cAMP Receptor Research Articles

Transcriptional regulation of fatty acid cis-trans isomerization in the solvent-tolerant soil bacterium, Pseudomonas putida F1.

One key to the success of Pseudomonas spp. is their ability to reside in hostile environments. Pseudomonas spp. possess a cis-trans isomerase (Cti) an enzyme that converts the cis-unsaturated fatty acids (FAs) of the membrane lipids to their trans-isomers to rigidify the membrane and thereby resist stresses. Whereas the posttranslational Cti regulation has been previously reported, transcriptional cti regulation remains to be studied in more details. Here, we have studied cti transcriptional regulation in the solvent-tolerant strain Pseudomonas putida F1. Two cti transcriptional start sites (cti-279 and cti-77) were identified with cti-279 transcript being dominant. Expression of cti was found to increase with temperature increase, addition of the organic solvent, octanol and in the stationary growth phase. We found that cti expression was repressed by the cyclic-AMP receptor protein (Crp) and repression required the cyclic-AMP ligand of Crp. Production of trans-unsaturated FAs was found to decrease after 24 h of growth. Although this decrease was accompanied by an increase in cyclopropane FA content, this was not at the expense of trans-unsaturated FAs demonstrating the absence of competition between Cti and Cfa in FA modification.

Plasticity of Promoter-Core Sequences Allows Bacteria to Compensate for the Loss of a Key Global Regulatory Gene

Transcription regulatory networks (TRNs) are of central importance for both short-term phenotypic adaptation in response to environmental fluctuations and long-term evolutionary adaptation, with global regulatory genes often being targets of natural selection in laboratory experiments. Here, we combined evolution experiments, whole-genome resequencing, and molecular genetics to investigate the driving forces, genetic constraints, and molecular mechanisms that dictate how bacteria can cope with a drastic perturbation of their TRNs. The crp gene, encoding a major global regulator in Escherichia coli, was deleted in four different genetic backgrounds, all derived from the Long-Term Evolution Experiment (LTEE) but with different TRN architectures. We confirmed that crp deletion had a more deleterious effect on growth rate in the LTEE-adapted genotypes; and we showed that the ptsG gene, which encodes the major glucose-PTS transporter, gained CRP (cyclic AMP receptor protein) dependence over time in the LTEE. We then further evolved the four crp-deleted genotypes in glucose minimal medium, and we found that they all quickly recovered from their growth defects by increasing glucose uptake. We showed that this recovery was specific to the selective environment and consistently relied on mutations in the cis-regulatory region of ptsG, regardless of the initial genotype. These mutations affected the interplay of transcription factors acting at the promoters, changed the intrinsic properties of the existing promoters, or produced new transcription initiation sites. Therefore, the plasticity of even a single promoter region can compensate by three different mechanisms for the loss of a key regulatory hub in the E. coli TRN.

CRP i.e. cAMP receptor protein provides a competitive edge to Salmonella Typhimurium in a microbial community set-up

The survival of enteric pathogens in the multi-nutrient environment involves interaction with other organisms. The fact that competition for nutrient resources has shaped the networking of metabolic pathways is well known. The cAMP receptor protein i.e. CRP is a keystone regulatory protein, connecting various metabolic pathways. Our aim was to study the importance of CRP in nutrient uptake and utilization in Salmonella Typhimurium under intra-species and inter-species nutritive competition. The crp gene knockout (Δcrp) was co-cultured with the wild-type or other pathogens. The Δcrp failed to compete with the wild-type Salmonella Typhimurium, Escherichia coli, Vibrio cholerae and Staphylococcus aureus in nutrient intensive media. However, the survival of the co-cultured Δcrp was unaffected in nutrient-poor media. These results suggest that CRP is necessary for the effective acquisition of readily available nutrients as found in rich media in co-culture. The role of released antimicrobials or surface proteins of the co-cultured strains was also overruled by culturing the mutant in the supernatant of these organisms and separating the cultures in the same media respectively. The co-cultured Δcrp showed an enhanced survival when overall metabolism was reduced with low temperature and antibiotics like chloramphenicol. A circumstantial evidence that CRP manages the global and limits the futile metabolism is provided by this study. The absence of CRP doesn’t affect the survival of the standalone culture. However, the role of this protein becomes obvious in a bacterial community setup. Therefore, CRP is crucial for Salmonella to survive in an intestinal and external environment.

