Pseudo Substrate Research Articles

A salt bridge of the C-terminal carboxyl group regulates PHPT1 substrate affinity and catalytic activity.

PHPT1 is a histidine phosphatase that modulates signaling in eukaryotes through its catalytic activity. Here, we present an analysis of the structure and dynamics of PHPT1 through a combination of solution NMR, molecular dynamics, and biochemical experiments. We identify a salt bridge formed between the R78 guanidinium moiety and the C-terminal carboxyl group on Y125 that is critical for ligand binding. Disruption of the salt bridge by appending a glycine residue at the C-terminus (G126) leads to a decrease in catalytic activity and binding affinity for the pseudo substrate, para-nitrophenylphosphate (pNPP), as well as the active site inhibitor, phenylphosphonic acid (PPA). We show through NMR chemical shift, 15N relaxation measurements, and analysis of molecular dynamics trajectories, that removal of this salt bridge results in an active site that is altered both structurally and dynamically thereby significantly impacting enzymatic function and confirming the importance of this electrostatic interaction.

Protein Kinase A Catalytic subunit:Smoothened interface ‐ Role of a parallel holoenzyme in negative feedback in Hedgehog Signaling

During signal transduction in the Hedgehog pathway, the G protein‐coupled receptor (GPCR) Smoothened (SMO) signals intracellularly via a novel mechanism involving sequestration of the Protein Kinase A (PKA) catalytic (C)‐subunit at the membrane. Activation of Hedgehog signaling leads to negative feedback regulation mediated through inhibition of PKA‐C by SMO at the ciliary membrane. While it is known that PKA‐C interacts with SMO at its C‐terminal proximal cytoplasmic tail domain (pCT), the dynamics of PKA‐C:SMO interactions is poorly understood. Here, we report a map of the PKA‐C:SMO interface by Amide Hydrogen Deuterium Exchange Mass spectrometry (HDXMS) over 1‐10 min timescales. SMO binding on PKA‐C resulted in global decreases in deuterium exchange (Dex). Peptides spanning the kinase activation loop (188‐198), P+1 loop (199‐211) and pseudo‐substrate recognition site between β5 and αD (129‐145) showed significant protection (>0.5 Da) when bound to SMO, identifying these regions as the SMO binding interface on PKA‐C. Interestingly, the footprint of SMO binding on PKA‐C was identical to that of the RIa regulatory subunit in PKA‐I holoenzymes. Furthermore, increased Dex (0.4 Da) was observed at the region spanning αC and β4 (83‐100), indicating that SMO binding destabilizes the active kinase conformation to allosterically inhibit PKA‐C. Correspondingly on SMO, PKA‐C binding elicited only low magnitudes of Dex protection (0.4 Da) in regions flanking the pseudo substrate site (573‐590, 622‐631). Increased Dex (0.4 Da) was observed at the sterol binding pocket (96‐112) in the extracellular Cysteine rich domain, indicating that PKA‐C binding may allosterically impact SMO activation by modulating its ability to bind sterols. A significant increase in Dex (0.9 Da) was observed at SMO helix 8 (537‐546) which is positioned parallel to the membrane, indicating that C binding promoted a disordered helix 8 conformation. We speculate that the pCT exists in a membrane‐bound or folded conformation in free SMO, but PKA‐C interaction switches the binding mode of pCT leading to a significantly Dex‐protected state. Thus, PKA‐C:SMO interactions reveal an unexpected intersection between cyclic nucleotide signaling and the Hedgehog pathway, defining a novel mechanism of PKA‐C regulation beyond the canonical PKA signalosome.

