Role of the dsRNA-dependent protein kinase (PKR) in the attenuation of protein loss from muscle by insulin and insulin-like growth factor-I (IGF-I)

Proteolysis-inducing factor (PIF), a tumour-produced cachectic factor, induced a dose-dependent decrease in protein synthesis in murine myotubes, together with an increase in phosphorylation of eucaryotic initiation factor 2 (eIF2) on the alpha-subunit. Both insulin (1 nM) and insulin-like growth factor I (IGF-I) (13.2 nM) attenuated the depression of protein synthesis by PIF and the increased phosphorylation of eIF2alpha, by inhibiting the activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) by induction of protein phosphatase 1. A low-molecular weight inhibitor of PKR also reversed the depression of protein synthesis by PIF to the same extent, as did insulin and IGF-I. Both insulin and IGF-I-stimulated protein synthesis in the presence of PIF, and this was attenuated by Salubrinal, an inhibitor of phospho eIF2alpha phosphatase, suggesting that at least part of this action was due to their ability to inhibit phosphorylation of eIF2alpha. Both insulin and IGF-I also attenuated the induction of protein degradation in myotubes induced by PIF, this effect was also attenuated by Salubrinal. These results suggest an alternative mechanism involving PKR to explain the effect of insulin and IGF-I on protein synthesis and degradation in skeletal muscle in the presence of catabolic factors.

Mechanism of attenuation of angiotensin-II-induced protein degradation by insulin-like growth factor-I (IGF-I)

Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin-proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Cα (PKCα), degradation of inhibitor-κB (I-κB) and nuclear migration of nuclear factor-κB (NF-κB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin-proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the α-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCα by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 μM), an inhibitor of eIF2α dephosphorylation, as was activation of PKCα. In addition myotubes transfected with a dominant-negative PKR (PKRΔ6) showed no release of arachidonate in response to Ang II, and no activation of PKCα. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA2/PKC pathway leading to activation of NF-κB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR. © 2007 Elsevier Inc. All rights reserved.

Uncovering changes in the redox protein interactome of PTEN

Phosphatase and tensin homolog (PTEN) is a redox-sensitive, dual-specificity protein phosphatase involved in regulating a number of cellular processes including metabolism, apoptosis, cell proliferation and survival. It acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of a redox regulation of PTEN downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on the PTEN interactome is still poorly defined. To investigate this, PTEN-GST fusion protein was prepared in its reduced form and an H2O2-oxidized form that was reversible by DTT treatment, and these were immobilized on a glutathione-sepharose-based support. The immobilized protein was incubated with cell lysate to capture interacting proteins. Captured proteins were eluted from the beads, analyzed by LC-MSMS and comparatively quantified using label-free methods. After subtraction of interactors that were also present in the resin and GST controls, 97 individual protein interactors were identified, including several that are novel. Fourteen interactors that varied significantly with the redox status of PTEN were identified, including thioredoxin and peroxiredoxin-1. Except for one interactor, their binding was higher for oxidized PTEN. Using western blotting, altered binding to PTEN was confirmed for 3 selected interactors (Prdx1, Trx, and Anxa2) and DDB1 was validated as a novel interactor with unaltered binding. Our results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome which is important for the cellular function of PTEN. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.

The anti-inflammatory action of tetracyclines

For six decades tetracyclines have been successfully used for their broad spectrum antibiotic effects. However, non-antibiotic effects of tetracyclines have been reported. The anti-inflammatory effects of tetracycline drugs have been investigated in the context of a range of inflammatory diseases including sepsis and a number of neurodegenerative diseases. This thesis investigates the effects of a range of clinically important tetracyclines (oxytetracycline, doxycycline, minocycline and tigecycline) on the ability of the J774.2 cell line to produce nitric oxide when stimulated with the bacterial cell wall component, LPS. The proteome of J774.2 cells was analysed in response to LPS stimulation (1 µg/ml) with and without prior treatment with minocycline (50µg/ml), this allows the unbiased analysis of the cellular proteome in response to minocycline and LPS, protein spots of interest were excised and identified by nano-electrospray ionisation-linear ion trap mass spectroscopy. All of the tetracyclines that were investigated inhibited LPS-induced nitric oxide production in a dose dependent manner and this was due to the inhibition of inducible nitric oxide synthase expression. This is the first report to show that tigecycline inhibits inducible nitric oxide expression and nitric oxide production. Using two-dimensional gel electrophoresis and total protein staining eleven proteins were identified as being modulated by LPS. Of these eleven proteins; expression of some, but not all was modulated when the cells received a prior treatment with minocycline suggesting that minocycline does not completely block LPS-induced macrophage activation but probably specifically acts on particular inflammatory signaling pathways in macrophages. Three protein spots with a similar molecular weight but different pI values identified in this proteomic study were identified as ATP synthase ß chain. These different protein spots probably correspond to different phosphorylation states of the protein, suggesting that minocycline affects the balance of protein kinase and protein phosphatase activity in the immune response.

Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins

Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.

The development of a model system and high throughput assay for the study of interactions between zinc finger proteins and DNA

It has been recognised for some time that a full code of amino acid-based recognition of DNA sequences would be useful. Several approaches, which utilise small DNA binding motifs called zinc fingers, are presently employed. None of the current approaches successfully combine a combinatorial approach to the elucidation of a code with a single stage high throughput screening assay. The work outlined here describes the development of a model system for the study of DNA protein interactions and the development of a high throughput assay for detection of such interactions. A zinc finger protein was designed which will bind with high affinity and specificity to a known DNA sequence. For future work it is possible to mutate the region of the zinc finger responsible for the specificity of binding, in order to observe the effect on the DNA / protein interactions. The zinc finger protein was initially synthesised as a His tagged product. It was not possible however to develop a high throughput assay using the His tagged zinc finger protein. The gene encoding the zinc finger protein was altered and the protein synthesised as a Glutathione S-Transferase (GST) fusion product. A successful assay was developed using the GST protein and Scintillation Proximity Assay technology (Amersham Pharmacia Biotech). The scintillation proximity assay is a dynamic assay that allows the DNA protein interactions to be studied in "real time". This assay not only provides a high throughput method of screening zinc finger proteins for potential ligands but also allows the effect of addition of reagents or competitor ligands to be monitored.

Effect of branched-chain amino acids on muscle atrophy in cancer cachexia

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2α (eukaryotic initiation factor 2α) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70S6k (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70S6k, caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation. © The Authors.

Synthesis and expression of a gene encoding a 48-residue repeat in the Pseudomonas syringae ice nucleation protein

The aim of this work was to construct short analogues of the repetitive water-binding domain of the Pseudomonas syringae ice nucleation protein, InaZ. Structural analysis of these analogues might provide data pertaining to the protein-water contacts that underlie ice nucleation. An artificial gene coding for a 48-mer repeat sequence from InaZ was synthesized from four oligodeoxyribonucleotides and ligated into the expression vector, pGEX2T. The recombinant vector was cloned in Escherichia coli and a glutathione S-transferase fusion protein obtained. This fusion protein displayed a low level of ice-nucleating activity when tested by a droplet freezing assay. The fusion protein could be cleaved with thrombin, providing a means for future recovery of the 48-mer peptide in amounts suitable for structural analysis by nuclear magnetic resonance spectroscopy.

Novel peptide antagonists of adrenomedullin and calcitonin gene-related peptide receptors:identification, pharmacological characterization, and interactions with position 74 in receptor activity-modifying protein 1/3

Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin peptide family, which also includes calcitonin gene-related peptide (CGRP) and AM2. The two AM receptors, AM(1) and AM(2), are calcitonin receptor-like receptor (CL)/receptor activity-modifying protein (RAMP) (RAMP2 and RAMP3, respectively) heterodimers. CGRP receptors comprise CL/RAMP1. The only human AM receptor antagonist (AM(22-52)) is a truncated form of AM; it has low affinity and is only weakly selective for AM(1) over AM(2) receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM(1), AM(2), and CGRP receptors. AM(22-52) was the framework for generating further AM fragments (AM(26-52) and AM(30-52)), novel AM/alphaCGRP chimeras (C1-C5 and C9), and AM/AM(2) chimeras (C6-C8). cAMP assays were used to screen the antagonists at all receptors to determine their affinity and selectivity. Circular dichroism spectroscopy was used to investigate the secondary structures of AM and its related peptides. The data indicate that the structures of AM, AM2, and alphaCGRP differ from one another. Our chimeric approach enabled the identification of two nonselective high-affinity antagonists of AM(1), AM(2), and CGRP receptors (C2 and C6), one high-affinity antagonist of AM(2) receptors (C7), and a weak antagonist selective for the CGRP receptor (C5). By use of receptor mutagenesis, we also determined that the C-terminal nine amino acids of AM seem to be responsible for its interaction with Glu74 of RAMP3. We provide new information on the structure-activity relationship of AM, alphaCGRP, and AM2 and how AM interacts with CGRP and AM(2) receptors.

Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A-class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg(2.39), His(2.43) and Glu(3.46), which makes a polar lock with T(6.37). These alignments and models provide useful tools for understanding class B GPCR function.

Receptor activity-modifying protein-dependent effects of mutations in the calcitonin receptor-like receptor:implications for adrenomedullin and calcitonin gene-related peptide pharmacology

Background and Purpose Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear. Experimental Approach Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants. Key Results An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide. Conclusions and Implications RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2. © 2013 The Authors. British Journal of Pharmacology published by John Wiley &. Sons Ltd on behalf of The British Pharmacological Society.

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

Background - Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Results - Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. Conclusions - The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.

Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors

The calcitonin gene-related peptide (CGRP) family of G protein- coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in aGαs-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gαs and Gαq but also identify a Gαi component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMPdependent signaling bias among the Gαs, Gαi, and Gαq/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.