AphA is a master regulator of natural competence in Vibrio cholerae

Vibrio cholerae is the etiological agent of cholera, a disease affecting 4.3 million people each year (WHO, 2015). The symptoms of cholera include profuse watery diarrhoea and vomiting, leading to coma and death in serious cases. The El Tor biotype of V. cholerae is globally predominant, and the success of this biotype to cause disease is thought to be due, in part, to the ability of the organism to successfully colonise two different environments; i) marine and brackish coastal waters where V. cholerae forms biofilms on the chitinous surfaces of shellfish, and ii) the human intestine. When bound to chitinous surfaces V. cholerae expresses genes required for biofilm formation, chitin utilisation and natural competence. Conversely, within the human host, V. cholerae switches on the expression of virulence genes. The transcription factor AphA was discovered as a regulator of genes encoding the toxin co-regulated pilus (TCP) of V. cholerae. It is also known that AphA is active when V. cholerae populations have a low cell density. Hence, AphA is generally considered a regulator of virulence and quorum sensing. We have mapped DNA binding by AphA across the entire V. cholerae genome. The majority of AphA target genes encode cell surface and cell envelope proteins. Unexpectedly, we show that AphA also targets key gene clusters within the natural competence regulon of V. cholerae. Using biochemistry and genetics we show that AphA is a master repressor of natural competence and acts to antagonise transcription activation by the cyclic-AMP receptor protein.

Some chemotactic mutants can be progress through development in chimeric populations

Cell migration in response to morphogen gradients affects morphogenesis. Chemotaxis towards adenosine 3', 5'-monophosphate (cAMP) is essential for the early stage of morphogenesis in the slime mold Dictyostelium discoideum. Here, we show that D. discoideum completes morphogenesis without cAMP-chemotaxis-dependent cell migration. The extracellular cAMP gradient is believed to cause cells to form a slug-shaped multicellular structure and fruiting body. The cAMP receptor, cAR1, was not expressed at the cell surface during these stages, correlating with reduced chemotactic activity. Gβ-null cells expressing temperature sensitive Gβ are unable to generate extracellular cAMP (Jin et al., 1998) and thus unable to aggregate and exhibit proper morphogenesis under restrictive temperature. However, when mixed with wild type cells ts-Gβ expressing gβ-null cells normally aggregated and exhibited normal morphogenesis under restrictive temperature. Furthermore, cells migrated after aggregation in a mixture containing wild-type cells. KI-5 cells, which do not show aggregation or morphogenesis, spontaneously migrated to a transplanted wild-type tip and underwent normal morphogenesis and cell differentiation; this was not observed in cells lacking tgrB1and tgrC1 cells adhesion molecules. Thus, cAMP gradient-dependent cell migration may not be required for multicellular pattern formation in late Dictyostelium development.

The GTPase BipA expressed at low temperature in Escherichia coli assists ribosome assembly and has chaperone-like activity

BPI-inducible protein A (BipA) is a conserved ribosome-associated GTPase in bacteria that is structurally similar to other GTPases associated with protein translation, including IF2, EF-Tu, and EF-G. Its binding site on the ribosome appears to overlap those of these translational GTPases. Mutations in the bipA gene cause a variety of phenotypes, including cold and antibiotics sensitivities and decreased pathogenicity, implying that BipA may participate in diverse cellular processes by regulating translation. According to recent studies, a bipA-deletion strain of Escherichia coli displays a ribosome assembly defect at low temperature, suggesting that BipA might be involved in ribosome assembly. To further investigate BipA's role in ribosome biogenesis, here, we compared and analyzed the ribosomal protein compositions of MG1655 WT and bipA-deletion strains at 20 °C. Aberrant 50S ribosomal subunits (i.e. 44S particles) accumulated in the bipA-deletion strain at 20 °C, and the ribosomal protein L6 was absent in these 44S particles. Furthermore, bipA expression was significantly stimulated at 20 °C, suggesting that it encodes a cold shock-inducible GTPase. Moreover, the transcriptional regulator cAMP receptor protein (CRP) positively promoted bipA expression only at 20 °C. Importantly, GFP and α-glucosidase refolding assays revealed that BipA has chaperone activity. Our findings indicate that BipA is a cold shock-inducible GTPase that participates in 50S ribosomal subunit assembly by incorporating the L6 ribosomal protein into the 44S particle during the assembly.