Impact of deleterious missense PRKCI variants on structural and functional dynamics of protein

Protein kinase C iota (PKCɩ) is a novel protein containing 596 amino acids and is also a member of atypical kinase family. The role of PKCɩ has been explored in neurodegenerative diseases, neuroblastoma, ovarian and pancreatic cancers. Single nucleotide polymorphisms (SNPs) have not been studied in PKCɩ till date. The purpose of the current study is to scrutinize the deleterious missense variants in PKCɩ and determine the effect of these variants on stability and dynamics of the protein. The structure of protein PKCɩ was predicted for the first time and post translational modifications were determined. Genetic variants of PKCɩ were retrieved from ENSEMBL and only missense variants were further analyzed because of its linkage with diseases. The pathogenicity of missense variants, effect on structure and function of protein, association with cancer and conservancy of the protein residues were determined through computational approaches. It is observed that C1 and the pseudo substrate region has the highest number of pathogenic SNPs. Variations in the kinase domain of the protein are predicted to alter overall phosphorylation of the protein. Molecular dynamic simulations predicted noteworthy change in structural and functional dynamics of the protein because of these variants. The study revealed that nine deleterious variants can possibly contribute to malfunctioning of the protein and can be associated with diseases. This can be useful in diagnostics and developing therapeutics for diseases related to these polymorphisms.

Identification of the Kinase-Substrate Recognition Interface between MYPT1 and Rho-Kinase.

Protein kinases exert physiological functions through phosphorylating their specific substrates; however, the mode of kinase–substrate recognition is not fully understood. Rho-kinase is a Ser/Thr protein kinase that regulates cytoskeletal reorganization through phosphorylating myosin light chain (MLC) and the myosin phosphatase targeting subunit 1 (MYPT1) of MLC phosphatase (MLCP) and is involved in various diseases, due to its aberrant cellular contraction, morphology, and movement. Despite the importance of the prediction and identification of substrates and phosphorylation sites, understanding of the precise regularity in phosphorylation preference of Rho-kinase remains far from satisfactory. Here we analyzed the Rho-kinase–MYPT1 interaction, to understand the mode of Rho-kinase substrate recognition and found that the three short regions of MYPT1 close to phosphorylation sites (referred to as docking motifs (DMs); DM1 (DLQEAEKTIGRS), DM2 (KSQPKSIRERRRPR), and DM3 (RKARSRQAR)) are important for interactions with Rho-kinase. The phosphorylation levels of MYPT1 without DMs were reduced, and the effects were limited to the neighboring phosphorylation sites. We further demonstrated that the combination of pseudosubstrate (PS) and DM of MYPT1 (PS1 + DM3 and PS2 + DM2) serves as a potent inhibitor of Rho-kinase. The present information will be useful in identifying new substrates and developing selective Rho-kinase inhibitors.

Demonstration of device-quality 60% relaxed In0.2Ga0.8N on porous GaN pseudo-substrates grown by PAMBE

Achieving high-quality, relaxed InGaN substrates for longer-wavelength light emitting diodes (LEDs) is of great interest for the development of micro-LED based display technology. This work demonstrates molecular beam epitaxy (MBE)-grown In0.2Ga0.8N with a strain relaxation of 60% corresponding to an equivalently fully relaxed In composition of 12%. This was done by growing on a GaN-on-porous GaN pseudo-substrate (PS). The surface morphology of this film was found to be free of V-defects on the surface and with a threading dislocation density comparable to that of the GaN layers beneath. While InGaN grown on planar GaN-on-sapphire substrates remained nearly strained to the GaN underlayer, InGaN grown under identical conditions on PS displayed elastic-like relaxation. Furthermore, an increase in indium (In) composition was observed for the InGaN grown on PS. Where past work of InGaN grown on porous GaN PS by metalorganic chemical vapor deposition also resulted in relaxed InGaN templates suitable for device application, the surfaces of these relaxed films exhibited V-defects for thicker layers. Employing MBE, thicker films with higher In composition can be achieved with smooth surface morphology, thus enabling pseudo-substrates with a wide range of lattice constants. These pseudo-substrates of varying in-plane lattice constant are attractive for III-nitride based optoelectronics, particularly for green, amber, and red micro-LEDs.

