Serum-Stable and Selective Backbone-N-Methylated Cyclic Peptides That Inhibit Prokaryotic Glycolytic Mutases.

N-Methylated amino acids (N-MeAAs) are privileged residues of naturally occurring peptides critical to bioactivity. However, de novo discovery from ribosome display is limited by poor incorporation of N-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the N-methyl-aminoacyl-tRNA. By reconfiguring the tRNA's T-stem region to compensate and tune the EF-Tu affinity, we conducted Random nonstandard Peptides Integrated Discovery (RaPID) display of a macrocyclic peptide (MCP) library containing six different N-MeAAs. We have here devised a "pool-and-split" enrichment strategy using the RaPID display and identified N-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate mutases. The enriched MCPs reached 57% N-methylation with up to three consecutively incorporated N-MeAAs, rivaling natural products. Potent nanomolar inhibitors ranging in ortholog selectivity, strongly mediated by N-methylation, were identified. Co-crystal structures reveal an architecturally related Ce-2 Ipglycermide active-site metal-ion-coordinating Cys lariat MCP, functionally dependent on two cis N-MeAAs with broadened iPGM species selectivity over the original nematode-selective MCPs. Furthermore, the isolation of a novel metal-ion-independent Staphylococcus aureus iPGM inhibitor utilizing a phosphoglycerate mimetic mechanism illustrates the diversity of possible chemotypes encoded by the N-MeAA MCP library.

Effect of voxel size on the accuracy of 3D reconstructions with cone beam CT.

OBJECTIVES: The various types of cone beam CT (CBCT) differ in several technical characteristics, notably their spatial resolution, which is defined by the acquisition voxel size. However, data are still lacking on the effects of voxel size on the metric accuracy of three-dimensional (3D) reconstructions. This study was designed to assess the effect of isotropic voxel size on the 3D reconstruction accuracy and reproducibility of CBCT data. METHODS: The study sample comprised 70 teeth (from the Institut d'Anatomie Normale, Strasbourg, France). The teeth were scanned with a KODAK 9500 3D® CBCT (Carestream Health, Inc., Marne-la-Vallée, France), which has two voxel sizes: 200 µm (CBCT 200 µm group) and 300 µm (CBCT 300 µm group). These teeth had also been scanned with the KODAK 9000 3D® CBCT (Carestream Health, Inc.) (CBCT 76 µm group) and the SCANCO Medical micro-CT XtremeCT (SCANCO Medical, Brüttisellen, Switzerland) (micro-CT 41 µm group) considered as references. After semi-automatic segmentation with AMIRA® software (Visualization Sciences Group, Burlington, MA), tooth volumetric measurements were obtained. RESULTS: The Bland-Altman method showed no difference in tooth volumes despite a slight underestimation for the CBCT 200 µm and 300 µm groups compared with the two reference groups. The underestimation was statistically significant for the volumetric measurements of the CBCT 300 µm group relative to the two reference groups (Passing-Bablok method). CONCLUSIONS: CBCT is not only a tool that helps in diagnosis and detection but it has the complementary advantage of being a measuring instrument, the accuracy of which appears connected to the size of the voxels. Future applications of such measurements with CBCT are discussed.

Accuracy of 3D reconstructions based on cone beam computed tomography.

Three-dimensional imaging of teeth will increase its impact in clinical practice if reconstructions are metrically accurate. We hypothesized that, with cone beam computed tomography (CBCT) data, three-dimensional images of teeth can be reconstructed with the same accuracy and precision as with in vitro micro-computed tomography (micro-CT) data acquisition, the current reference standard. We used a sample of CBCT and micro-CT data taken of tooth germs. Volumes obtained with CBCT and micro-CT devices were statistically similar (n = 120, Passing-Bablok regression). Geometric deviations between CBCT and micro-CT three-dimensional surface reconstructions did not show any areas of important and systematic errors. Future investigations with the use of larger samples may also demonstrate that CBCT data will be helpful for a more in-depth study of other aspects of dental morphology--for example, assessing tooth development. With sufficient accuracy for clinical situations, potential future medical applications of such measurements with CBCT are envisaged.

Miniaturization of a hepatitis C virus RNA polymerase assay using a -102 degrees C cooled CCD camera-based imaging system.