Host lipid sensing promotes invasion of cells with pathogenic Salmonella

Pathogenic Salmonella species initiate infection by invading non-phagocytic intestinal epithelial cells (IEC). This invasion is brought about by a number of Salmonella invasion promoting molecules (Sips) encoded by the Salmonella Pathogenicity Island - 1 (SPI-1). Intracellular delivery of some of these molecules also brings about caspase-1 – mediated pyroptotic cell death that contributes to pathogen clearance. These molecules are secreted and delivered inside cells upon contact of Salmonella with one or more host signals whose identity has not been established. We show that lysophosphatidylcholine (LPC) released following activation of caspase-1 in Salmonella – infected cells and abundant in plasma amplifies production of Sips from this pathogen and promotes its cellular invasion. LPC brings about adenylate cyclase and cAMP receptor protein (CRP) - dependent de novo synthesis of SipC that is accompanied by its translocation to bacterial cell surface and release into the outside milieu. Treatment of Salmonella with LPC produces sustained induction of SPI - 1 transcriptional regulator, hilA. Our findings reveal a novel host lipid sensing - driven regulatory mechanism for Salmonella invasion.

Escherichia coli OxyS RNA triggers cephalothin resistance by modulating the expression of CRP-associated genes

Antibiotics have been one of the most successful forms of therapy in medicine. However, the efficiency of antibiotics is compromised by the emergence of antibiotic-resistant pathogens. To reduce antibiotic resistance, complete understanding of bacterial tactics to defend themselves against antibiotics is necessary. Small-noncoding RNAs (sRNAs) modulate gene expression by base-pairing with multiple target mRNAs. Cellular levels of Hfq-dependent sRNAs influence antibiotic resistance by modulating expression of specific target genes; therefore, such sRNAs could be a good tool to identify target mRNAs that modulate antibiotic susceptibility and may themselves be used as druggable molecules. Here, we report the identification of genes and pathways associated with OxyS RNA-mediated cephalothin resistance using phenotypic and expression analyses of OxyS-regulated genes identified by RNA-seq, literature mining, or predictions. From our studies we found that the differential expression of 27 OxyS-regulated genes was involved in cephalothin susceptibility. Among them, 17 gene knockouts showed resistance to the drug and nine from them is associated with cAMP receptor protein (CRP), a transcriptional dual regulator in E. coli. Moreover, levels of OxyS and OxyS-modulated genes (cycA and cysH) were also altered in multidrug-resistant (MDR) E. coli strains. Together, our data suggest that OxyS extensively modulates gene expression in multiple pathways to develop cephalothin resistance. In addition, OxyS and its regulated target genes, either individually or in combination, could be used as molecular markers and targets for the identification and eradication of cephalothin-resistant strains.

CAMP Receptor Protein of Salmonella enterica Serovar Typhimurium Modulate Glycolysis in Macrophages to Induce Cell Apoptosis.

We studied the role of glycolysis in the mechanism of cAMP receptor protein-induced macrophage cell death of Salmonella enterica serovar Typhimurium (S. Typhimurium). Cell apoptosis, caspase-3, -8, -9 enzyme activity, and pyruvic acid, lactic acid, ATP, and hexokinase (HK) contents were determined after infection of macrophages with S. Typhimurium SL1344 wild-type and a cAMP receptor protein mutant strain. While cell apoptosis, caspase-3, -8, -9 enzyme activity, lactic acid, hexokinase, and ATP levels significantly changed by infection with crp mutants compared to the wild-type strain (P < 0.05). Our data suggest that the cAMP receptor protein of S. Typhimurium can modulate macrophage death by effecting glycolysis levels. This finding may help to elucidate the mechanisms of S. Typhimurium pathogenesis.