The p105 NF-ĸB precursor is a pseudo substrate of the ubiquitin ligase FBXO7, and its binding to the ligase stabilizes it and results in stimulated cell proliferation

The NF-κB transcription factor is involved in inflammation and cell proliferation, survival, and transformation. It is a heterodimer made of p50 or p52 and a member of the Rel family of proteins. p50 and p52 are derived from limited ubiquitin- and proteasome-mediated proteolytic processing of the larger precursors p105 and p100, respectively. Both precursors can be either processed or completely degraded by the ubiquitin-proteasome system. Previous work in our laboratory identified KPC1 as a ubiquitin ligase that mediates processing of p105 to the p50 subunit. Overexpression of the ligase leads to increased level of p50 with a resultant marked tumor-suppressive effect. In the present study, we identify FBXO7, a known ubiquitin ligase that binds to p105 and ubiquitinates it, but surprisingly, leads to its accumulation and to that of p65 - the Rel partner of p50 - and to increased cell proliferation. Importantly, a ΔF-Box mutant of FBXO7 which is inactive has similar effects on accumulation of p105 and cell proliferation, strongly suggesting that p105 is a pseudo substrate of FBXO7.

Blue-light induced breakdown of barrier function on human retinal epithelial cells is mediated by PKC-ζ over-activation and oxidative stress

We aimed to study the time course decrease of human retinal pigment epithelium (RPE) barrier function when exposed to blue light. To this end, we cultured ARPE-19 cells on Electrical Cell-substrate Impedance Sensing (ECIS) multi-well arrays. Using an ad hoc light emitting diode (LED) array illumination system together with a set of neutral density filters and a 3-dimensional (3D) printed filter holder, cells were exposed to a gradient of irradiances of blue-light with a measured peak at 468 nm. The electrical resistance between 4 kHz and 64 kHz was recorded during the exposure. Blue light exposure induced a dose-dependent decrease in the resistances at 4 kHz, however the time course resistance at 64 kHz did not show any decrease before t = 52 h. Quantification of the barrier function using mathematical model integrated in the ECIS software showed that blue-light exposure induced a dose-dependent decrease in the barrier function associated with tight junction formation (P < 0.05). This was confirmed by the immunostaining of the tight-junction associated structural protein, Zonula occludens-1 (ZO-1). The detection of reactive oxygen species by carboxy-H2DCFDA confirmed that the blue light induced dose-dependent decrease in the barrier function is mediated by oxidative stress. On a separate experiment, blue-light exposed ARPE-19 cells were treated with 100 nM Protein Kinase C zeta (PKC-ζ) pseudo substrate inhibitor to identify underlying pathway for blue-light induced damage on the barrier function. The treatment with 100 nM PKC-ζ pseudo substrate inhibitor induced faster recovery of the barrier function compared to no treatment. Altogether our results document that blue LED light exposure decreased RPE barrier function in-vitro in a dose-dependent manner, before any cell death occurred. This damage induced by blue-light on tight junctions is mediated by oxidative stress through PKC-ζ activation. The quantification of the healing effect observed by inhibition of PKC-ζ might lead to development of high throughput wound healing assays through ECIS in the future.

Corona Assisted Ga Based Nanowire Growth on 3C-SiC(111)/Si(111) Pseudosubstrates

Gallium oxide nanowires were grown on different substrates using a corona plasma assisted vapor phase epitaxy process and gold catalyst. It is shown that the silicon carbide pseudo substrate in combination with the plasma excitation of the gas phase supports the growth of the gallium oxide nanowires. Analyzing the orientation of the nanowires with respect to the growth surface, it is concluded that the nanowires growth proceed along the fast growth direction of gallium oxide.

