blocks RGS binding (Huang et al, Mol, Cell. Biol. 26:6870–6879, 2006). The mice have short body length, cardiac hypertrophy, a triangular face with wide-set eyes and ears, and hematologic alterations. There is a slight increase in ERK activation and a pronounced enhancement of PI3K/Akt phosphorylation in MEFs from these mice suggesting that abnormal increases in Gαi2 signaling could represent a novel upstream mechanism for NS. This suggests a novel set of candidate genes for NS (GNAI2 and RGS proteins) and if validated could have important implications for therapy as well.
Neurotropic alphaviruses such as western, eastern, and Venezuelan equine encephalitis viruses cause serious and potentially fatal central nervous system infections in humans and are high-priority potential bioterrorism agents. There are currently no widely available vaccines or licensed therapies for these virulent pathogens. To identify potential novel antiviral drugs, we developed a cell-based assay with a western equine encephalitis virus replicon that expresses a luciferase reporter gene and screened a small molecule diversity library of 51,028 compounds. We identified and validated a thieno[3,2-b]pyrrole compound with a half maximal inhibitory concentration of <10 µmol/L, a selectivity index >20, and potent activity against live virus in cultured neuronal cells. Furthermore, a structure-activity relationship analysis with 20 related compounds identified several with enhanced activity profiles, including 6 with submicromolar half maximal inhibitory concentrations. In conclusion, we have identified a novel class of promising inhibitors with potent activity against virulent neurotropic alphaviruses.
A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 × 7.62 cm glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial CCD camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menton constants. A chip with 36 channels was used for screening for modulators of the G protein: RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. 36 electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4,320 assays per hour with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11% respectively in the 16 and 36-channel designs.
Regulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs). The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We recently described a focused one-bead, one-compound (OBOC) library screen to identify peptide inhibitors of RGS4. Here we extend our observations to include another peptide with a different mechanism of action.
Peptide 5nd (Tyr-Trp-c [Cys-Lys-Gly-Leu-Cys]-Lys-NH2, S-S) blocks the RGS4-Gαo interaction with an IC50 of 28 μM. It forms a covalent, dithiothreitol (DTT) sensitive adduct with a mass consistent with the incorporation of one peptide per RGS. Peptide 5nd activity is abolished by either changing its disulfide bridge to a methylene dithioether bridge, which cannot form disulfide bridges to the RGS, or by removing all cysteines from the RGS protein. However, no single cysteine in RGS4 is completely necessary or sufficient for 5nd activity.
Though it has some RGS selectivity, 5nd appears to be a partially random cysteine modifier. These data suggest that it inhibits RGS4 by forming disulfide bridges with the protein.
Signal transduction via guanine nucleotide binding proteins (G proteins) is involved in cardiovascular, neural, endocrine, and immune cell function. Regulators of G protein signaling (RGS proteins) speed the turn-off of G protein signals and inhibit signal transduction, but the in vivo roles of RGS proteins remain poorly defined. To overcome the redundancy of RGS functions and reveal the total contribution of RGS regulation at the Gαi2 subunit, we prepared a genomic knock-in of the RGS-insensitive G184S Gnai2 allele. The Gαi2G184S knock-in mice show a dramatic and complex phenotype affecting multiple organ systems (heart, myeloid, skeletal, and central nervous system). Both homozygotes and heterozygotes demonstrate reduced viability and decreased body weight. Other phenotypes include shortened long bones, a markedly enlarged spleen, elevated neutrophil counts, an enlarged heart, and behavioral hyperactivity. Heterozygous Gαi2+/G184S mice show some but not all of these abnormalities. Thus, loss of RGS actions at Gαi2 produces a dramatic and pleiotropic phenotype which is more evident than the phenotype seen for individual RGS protein knockouts.
Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80μM. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10μM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.
unfolded protein response; endoplasmic reticulum stress; cell-based assay; luciferase reporter; natural products