Sycrp2 Is Essential for Twitching Motility in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Two cAMP receptor proteins (CRPs), Sycrp1 (encoded by sll1371) and Sycrp2 (encoded by sll1924), exist in the cyanobacterium Synechocystis sp. strain PCC 6803. Previous studies have demonstrated that Sycrp1 has binding affinity for cAMP and is involved in motility by regulating the formation of pili. However, the function of Sycrp2 remains unknown. Here, we report that sycrp2 disruption results in the loss of motility of Synechocystis sp. PCC 6803, and that the phenotype can be recovered by reintroducing the sycrp2 gene into the genome of sycrp2-disrupted mutants. Electron microscopy showed that the sycrp2-disrupted mutant lost the pilus apparatus on the cell surface, resulting in a lack of cell motility. Furthermore, the transcript level of the pilA9-pilA11 operon (essential for cell motility and regulated by the cAMP receptor protein Sycrp1) was markedly decreased in sycrp2-disrupted mutants compared with the wild-type strain. Moreover, yeast two-hybrid analysis and a pulldown assay demonstrated that Sycrp2 interacted with Sycrp1 to form a heterodimer and that Sycrp1 and Sycrp2 interacted with themselves to form homodimers. Gel mobility shift assays revealed that Sycrp1 specifically binds to the upstream region of pilA9 Together, these findings indicate that in Synechocystis sp. PCC 6803, Sycrp2 regulates the formation of pili and cell motility by interacting with Sycrp1.IMPORTANCE cAMP receptor proteins (CRPs) are widely distributed in cyanobacteria and play important roles in regulating gene expression. Although many cyanobacterial species have two cAMP receptor-like proteins, the functional links between them are unknown. Here, we found that Sycrp2 in the cyanobacterium Synechocystis sp. strain PCC 6803 is essential for twitching motility and that it interacts with Sycrp1, a known cAMP receptor protein involved with twitching motility. Our findings indicate that the two cAMP receptor-like proteins in cyanobacteria do not have functional redundancy but rather work together.

Transcriptional Regulation Between the Two Global Regulators RovA and CRP in Yersinia pestis biovar Microtus.

Yersinia pestis is a dangerous bacterial pathogen that can cause plague. Both RovA and cyclic AMP receptor protein (cAMP-CRP) are required for regulating biofilm- and virulence-related genes in Y. pestis. In this study, the transcriptional regulation between RovA and cAMP-CRP were analyzed by using primer extension, quantitative RT-PCR, LacZ fusion, and electrophoretic mobility shift assay. The results indicated that RovA repressed crp transcription in an indirect manner, while that RovA had no regulatory action on cyaA at the transcriptional level. In addition, cAMP-CRP did not regulate the transcription of rovA. Taken together with our previous results, complex regulatory interactions of RovA, cAMP-CRP, and PhoP/PhoQ in Y. pestis were revealed, which would promote us gain deeper understanding about coordinative modulation of biofilm- and virulence-related regulator genes.

Theoretical analysis of inducer and operator binding for cyclic-AMP receptor protein mutants

Allosteric transcription factors undergo binding events at inducer binding sites as well as at distinct DNA binding domains, and it is difficult to disentangle the structural and functional consequences of these two classes of interactions. We compare the ability of two statistical mechanical models—the Monod-Wyman-Changeux (MWC) and the Koshland-Némethy-Filmer (KNF) models of protein conformational change—to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. We use these models to examine a beautiful recent experiment that created a single-chain version of the CRP homodimer, creating six mutants using all possible combinations of the wild type, D53H, and S62F subunits. We demonstrate that the MWC model can explain the behavior of all six mutants using a small, self-consistent set of parameters whose complexity scales with the number of subunits, providing a significant benefit over previous models. In comparison, the KNF model not only leads to a poorer characterization of the available data but also fails to generate parameter values in line with the available structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.

Assessing real-time signaling and agonist-induced CRHR1 internalization by optical methods.