Metamorphic Al0.5Ga0.5N:Si on AlN/sapphire for the growth of UVB LEDs

In this paper we investigate the growth of metamorphic Al0.5Ga0.5N:Si on c-plane AlN/sapphire. The structural properties of the AlGaN:Si pseudo substrates and the electro-optical characteristics of subsequently grown UVB LEDs are being examined. We demonstrate, that superlattices allow the controlled strain relaxation of Al0.5Ga0.5N by rearrangement of threading dislocations, thus preventing the formation of cracks. This study investigates AlN/GaN superlattices with a nominal GaN layer thickness between 1.0nm and 2.5nm at a fixed AlN layer thickness of 2.5nm. The number of superlattice-periods was also varied between 20 and 120. It was found that beyond a GaN layer thickness of 1.5nm three-dimensional structures are formed. Additionally, these three-dimensional structures reduce the local defect density of the subsequently grown Al0.5Ga0.5N layer. Although the Al0.5Ga0.5N layer appears to be almost fully relaxed, the relaxation state of this pseudo substrate, was found to be dependent on the GaN layer thickness in the superlattice. After optimizing the superlattice structure we were able to grow crack free 4μm thick Si-doped Al0.5Ga0.5N layers and on top UVB LEDs with a fully strained active region emitting at 310nm with output powers of more than 18mW at 500mA.

Su1885 Prolonged High-Fat Feeding Decreases SGLT1 and GLUT2 Activity and Blunts Their Upregulation by Glucose-Dependent Insulinotropic Polypeptide (GIP) in Obese Mice

generate PCTV to transport the chylomicron from ER to Golgi. The chylomicron output into lymph is correlated to intestinal luminal phosphatidylcholine (PC). Luminal PC is absorbed as lyso-phosphatidylcholine (lyso-PC). We previously showed that the dietary lipids are absorbed from the apical membrane by Caveolin-1 containing Cytosolic Endocytic Vesicles (CEV). We tested the hypothesis that lyso-PC activates the PKCζ; detach it from CEV, enabling PKCζ to phosphorylate Sar1b. Methods: Cytosol was isolated from rats whose intestinal PC was altered by (A) bile diversion, no PC (B) saline infusion, low PC (C) chow fed, normal PC (D) fat fed, high PC and (E) PC infusion, very high PC. PKCζ activity was measured by phosphorylation of PKCζ pseudo substrate. Lyso-PC was measured by HPLC. Results: PKCζ was activated by lyso-PC, non-linear regression curve for PKCζ vs. lyso-PC, calculated as Km=1.49 ± 0.244 nM and Vmax = 1.12 ± 1.058 nM. The amount of cytosolic lyso-PC in A to E ranges from 0 to 0.45 nM, suggesting that the amount of cytosolic lysoPC is always within a range to control PKCζ activation. PKCζ activity ranges from 0 to 0.7 Arbitrary Unit in A to E. Post absorptive CEV contain PKCζ by western blot but PKCζ detachment from the CEV is proportional to cytosolic lyso-PC as estimated by western blot of CEV. Biotinylation of r-PKCζ showed a conformational change on activation by lyso-PC. We conclude that PKCζ on CEV was activated by lyso-PC, changes its conformation and eluted from vesicles, enter into the cytosol and phosphorylate Sar1b, split the heteroquatramer protein complex to release FABP1. Now free FABP1 can bind to ER membrane for PCTV formation, which transports chylomicrons from ER to Golgi. Fig. 1 illustrated the scheme for control of dietary lipid transport.

Su1887 Mechanism of Inhibition of Na-Glutamine Co-Transport by Prostaglandin E2 in Intestinal Epithelial Cells

