Improving recombinant human adenosine A2A receptor production in yeast

Over 50% of clinically-marketed drugs target membrane proteins; in particular G protein-coupled receptors (GPCRs). GPCRs are vital to living cells, performing an active role in many processes, making them integral to drug development. In nature, GPCRs are not sufficiently abundant for research and their structural integrity is often lost during extraction from cell membranes. The objectives of this thesis were to increase recombinant yield of the GPCR, human adenosine A2A receptor (hA2AR) by investigating bioprocess conditions in large-scale Pichia pastoris and small-scale Saccharomyces cerevisiae cultivations. Extraction of hA2AR from membranes using novel polymers was also investigated. An increased yield of hA2AR from P. pastoris was achieved by investigating the methanol feeding regime. Slow, exponential feed during induction (μlow) was compared to a faster, exponential feed (μhigh) in 35 L pilot-scale bioreactors. Overall hA2AR yields were increased for the μlow cultivation (536.4pmol g-1) compared to the μhigh148.1 pmol g-1. hA2AR levels were maintained in cytotoxic methanol conditions and unexpectedly, pre-induction levels of hA2AR were detected. Small-scale bioreactor work showed that Design of Experiments (DoE) could be applied to screen for bioprocess conditions to give optimal hA2AR yields. Optimal conditions were retrieved for S. cerevisiae using a d-optimal screen and response surface methodology. The conditions were 22°C, pH 6.0, 30% DO without dimethyl sulphoxide. A polynomial equation was generated to predict hA2AR yields if conditions varied. Regarding the extraction, poly (maleic anhydride-styrene) or PMAS was successful in solubilising hA2AR from P. pastoris membranes compared with dodcecyl-β-D-maltoside (DDM) detergent. Variants of PMAS worked well as solubilising agents with either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or cholesteryl hemisuccinate (CHS). Moreover, esterification of PMAS improved solubilisation, suggesting that increased hydrophobicity stabilises hA2AR during extraction. Overall, hA2AR yields were improved in both, P. pastoris and S. cerevisiae and the use of novel polymers for efficient extraction was achieved.

Activation of protein kinase B by adenosine A1 and A3 receptors in newborn rat cardiomyocytes

It is well established that adenosine receptors are involved in cardioprotection and that protein kinase B (PKB) is associated with cell survival. Therefore, in this study we have investigated whether adenosine receptors (A1, A2A and A3) activate PKB by Western blotting and determined the involvement of phosphatidylinositol 3-kinase (PI-3K)/PKB in adenosine-induced preconditioning in cultured newborn rat cardiomyocytes. Adenosine (non-selective agonist), CPA (A1 selective agonist) and Cl-IB-MECA (A(3) selective agonist) all increased PKB phosphorylation in a time- and concentration-dependent manner. The combined maximal response to CPA and Cl-IB-MECA was similar to the increase in PKB phosphorylation induced by adenosine alone. CGS 21680 (A2A selective agonist) did not stimulate an increase in PKB phosphorylation. Adenosine, CPA and Cl-IB-MECA-mediated PKB phosphorylation were inhibited by pertussis toxin (PTX blocks G(i)/G(o)-protein), genistein (tyrosine kinase inhibitor), PP2 (Src tyrosine kinase inhibitor) and by the epidermal growth factor (EGF) receptor tyrosine kinase inhibitor AG 1478. The PI-3K inhibitors wortmannin and LY 294002 blocked A(1) and A(3) receptor-mediated PKB phosphorylation. The role of PI-3K/PKB in adenosine-induced preconditioning was assessed by monitoring Caspase 3 activity and lactate dehydrogenase (LDH) release induced by exposure of cardiomyocytes to 4 h hypoxia (0.5% O2) followed by 18 h reoxygenation (HX4/R). Pre-treatment with wortmannin had no significant effect on the ability of adenosine-induced preconditioning to reduce the release of LDH or Caspase 3 activation following HX4/R. In conclusion, we have shown for the first time that adenosine A1 and A3 receptors trigger increases in PKB phosphorylation in rat cardiomyocytes via a G1/G0-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning Adenosine A1 receptor .

Site-selective direct arylation of unprotected adenine nucleosides mediated by palladium and copper:insights into the reaction mechanism

Reaction conditions facilitating the site-selective direct aryl functionalisation at the C-8 position of adenine nucleosides have been identified. Many different aromatic components may be effectively cross-coupled to provide a diverse array of arylated adenine nucleoside products without the need for ribose or adenine protecting groups. The optimal palladium catalyst loading lies between 0.5 and 5 mol %. Addition of excess mercury to the reaction had a negligible affect on catalysis, suggesting the involvement of a homogeneous catalytic species. A study by transmission electron microscopy (TEM) shows that metal containing nanoparticles, ca. 3 nm with good uniformity, are formed during the latter stages of the reaction. Stabilised PVP palladium colloids (PVP=N-polyvinylpyrrolidone) are catalytically active in the direct arylation process, releasing homogenous palladium into solution. The effect of various substituted 2-pyridine ligand additives has been investigated. A mechanism for the site-selective arylation of adenosine is proposed. © 2008 Elsevier Ltd. All rights reserved.

Nucleoside diphosphate kinase--a component of the [Na+1]- and [Cl-]-sensitive phosphorylation cascade in human and murine airway epithelium

We have shown that proteins within apically enriched fractions of human nasal respiratory epithelium vary their phosphohistidine content with ambient [Cl-] and other anion concentrations. This membrane-delimited phosphorylation cascade includes a multifunctional protein histidine kinase - nucleoside diphosphate kinase (NDPK). NDPK is itself a cascade component in both human and ovine airway, the self-phosphorylation of which is inhibited selectively by [Na+] in the presence of ATP (but not GTP). These findings led us to propose the existence of a dual anion-/cation-controlled phosphorylation-based "sensor" bound to the apical membrane. The present study showed that this cascade uses ATP to phosphorylate a group of proteins above 45 kDa (p45-group, identities unknown). Additionally, the Cl- dependence of ATP (but not GTP) phosphorylation is conditional on phosphatase activity and that interactions exist between the ATP- and GTP-phosphorylated components of the cascade under Cl--free conditions. As a prelude to studies in cystic fibrosis (CF) mice, we showed in the present study that NDPK is present and functionally active in normal murine airway. Since NDPK is essential for UTP synthesis and regulates fetal gut development, G proteins, K+channels, neutrophil-mediated inflammation and pancreatic secretion, the presence of ion-regulated NDPK protein in mouse airway epithelium might aid understanding of the pathogenesis of CF.

Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems. © 2010 Elsevier Inc.