Innovations in detection technologies have allowed us to develop a novel assay in 1536-well plate format and assess the advantages of screen miniaturization compared with conventional high-throughput compound screening in 96- or 384-well plates. An HCV RNA polymerase assay has been miniaturized in 1536-well plates by using a new detection technology known as LEADseeker homogeneous imaging system. It uses a -102 degrees C cooled charge-coupled device (CCD) camera and newly designed scintillation proximity microparticles. The miniaturized assay used europium-doped streptavidin-coated yttrium oxide (YO(x)) or polystyrene (PS) microspheres to capture biotin-labeled [(3)H]RNA product transcripts. Beads in proximity to the radioisotope convert the emitted beta(-) particles into photons having wavelengths in the red region of the visible spectrum, optimal for detection by the CCD camera. Because the camera collects light from all wells of the plate simultaneously, 1536-well plates are imaged as rapidly as 384-well plates, on the order of 10 min per plate. The assay has a signal to background of approximately 20-fold, satisfactory for high-throughput robotics screening. The enzyme kinetics and potency of a known inhibitor were similar to those obtained from the conventional assay using scintillation proximity assay (SPA) beads and a scintillation plate counter. Furthermore, the newly developed microbeads (emitting at 610 to 620 nm) are less prone to quenching effects caused by yellow-colored compounds, than conventional SPA beads or scintillation fluid (emitting at 400 to 480 nm region). Thus, the LEADseeker imaging system is a useful new tool for miniaturization of assays for high-throughput screening.

Radiolabeling of receptor ligands by chemical incorporation of phosphorylation sites.

A chemical reagent, N-acetyl-cys((succinimidyl-6-(thioacetyl)amino) hexanoate)-ser-arg-arg-ala-ser-val-tyr-amide ("phosite NHS ester"), allowing the introduction of phosphorylation sites into proteins, peptides, or small molecules, has been synthesized and characterized. The phosite reagent enables the enzymatic radiolabeling of any protein, peptide, or small molecule containing a reactive amine using [(32)P] or [(33)P]ATP and protein kinase A. The utility of the reagent has been demonstrated in cytokine and G protein-coupled radioligand receptor binding assays using whole cell and immobilized receptor formats. Use of the reagent does not require genetic manipulation of the target ligand.

Ligand-receptor binding measured by laser-scanning imaging.

This report describes the integration of laser-scanning fluorometric cytometry and nonseparation ligand-binding techniques to provide new assay methods adaptable to miniaturization and high-throughput screening. Receptor-bound, cyanine dye-labeled ligands, [Cy]ligands, were discriminated from those free in solution by measuring the accumulated fluorescence associated with a receptor-containing particle. To illustrate the various binding formats accommodated by this technique, saturation- and competition-binding analyses were performed with [Cy]ligands and their cognate receptors expressed in CHO cells or as fusion proteins coated on polystyrene microspheres. We have successfully applied this technique to the analysis of G protein-coupled receptors, cytokine receptors, and SH2 domains. Multiparameter readouts from ligands labeled separately with Cy5 and Cy5.5 demonstrate the simultaneous analysis of two target receptors in a single well. In addition, laser-scanning cytometry has been used to assay enzymes such as phosphatases and in the development of single-step fluorescent immunoassays.

Palmitoylation increases the kinase activity of the G protein-coupled receptor kinase, GRK6.

The G protein-coupled receptor kinase GRK6 undergoes posttranslational modification by palmitoylation. Palmitoylated GRK6 is associated with the membrane, while nonpalmitoylated GRK6 remains cytosolic. We have separated palmitoylated from nonpalmitoylated GRK6 to assess their relative kinase activity. Palmitoylated GRK6 is 10-fold more active at phosphorylating beta2-adrenergic receptor than nonpalmitoylated wild-type GRK6 or a nonpalmitoylatable mutant GRK6. A nonpalmitoylatable mutant GRK6 which has been further mutated to undergo posttranslational geranylgeranylation is also more active, recovering most of the activity of the palmitoylated enzyme. This activity increase by lipid modification is expected, as the lipid helps GRK6 localize to cellular membranes where its receptor substrates are found. However, when assayed using a soluble protein (casein) as a substrate, both palmitoylated and prenylated GRK6 display significantly higher activity than nonpalmitoylated wild-type or nonpalmitoylatable mutant GRK6 kinases. This increased activity is not altered by addition of exogenous palmitate or phosphatidycholine vesicles, arguing that it is not due to direct activation of GRK6 by binding palmitate, nor to nonspecific association of the GRK6 with casein. Further, chemical depalmitoylation reduces the casein phosphorylation activity of the palmitoylated, but not prenylated, GRK6 kinase. Thus, palmitoylation of GRK6 appears to play a dual role in increasing the activity of GRK6: it increases the hydrophobicity and membrane association of the GRK6 protein, which helps bring the GRK6 to its membrane-bound substrates, and it increases the kinase catalytic activity of GRK6.