The development of live-cell sensors for real-time measurement of signaling responses, with improved spatial and temporal resolution with respect to classical biochemical methods, has changed our understanding of cellular signaling. Examination of cAMP generation downstream activated GPCRs has shown that signaling responses can be short-lived (generated from the cell surface) or prolonged after receptor internalization. Class B secretin-like Corticotropin-releasing hormone receptor 1 (CRHR1) is a key player in stress pathophysiology. By monitoring real-time signaling in living cells, we uncovered cell context-dependent temporal characteristics of CRHR1-elicited cAMP responses and disclosed a specific link between cAMP generation and receptor signaling from internal compartments. We describe technical aspects and elaborate the protocols for cell line expression of Förster resonance energy transfer (FRET)-based biosensors to study the dynamics of cAMP and calcium signaling responses downstream activated CRHR1, live-cell imaging and analysis, and fluorescence flow cytometry to determine receptor levels at the cell surface.

The Molecular Basis for Specificity at the Level of the Protein Kinase a Catalytic Subunit

Assembly of multi enzyme complexes at subcellular localizations by anchoring- and scaffolding proteins represents a pivotal mechanism for achieving spatiotemporal regulation of cellular signaling after hormone receptor targeting [for review, see (1)]. In the 3′ 5′-cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) signaling pathway it is generally accepted that specificity is secured at several levels. This includes at the first level stimulation of receptors coupled to heterotrimeric G proteins which through stimulation of adenylyl cyclase (AC) forms the second messenger cAMP. Cyclic AMP has several receptors including PKA. PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. The R subunit is the receptor for cAMP and compartmentalizes cAMP signals through binding to cell and tissue-specifically expressed A kinase anchoring proteins (AKAPs). The current dogma tells that in the presence of cAMP, PKA dissociates into an R subunit dimer and two C subunits which are free to phosphorylate relevant substrates in the cytosol and nucleus. The release of the C subunit has raised the question how specificity of the cAMP and PKA signaling pathway is maintained when the C subunit no longer is attached to the R subunit-AKAP complex. An increasing body of evidence points toward a regulatory role of the cAMP and PKA signaling pathway by targeting the C subunits to various C subunit binding proteins in the cytosol and nucleus. Moreover, recent identification of isoform specific amino acid sequences, motifs and three dimensional structures have together provided new insight into how PKA at the level of the C subunit may act in a highly isoform-specific fashion. Here we discuss recent understanding of specificity of the cAMP and PKA signaling pathway based on C subunit subcellular targeting as well as evolution of the C subunit structure that may contribute to the dynamic regulation of C subunit activity.

Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria.

Horizontal gene transfer (HGT) plays an important role in bacterial evolution. It is well accepted that DNA is pulled/pushed into recipient cells by conserved membrane-associated DNA transport systems, which allow the entry of only single-stranded DNA (ssDNA). However, recent studies have uncovered a new type of natural bacterial transformation in which double-stranded DNA (dsDNA) is taken up into the cytoplasm, thus complementing the existing methods of DNA transfer among bacteria. Regulated by the stationary-phase regulators RpoS and cAMP receptor protein (CRP), Escherichia coli establishes competence for natural transformation with dsDNA, which occurs in agar plates. To pass across the outer membrane, a putative channel, which may compete for the substrate with the porin OmpA, may mediate the transfer of exogenous dsDNA into the cell. To pass across the inner membrane, dsDNA may be bound to the periplasmic protein YdcS, which delivers it into the inner membrane channel formed by YdcV. The discovery of cell-to-cell contact-dependent plasmid transformation implies the presence of additional mechanism(s) of transformation. This review will summarize the current knowledge about mechanisms of HGT with an emphasis on recent progresses regarding non-canonical mechanisms of natural transformation. Fully understanding the mechanisms of HGT will provide a foundation for monitoring and controlling multidrug resistance.

Site-directed spin label electron paramagnetic resonance spectroscopy as a probe of conformational dynamics in the Fe(III) "locked-off" state of the CO-sensing transcription factor CooA.