generate PCTV to transport the chylomicron from ER to Golgi. The chylomicron output into lymph is correlated to intestinal luminal phosphatidylcholine (PC). Luminal PC is absorbed as lyso-phosphatidylcholine (lyso-PC). We previously showed that the dietary lipids are absorbed from the apical membrane by Caveolin-1 containing Cytosolic Endocytic Vesicles (CEV). We tested the hypothesis that lyso-PC activates the PKCζ; detach it from CEV, enabling PKCζ to phosphorylate Sar1b. Methods: Cytosol was isolated from rats whose intestinal PC was altered by (A) bile diversion, no PC (B) saline infusion, low PC (C) chow fed, normal PC (D) fat fed, high PC and (E) PC infusion, very high PC. PKCζ activity was measured by phosphorylation of PKCζ pseudo substrate. Lyso-PC was measured by HPLC. Results: PKCζ was activated by lyso-PC, non-linear regression curve for PKCζ vs. lyso-PC, calculated as Km=1.49 ± 0.244 nM and Vmax = 1.12 ± 1.058 nM. The amount of cytosolic lyso-PC in A to E ranges from 0 to 0.45 nM, suggesting that the amount of cytosolic lysoPC is always within a range to control PKCζ activation. PKCζ activity ranges from 0 to 0.7 Arbitrary Unit in A to E. Post absorptive CEV contain PKCζ by western blot but PKCζ detachment from the CEV is proportional to cytosolic lyso-PC as estimated by western blot of CEV. Biotinylation of r-PKCζ showed a conformational change on activation by lyso-PC. We conclude that PKCζ on CEV was activated by lyso-PC, changes its conformation and eluted from vesicles, enter into the cytosol and phosphorylate Sar1b, split the heteroquatramer protein complex to release FABP1. Now free FABP1 can bind to ER membrane for PCTV formation, which transports chylomicrons from ER to Golgi. Fig. 1 illustrated the scheme for control of dietary lipid transport.

Dynamics of Bcl-xL in Water and Membrane: Molecular Simulations

The Bcl2 family of proteins is capable of switching the apoptotic machinery by directly controlling the release of apoptotic factors from the mitochondrial outer membrane. They have ‘pro’ and ‘anti’-apoptotic subgroups of proteins which antagonize each other’s function; however a detailed atomistic understanding of their mechanisms based on the dynamical events, particularly in the membrane, is lacking. Using molecular dynamics simulations totaling 1.6µs we outline the major differences between the conformational dynamics in water and in membrane. Using implicit models of solvent and membrane, the simulated results reveal a picture that is in agreement with the ‘hit-and run’ concept which states that BH3-only peptides displace the tail (which acts as a pseudo substrate of the protein itself) from its binding pocket; this helps the membrane association of the protein after which the BH3 peptide becomes free. From simulations, Bcl-xL appears to be auto-inhibited by its C-terminal tail that embeds into and covers the hydrophobic binding pocket. However the tail is unable to energetically compete with BH3-peptides in water. In contrast, in the membrane, neither the tail nor the BH3-peptides are stable in the binding pocket and appear to be easily dissociated off as the pocket expands in response to the hydrophobic environment. This renders the binding pocket large and open, thus receptive to interactions with other protein partners. Principal components of the motions are dramatically different in the aqueous and in the membrane environments and provide clues regarding the conformational transitions that Bcl-xL undergoes in the membrane, in agreement with the biochemical data.

Contact pattern sensitivity and precision machine control in roll-to-roll microcontact printing

ABSTRACTScaling contact lithography (microcontact printing, microflexography, and nanoimprint lithography) to large roll-to-roll platforms will enable high speed, low cost lithographic patterning of surfaces. However, many details of robust implementations at the roll-to-roll scale remain an engineering challenge, including precise regulation of printing pressures and the stamp-substrate interaction. This paper introduces a method for precise control of contact pressure that can accommodate large dimensional variations, i.e. varying stamp and substrate thicknesses. This control algorithm is implemented on a simply supported roll positioning stage. Experimental results for microcontact printing and microflexography are shown both with in situ contact measurements on a pseudo substrate and with 5 um silver nanoparticle prints. Ultimately, this approach enables robust printing despite sensitive stamp patterns and large dimensional variations (&gt; 10 μm) in substrates, stamps, and roll equipment.