Chemokine receptor-ligand interactions measured using time-resolved fluorescence.

Two G protein-coupled receptor subtypes (CXCR1 and CXCR2) mediate Interleukin-8 (IL8) action in cells. A nonradioactive lanthanide-chelate derivatized IL8 ligand was developed to measure the binding activity of the chemokine receptors, CXCR1 and CXCR2. Site-specific mutagenesis of the carboxyl-terminal serine of IL8 to cysteine resulted in a mutant IL8 (IL8-S72C) having a single free sulfhydryl. Using an iodoacetamide derivative of the Eu3+-chelate of N-(p-benzoic acid)diethylenetriamine-N,N',N"-tetraacetic acid (DTTA), incorporation of one Eu3+ per IL8 molecule ([Eu3+]IL8-S72C) was achieved. The dissociation constant for this conjugate was similar to that measured for [125I]IL8 ( approximately 2 nM) when measured by time-resolved fluorometry using CHO cell lines stably expressing CXCR1 or CXCR2 receptors. The sensitivity, stability, and high specific activity of europium-labeled IL8 demonstrate the usefulness of lanthanide-labeled proteins in the measurement of receptor-ligand interactions and may be extended to other peptide ligands.

Rhodopsin kinase: expression in baculovirus-infected insect cells, and characterization of post-translational modifications.

Structure-function studies of rhodopsin kinase (RK; EC 2.7.1.125) require a variety of mutants. Therefore, there is need for a suitable system for the expression of RK mutant genes. Here we report on a study of expression of the RK gene in baculovirus-infected Sf21 cells and characterization of the enzyme produced as purified to near homogeneity. Particular attention has been paid to the post-translational modifications, autophosphorylation and isoprenylation, found in the native bovine RK. The protein produced has been purified using, successively, heparin-Sepharose, Mono Q, and Mono S FPLC (fast protein liquid chromatography) and was obtained in amounts of about 2 mg from 1 liter of cell culture. The enzyme from the last step of purification was obtained in two main fractions that differ in the level of phosphorylation. The protein peak eluted first carries two phosphate groups per protein, whereas the second protein peak is monophosphorylated. Further, while both peaks are isoprenylated, the isoprenyl groups consist of mixtures of C5, C10, C15, and C20 isoprenyl moieties. From these results, we conclude that the above expression system is suitable for some but not all aspects of structure-function studies.

Phosducin, potential role in modulation of olfactory signaling.

Phosducin, which tightly binds betagamma-subunits of heterotrimeric G-proteins, has been conjectured to play a role in regulating second messenger signaling cascades, but to date its specific function has not been elucidated. Here we demonstrate a potential role for phosducin in regulating olfactory signal transduction. In isolated olfactory cilia certain odorants elicit a rapid and transient cAMP response, terminated by a concerted process which requires the action of two protein kinases, protein kinase A (PKA) and a receptor-specific kinase (GRK3) (Schleicher, S., Boekhoff, I. Arriza, J., Lefkowitz, R. J., and Breer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1420-1424). The mechanism of action of GRK3 involves a Gbetagamma-mediated translocation of the kinase to the plasma membrane bound receptors (Pitcher, J. A., Inglese, J., Higgins, J. B. , Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M. , Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267). A protein with a molecular mass of 33 kDa that comigrates on SDS gels with recombinant phosducin and which is immunoreactive with phosducin antibodies is present in olfactory cilia. Recombinant phosducin added to permeabilized olfactory cilia preparations strongly inhibits termination of odorant-induced cAMP response and odorant-induced membrane translocation of GRK3. In addition, the cAMP analogue dibutyryl cAMP stimulates membrane targeting of the receptor kinase. This effect is presumably due to PKA-mediated phosphorylation of phosducin, which diminishes its affinity for binding to the Gbetagamma-subunit, thereby making Gbetagamma available to function as a membrane anchor for GRK3. A specific PKA inhibitor blocks the odorant-induced translocation of the receptor kinase. Consistent with this formulation, a non-phosphorylatable mutant of phosducin (phosducin Ser-73 --> Ala) is an even more effective inhibitor of desensitization and membrane targeting of GRK3 than the wild-type protein. A phosducin mutant that mimics phosphorylated phosducin (phosducin Ser-73 --> Asp) lacks this property and in fact recruits GRK3 to the membrane and potentiates desensitization. These results suggest that phosducin may act as a phosphorylation-dependent switch in second messenger signaling cascades, regulating the kinetics of desensitization processes by controlling the activity of Gbetagamma-dependent GRKs.