The transcriptional activator CooA belongs to the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase) superfamily of transcriptional regulators and uses heme to sense carbon monoxide (CO). Effector-driven allosteric activation is well understood in CRP, a CooA homologue. A structural allosteric activation model for CooA exists which parallels that of CRP; however, the role of protein dynamics, which is crucial in CRP, is not well understood in CooA. We employed site-directed spin labeling electron paramagnetic resonance spectroscopy to probe CooA motions on the μs-ms timescale. We created a series of Cys substitution variants, each with a cysteine residue introduced into a key functional region of the protein: K26C, E60C, F132C, D134C, and S175C. The heme environment and DNA binding affinity of each variant were comparable to those of wild-type CooA, with the exception of F132C, which displayed reduced DNA binding affinity. This observation confirms a previously hypothesized role for Phe132 in transmitting the allosteric CO binding signal. Osmolyte perturbation studies of Fe(III) "locked-off" CooA variants labeled with either MTSL or MAL-6 nitroxide spin labels revealed that multicomponent EPR spectra report on conformational flexibility on the μs-ms timescale. Multiple dynamic populations exist at every site examined in the structurally uncharacterized Fe(III) "locked-off" CooA. This observation suggests that, in direct contrast to effector-free CRP, Fe(III) "locked-off" CooA undergoes conformational exchange on the μs-ms timescale. Importantly, we establish MAL-6 as a spin label with a redox-stable linkage that may be utilized to compare conformational dynamics between functional states of CooA.

Large-Scale Functional Analysis of CRP-Mediated Feed-Forward Loops.

Feed-forward loops (FFLs) represent an important and basic network motif to understand specific biological functions. Cyclic-AMP (cAMP) receptor protein (CRP), a transcription factor (TF), mediates catabolite repression and regulates more than 400 genes in response to changes in intracellular concentrations of cAMP in Escherichia coli. CRP participates in some FFLs, such as araBAD and araFGH operons and adapts to fluctuating environmental nutrients, thereby enhancing the survivability of E. coli. Although computational simulations have been conducted to explore the potential functionality of FFLs, a comprehensive study on the functions of all structural types on the basis of in vivo data is lacking. Moreover, the regulatory role of CRP-mediated FFLs (CRP-FFLs) remains obscure. We identified 393 CRP-FFLs in E. coli using EcoCyc and RegulonDB. Dose–response genomic microarray of E. coli revealed dynamic gene expression of each target gene of CRP-FFLs in response to a range of cAMP dosages. All eight types of FFLs were present in CRP regulon with various expression patterns of each CRP-FFL, which were further divided into five functional groups. The microarray and reported regulatory relationships identified 202 CRP-FFLs that were directly regulated by CRP in these eight types of FFLs. Interestingly, 34% (147/432) of genes were directly regulated by CRP and CRP-regulated TFs, which indicates that these CRP-regulated genes were also regulated by other CRP-regulated TFs responding to environmental signals through CRP-FFLs. Furthermore, we applied gene ontology annotation to reveal the biological functions of CRP-FFLs.

CAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization.

ABSTRACTThe bacterium Vibrio cholerae is native to aquatic environments and can switch lifestyles to cause disease in humans. Lifestyle switching requires modulation of genetic systems for quorum sensing, intestinal colonization, and toxin production. Much of this regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, we have mapped the binding of cAMP receptor protein (CRP) and RNA polymerase across the V. cholerae genome. We show that CRP is an integral component of the regulatory network that controls lifestyle switching. Focusing on a locus necessary for toxin transport, we demonstrate CRP-dependent regulation of gene expression in response to host colonization. Examination of further CRP-targeted genes reveals that this behavior is commonplace. Hence, CRP is a key regulator of many V. cholerae genes in response to lifestyle changes.