Chemical, genetic and structural assessment of pyridoxal kinase as a drug target in the African trypanosome

Pyridoxal-5′-phosphate (vitamin B6) is an essential cofactor for many important enzymatic reactions such as transamination and decarboxylation. African trypanosomes are unable to synthesise vitamin B6de novo and rely on uptake of B6 vitamers such as pyridoxal and pyridoxamine from their hosts, which are subsequently phosphorylated by pyridoxal kinase (PdxK). A conditional null mutant of PdxK was generated in Trypanosoma brucei bloodstream forms showing that this enzyme is essential for growth of the parasite in vitro and for infectivity in mice. Activity of recombinant T. brucei PdxK was comparable to previously published work having a specific activity of 327 ± 13 mU mg−1 and a Kmapp with respect to pyridoxal of 29.6 ± 3.9 µM. A coupled assay was developed demonstrating that the enzyme has equivalent catalytic efficiency with pyridoxal, pyridoxamine and pyridoxine, and that ginkgotoxin is an effective pseudo substrate. A high resolution structure of PdxK in complex with ATP revealed important structural differences with the human enzyme. These findings suggest that pyridoxal kinase is an essential and druggable target that could lead to much needed alternative treatments for this devastating disease.

Synthesis and Evaluation of a New Series of Tri-, Di-, and Mono-N-alkylcarbamylphloroglucinols as Bulky Inhibitors of Acetylcholinesterase

1,3,5-Tri-N-alkylcarbamylphloroglucinols (1-4) are synthesized as a new series of bulky inhibitors of acetylcholinesterase that may block the catalytic triad, the anionic substrate binding site, and the entrance of the enzyme simultaneously. Among three series of phloroglucinol-derived carbamates, tridentate inhibitors 1,3,5-tri-N-alkylcarbamylphloroglucinols (1-4), bidentate inhibitors 3,5-di-N-n-alkylcarbamyloxyphenols (5-8), and monodentate inhibitors 5-N-n-alkylcarbamyloxyresorcinols (9-12), tridentate inhibitors 1-4 are the most potent inhibitors of mouse acetylcholinesterase. When different n-alkylcarbamyl substituents in tridentate inhibitors 1-4 are compared, n-octylcarbamate 1 is the most potent inhibitor of the enzyme. All inhibitors 1-12 are characterized as the pseudo substrate inhibitors of acetylcholinesterase. Thus, tridentate inhibitors 1-4 are supposed to be hydrolyzed to bidentate inhibitors 5-8 after the enzyme catalysis. Subsequently, bidentate inhibitors 5-8 and monodentate inhibitors 9-12 are supposed to yield monodentate inhibitors 9-12 and phloroglucinol, respectively, after the enzyme catalysis. This means that tridentate inhibitors 1-4 may act as long period inhibitors of the enzyme. Therefore, inhibitors 1-4 may be considered as a new methodology to develop the long-acting drug for Alzheimer's disease. Automated dockings of inhibitor 1 into the X-ray crystal structure of acetylcholinesterase suggest that the most suitable configuration of inhibitor 1 to the enzyme binding is the (1,3,5)- (cis,trans,trans)-tricarbamate rotamer. The cis-carbamyl moiety of this rotamer does not bind into the acetyl group binding site of the enzyme but stretches out itself to the entrance. The other two trans-carbmayl moieties of this rotamer bulkily block the tryptophan 86 residue of the enzyme.