G protein-coupled receptor kinase mediates desensitization of norepinephrine-induced Ca2+ channel inhibition.

G protein-coupled receptors are essential signaling molecules at sites of synaptic transmission. Here, we explore the mechanisms responsible for the use-dependent termination of metabotropic receptor signaling in embryonic sensory neurons. We report that the inhibition of voltage-dependent Ca2+ channels mediated by alpha2-adrenergic receptors desensitizes slowly with prolonged exposure to the transmitter and that the desensitization is mediated by a G protein-coupled receptor kinase (GRK). Intracellular introduction of recombinant, purified kinases or synthetic blocking peptides into individual neurons demonstrates the specific involvement of a GRK3-like protein. These results suggest that GRK-mediated termination of receptor-G protein coupling is likely to regulate synaptic strength and, as such, may provide one effective mechanism for depression of synaptic transmission.

Characterization of the G protein-coupled receptor kinase GRK4. Identification of four splice variants.

A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.

Heterotrimeric G proteins interact with the small GTPase ARF. Possibilities for the regulation of vesicular traffic.

Trimeric G proteins have emerged as important regulators of membrane trafficking. To explore a role for G beta gamma in endosome fusion, we have taken advantage of beta-adrenergic receptor kinase (beta ARK), an enzyme translocated to membranes by interaction with G beta gamma. The COOH terminus of beta ARK (beta ARKct) has a G beta gamma-binding domain which blocks some G beta gamma-mediated processes. We found that beta ARKct and peptide G, a peptide derived from beta ARKct, inhibit in vitro endosome fusion. Interestingly, peptide G and ARF share sequence similarity. Peptide G and beta ARKct reversed ARF-mediated inhibition of endosome fusion and blocked ARF binding to membranes. Using an ARF fusion protein, we show that both G beta gamma and G alpha s interact with the small GTPase ARF, an interaction that is regulated by nucleotide binding. We conclude that G proteins may participate in the regulation of vesicular trafficking by directly interacting with ARF, a cytosolic factor required for transport.

Ca(2+)-dependent interaction of recoverin with rhodopsin kinase.

Recoverin (Rv) is a myristoylated Ca(2+)-binding protein present primarily in bovine photoreceptors. It represents a newly identified family of neuronal specific Ca(2+)-binding proteins that includes neurocalcin, hippocalcin, and guanylyl cyclase-activating protein. To investigate the function of Rv in photoreceptors, we identified proteins that bind immobilized Rv in a Ca(2+)-dependent manner. Rhodopsin kinase (RK), interphotoreceptor retinoid-binding protein, and tubulin interact with Rv in the presence of Ca2+. The importance of the Rv/RK interaction was further characterized. RK, purified using immobilized Rv as an affinity matrix, catalyzed the light-dependent and Ca(2+)-independent incorporation of phosphates into rhodopsin when reconstituted with urea-stripped rod outer segment membranes. When only a small fraction (0.04%) of rhodopsin was photolyzed, as many as 700 phosphates were incorporated per photolyzed rhodopsin, a phenomenon known as "high gain" phosphorylation. When recoverin was added, the activity of RK became sensitive to free Ca2+, with EC50 = 3 microM. The N-terminal myristoyl residue of Rv enhances the inhibitory effect of Rv and introduces cooperativity to the Ca(2+)-dependent inhibition of rhodopsin phosphorylation. Rv neither interacts with other members of the G-protein-coupled receptor kinase family such as beta-adrenergic receptor kinase 1 nor inhibits beta-adrenergic receptor kinase 1 activity. The specific and Ca(2+)-dependent Rv/RK interaction is necessary for the inhibitory effect of Rv on rhodopsin phosphorylation and may play an important role in photoreceptor light adaptation.

Rhodopsin kinase autophosphorylation. Characterization of site-specific mutations.