An Allosteric Glucagon Receptor Antagonist, LGD-6972, Displays Biased Receptor Signaling

Glucagon receptor antagonists (GRA) are novel molecules in development for the treatment of type 1 and type 2 (T2DM) diabetes. LGD-6972 recently demonstrated robust efficacy in a 12-week, Phase 2 study in T2DM, reducing HbA1c by 1.2% from baseline, with a safety profile more favorable than other GRAs at this stage of development. LGD-6972 is structurally distinct from other small molecule GRAs, containing a sulphonic acid tail (SAT) rather than a carboxylic acid tail (CAT), potentially explaining this finding. We performed computational modeling of LGD-6972 bound to the glucagon receptor (GCGR) and compared it to x-ray crystallography data with receptor bound MK-0893. The SAT of LGD-6972 creates a larger pocket between helix 6 and 7 in the GCGR transmembrane domain, and alters the orientation of helix 8. Activity of LGD-6972, MK-0893 and other SAT and CAT GRAs were examined in cellular models of glucagon-stimulated cAMP accumulation, β-arrestin recruitment, and receptor internalization. All tested GRAs were full (&amp;gt;95%) antagonists of cAMP accumulation. CAT GRAs were full antagonists of β-arrestin (≥96%) and receptor internalization (≥94%). In contrast, SAT GRAs had reduced antagonism of β-arrestin (58-87%) and receptor internalization (6-52%). The relative potency was also altered with greater potency for CAT GRAs on β-arrestin vs. cAMP, whereas the opposite was true for SAT GRAs. Furthermore, a LGD-6972 analog with a CAT and a MK-0893 analog with a SAT displayed CAT- and SAT-like activity, respectively, indicating a clear structure-activity relationship exists, with LGD-6972 displaying receptor-biased signaling. To further investigate the potential differences among compounds, primary human hepatocytes were treated with glucagon or glucagon with MK-0893 or LGD-6972 and gene expression was analyzed by microarray. Broad differences between glucagon and the GRAs were observed and future studies will investigate the effects of SAT and CAT compounds. Disclosure E.G. Vajda: Employee; Self; Ligand Pharmaceuticals, Inc. L. Zhi: Employee; Self; Ligand Pharmaceuticals, Inc. K. Marschke: Employee; Self; Ligand Pharmaceuticals, Inc..

DdrI, a cAMP Receptor Protein Family Member, Acts as a Major Regulator for Adaptation of Deinococcus radiodurans to Various Stresses

The DNA damage response ddrI gene encodes a transcription regulator belonging to the cAMP receptor protein (CRP) family. Cells devoid of the DdrI protein exhibit a pleiotropic phenotype, including growth defects and sensitivity to DNA-damaging agents and to oxidative stress. Here, we show that the absence of the DdrI protein also confers sensitivity to heat shock treatment, and several genes involved in heat shock response were shown to be upregulated in a DdrI-dependent manner. Interestingly, expression of the Escherichia coli CRP partially compensates for the absence of the DdrI protein. Microscopic observations of ΔddrI mutant cells revealed an increased proportion of two-tetrad and anucleated cells in the population compared to the wild-type strain, indicating that DdrI is crucial for the completion of cell division and/or chromosome segregation. We show that DdrI is also involved in the megaplasmid MP1 stability and in efficient plasmid transformation by facilitating the maintenance of the incoming plasmid in the cell. The in silico prediction of putative DdrI binding sites in the D. radiodurans genome suggests that hundreds of genes, belonging to several functional groups, may be regulated by DdrI. In addition, the DdrI protein absolutely requires cAMP for in vitro binding to specific target sequences, and it acts as a dimer. All these data underline the major role of DdrI in D. radiodurans physiology under normal and stress conditions by regulating, both directly and indirectly, a cohort of genes involved in various cellular processes, including central metabolism and specific responses to diverse harmful environments.IMPORTANCEDeinococcus radiodurans has been extensively studied to elucidate the molecular mechanisms responsible for its exceptional ability to withstand lethal effects of various DNA-damaging agents. A complex network, including efficient DNA repair, protein protection against oxidation, and diverse metabolic pathways, plays a crucial role for its radioresistance. The regulatory networks orchestrating these various pathways are still missing. Our data provide new insights into the crucial contribution of the transcription factor DdrI for the D. radiodurans ability to withstand harmful conditions, including UV radiation, mitomycin C treatment, heat shock, and oxidative stress. Finally, we highlight that DdrI is also required for accurate cell division, for maintenance of plasmid replicons, and for central metabolism processes responsible for the overall cell physiology.