Annealing, Passivation and Contacting of Ion Implanted Phosphorus Emitter Solar Cells

Recently, ion implantation doping technique has shown potential to improve silicon solar cell efficiency while reducing the number of process steps. In this context, we developed and studied a p-type silicon solar cells fabrication process with a phosphorus implanted emitter. The process is similar to the POCl3 homogeneous diffusion process however replaces the high temperature gaseous diffusion and associated steps by an implantation step followed by an annealing to activate phosphorus dopants. We employed simulation results and advanced characterization to optimize the annealing temperature. The influence of this annealing temperature on the activated dopant profiles and on the crystal damage was highlighted. By adding an O2 flow during the annealing step, a SiO2 layer was grown and an optimum SiO2/SiNx stack was used to improve passivation. Its benefits were quantified by comparison with the standard SiNx passivation. The influence of the implantation dopant density on the passivation quality was also studied. By using an adapted front screen-printed silver paste to contact this new implanted emitter profile we demonstrated an 18,73% certified solar cell efficiency on industrial 156x156 mm2 pseudo square substrate.

Kinetics and Protein‐Inhibitor Docking Studies of Enantiomers of exo‐2‐Norbornyl‐N‐n‐butylcarbamates as Pseudomonas Lipase Inhibitors to Probe the Enantioselectivity of the Enzyme

AbstractThe Pseudomonas species lipase inhibition shows enantioselectivity for R‐enantiomer over S‐enantiomer of exo‐2‐norbornyl‐N‐n‐butylcarbamates. R‐, S‐, and racemic‐exo‐2‐norbornyl‐N‐n‐butylcarbamates are all characterized as pseudo substrate inhibitors of the enzyme. Thus, the mechanism for Pseudomonas species lipase‐catalyzed hydrolysis of the inhibitor is formation of the first enzyme‐inhibitor Michaelis complex via nucleophilic attack of the active site serine to the inhibitor (Ki step) then formation of the butylcarbamyl enzyme intermediate from this complex (k2 step). Comparison of bimolecular rate constants (ki = k2 / Ki) of the inhibitors indicates that R‐enantiomer is 1.8 times more potent than S‐enantiomer. Thus, Pseudomonas species lipase shows enantioselectivity of 1.8 for R‐exo‐2‐norbornyl‐N‐n‐butyl‐carbamate over S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate. Protein‐ligand interaction studies on both enantiomers of exo‐2‐norbornyl‐N‐n‐butylcarbamate as inhibitors of Pseudomonas species lipase using AutoDock suggest that R‐enantiomer binds more tightly into the active site of the enzyme than S‐enantiomer. The norbornyl ring of S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate is repulsive to Ser 82 and His 251 of the catalytic triad as well as to Met 16 of the oxyanion hole. These repulsions may create few unfavorable interactions between S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate and the enzyme and make this inhibitor a less potent one.

Similarities and Differences in Signal Transduction and Crosstalk of the G Protein-Coupled Receptors CXCR4 and CysLT1 in CD34+ Hematopoietic Stem/Progenitor Cells,