Upon illumination, rhodopsin kinase (RK) phosphorylates the visual pigment rhodopsin, which is thought to partially terminate the biochemical events that follow photon absorption. RK enzymology was explored by mutagenesis of the residues Ser488, Thr489 (major autophosphorylation sites), and Lys491 (a distal residue). We found the following to be true. (i) Double mutations at residues Ser488 and Thr489 to Ala or Asp decrease autophosphorylation to substoichiometrical levels, while single mutations at either residue independently reduce autophosphorylation by half. (ii) Phosphorylation of residue Ser488 influences the affinity of RK for heparin-Sepharose only moderately, whereas Thr489 and Lys491 are important for this interaction. RK K491A does not phosphorylate acidic peptides, suggesting that this residue participates in substrate binding. (iii) Mutations in the autophosphorylation region affect the Km for ATP, suggesting that this region is involved in binding of ATP to the catalytic site. (iv) RK mutants S488A or S488D and RK S488A and T489A have an increased ability to phosphorylate Rho in the dark. (v) Mutations at the autophosphorylation region change the initial site of phosphorylation on photolyzed rhodopsin (Rho*), implying that this region may regulate selectivity of the site of phosphorylation.

Cardiac muscarinic potassium channel activity is attenuated by inhibitors of G beta gamma.

The cardiac muscarinic potassium channel (IK.ACh) is activated by a G protein upon receptor stimulation with acetylcholine. The G protein subunit responsible for activation (G alpha versus G beta gamma) has been disputed. We used G beta gamma inhibitors derived from the beta-adrenergic kinase 1 (beta ARK1) to assess the relative importance of G beta gamma in IK.ACh activation. In rabbit atrial myocytes, IK.ACh had a conductance of 49 +/- 6.2 pS. In inside-out patches, the mean open time was 1.60 +/- 0.57 ms, mean time constant (tau o) was 1.59 +/- 0.53 ms, and mean closed time was 3.02 +/- 1.35 ms (n = 38). beta ARK1 is a G beta gamma-sensitive enzyme that interacts with G beta gamma through a defined sequence near its carboxyl terminus. A 28-amino-acid peptide derived from the carboxyl terminus of beta ARK1 (peptide G) increased the closed time to 10.04 ms (P < .001) and decreased opening probability (NPo) by 71% (P < .001). Fusion proteins containing the entire carboxyl terminus of beta ARK1, glutathione S-transferase beta ARK1ct and hexahistidine beta ARK1ct, decreased NPo by 67% (P = .03) and 48% (P = .009), respectively. They also both significantly increased the closed time. None of the inhibitors affected mean open time or channel amplitude. A control peptide derived from a neighboring region of beta ARK1 had no significant effect on IK.ACh activity. These results provide further evidence for the role of G beta gamma in the activation of IK.ACh.

G beta gamma interactions with PH domains and Ras-MAPK signaling pathways.

G-protein-coupled receptor signaling and receptor tyrosine kinase (RTK) signaling are two mechanisms of transmembrane communication used by numerous extracellular agents and stimuli. The beta gamma-subunit complex of G proteins mediates many of the functions associated with G-protein-coupled receptor signaling and may even provide a means to link G proteins to RTK-initiated cascades. This connection may be mediated by the pleckstrin homology domain, a modular domain found in many signaling proteins that interact with G beta gamma.

Protein kinases that phosphorylate activated G protein-coupled receptors.

G protein-coupled receptor kinases (GRKs) are a family of serine/threonine protein kinases that specifically recognize agonist-occupied, activated G protein-coupled receptor proteins as substrates. Phosphorylation of an activated receptor by a GRK terminates signaling by that receptor, by initiating the uncoupling of the receptor from heterotrimeric G proteins. Six distinct mammalian GRKs are known, which differ in tissue distribution and in regulatory properties. The intracellular localization of GRKs to membrane-bound receptor substrates is the most important known regulatory feature of these enzymes. Rhodopsin kinase (GRK1) requires a post-translationally added farnesyl isoprenoid to bind to light-activated rhodopsin. The beta-adrenergic receptor kinases (GRK2 and GRK3) associate with heterotrimeric G protein beta gamma-subunits, released upon receptor activation of G proteins, for membrane anchorage. The recently-described GRKs 4, 5, and 6 comprise a distinct subgroup of GRKs. These kinases utilize distinct mechanisms for membrane localization, which are just beginning to be defined. All GRKs appear to play the same general cellular role of desensitizing activated G protein-coupled receptors, but utilize distinctly individual means to the same end.