Abstract 3392CD34+ hematopoietic stem- and progenitor cells (HPC) express high levels of the G protein-coupled receptors (GPCR) CXCR4 and CysLT1. In contrast to the established role of CXCR4 in stem cell homing, the function of CysLT1 in HPC remains only partially understood. We found that upon stimulation of peripheral blood CD34+ HPC in vitro with the respective ligands (CXCL12/SDF1 for CXCR4 and the cysteinyl-leukotriene LTD4 for CysLT1), both receptors similarly mediate the following functional activities: intracellular calcium fluxes, actin polymerization, adhesion to endothelium in vitro and chemotaxis. By Westernblot analysis, we could demonstrate Pyk2- and MAP-kinase phosphorylation as pivotal elements of CysLT1 signaling. These pathways have previously been identified also in CXCR4 signaling.To further analyze signal transduction pathways of both receptors, CD34+ cells were pretreated with pertussis toxin (PTX) or with a specific PKC inhibitor, which is an isoform-specific inhibitory myristoylated peptide derived from the pseudosubstrate (PS) region of PKCzeta, mimics the substrate, and maintains PKC in its nonactive isoform. Subsequently, we determined actin polymerization by flow cytometry using phalloidin-FITC, and adhesion to IL-1 stimulated endothelial cells (HUVEC). CXCR4 signaling leading to actin polymerization was found to be completely blocked by preincubation with pertussis toxin (PTX) and therefore is mediated exclusively by Gi proteins, while CysLT1 also involves Gq proteins as reflected by only partial inhibition by PTX. For both receptors, the Pyk2 signaling pathway leading to actin polymerization and adhesion was completely suppressed by preincubation with PSzeta and therefore dependent on atypical PKCzeta, which is calcium and DAG independent.We further examined whether these two similarly functioning receptors maintain any crosstalk, as has been reported for GPCR. Their possible interaction was explored using actin polymerization as a functional read-out. CXCR4- and CysLT1-mediated actin polymerization in response to their respective ligand was induced within 10 sec and returned to basal levels after 4 min. A second challenge after 4 min with the same ligand resulted in a complete suppression, demonstrating self-desensitization of both receptors. Interestingly, restimulating CXCL12-induced cells with LTD4 after 4 min resulted in complete suppression of actin polymerization, whereas restimulating LTD4-induced cells with CXCL12 lead to F-actin levels comparable to those achieved with the first challenge. These data show for the first time that CXCR4 can cross-desensitize CysLT1 while there is no crosstalk from CysLT1 to CXCR4.In conclusion, CXCR4 and CysLT1 share major signaling pathways. However, the ability to desensitize other GPCR is strikingly different. The finding that CXCR4 cross-desensitizes CysLT1 but not vice versa could explain our observation that a CysLT1 antagonist (montelukast) did not mobilize HPC in vivo, as the presence of CXCL12 in the stem cell niche may result in desensitization of CysLT1. In contrast, CXCR4-dependent bone marrow homing may not be influenced by conditions with high local and systemic cysteinyl-leukotriene concentrations, e.g. during allergy and inflammation. In the absence of CXCR4 activation however, CysLT1 could be important for homing of stem and progenitor cells in areas other than the bone marrow with a high local concentration of cysteinyl leukotrienes, e.g. in inflamed tissues. Disclosures:No relevant conflicts of interest to declare.

Protein Kinase Cζ Mediates μ-Opioid Receptor-induced Cross-desensitization of Chemokine Receptor CCR5

We have previously shown that the μ-opioid receptor (MOR) is capable of mediating cross-desensitization of several chemokine receptors including CCR5, but the biochemical mechanism of this process has not been fully elucidated. We have carried out a series of functional and biochemical studies and found that the mechanism of MOR-induced cross-desensitization of CCR5 involves the activation of PKCζ. Inhibition of PKCζ by its pseudosubstrate inhibitor, or its siRNA, or dominant negative mutants suppresses the cross-desensitization of CCR5. Our results further indicate that the activation of PKCζ is mediated through a pathway involving phosphoinositol-dependent kinase-1 (PDK1). In addition, activation of MOR elevates the phosphorylation level and kinase activity of PKCζ. The phosphorylation of PKCζ can be suppressed by a dominant negative mutant of PDK1. We observed that following MOR activation, the interaction between PKCζ and PDK1 is immediately increased based on the analysis of fluorescent resonance energy transfer in cells with the expression of PKCζ-YFP and PDK1-CFP. In addition, cells expressing PKCζ kinase motif mutants (Lys-281, Thr-410, Thr-560) fail to exhibit full MOR-induced desensitization of CCR5 activity. Taken together, we propose that upon DAMGO treatment, MOR activates PKCζ through a PDK1-dependent signaling pathway to induce CCR5 phosphorylation and desensitization. Because CCR5 is a highly proinflammatory receptor, and a critical coreceptor for HIV-1, these results may provide a novel approach for the development of specific therapeutic agents to treat patients with certain inflammatory diseases or AIDS.

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of exo- and endo-2-Norbornyl-N–n-butylcarbamates

Four stereoisomers of 2-norbornyl-N-n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N-n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(-)-exo-2-norbornyl-N-n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(-)-endo-2-norbornyl-N-n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.