Abstract-Adenosine plays multiple roles in the efficient functioning of the heart by regulating coronary blood flow, cardiac pacemaking, and contractility. Previous studies have implicated the equilibrative nucleoside transporter family member equilibrative nucleoside transporter-1 (ENT1) in the regulation of cardiac adenosine levels. We report here that a second member of this family, ENT4, is also abundant in the heart, in particular in the plasma membranes of ventricular myocytes and vascular endothelial cells but, unlike ENT1, is virtually absent from the sinoatrial and atrioventricular nodes. Originally described as a monoamine/organic cation transporter, we found that both human and mouse ENT4 exhibited a novel, pH-dependent adenosine transport activity optimal at acidic pH (apparent K m values 0.78 and 0.13 mmol/L, respectively, at pH 5.5) and absent at pH 7.4. In contrast, serotonin transport by ENT4 was relatively insensitive to pH. ENT4-mediated nucleoside transport was adenosine selective, sodium independent and only weakly inhibited by the classical inhibitors of equilibrative nucleoside transport, dipyridamole, dilazep, and nitrobenzylthioinosine. We hypothesize that ENT4, in addition to playing roles in cardiac serotonin transport, contributes to the regulation of extracellular adenosine concentrations, in particular under the acidotic conditions associated with ischemia. Key Words: nucleoside Ⅲ adenosine Ⅲ transport Ⅲ ischemia Ⅲ pH T he purine nucleoside adenosine is produced by the action of both endo-and ecto-nucleotidases on adenine nucleotides in the heart and plays key roles in the regulation of coronary blood flow and myocardial O 2 supply-demand balance. 1-4 For example, action of adenosine on A 2A receptors on vascular smooth muscle and endothelial cells causes coronary vasodilatation. 1,5 In contrast, the negative inotropic and dromotropic effects of adenosine on the heart are mediated primarily by A 1 receptors. 2 Similarly, the negative chromotropic effect of adenosine involves action of A 1 receptors in the sinoatrial (SA) node on the inwardly rectifying potassium channel current I K-Ado and the hyperpolarization-activated pacemaker current I f . 2,6 Endogenous adenosine, acting on mitochondrial K ATP channels via A 1 and A 3 receptors, also makes a major contribution to the phenomenon of ischemic preconditioning. 5,7 Extracellular adenosine concentrations in the heart are governed both by action of ecto-5Ј-nucleotidase on adenine nucleotides released from cells and by transporter-mediated flux of adenosine across cell membranes. 3,4 Although most adenosine production occurs intracellularly, under normoxic conditions, metabolism maintains a low intracellular concentration and, therefore, the net flux of adenosine is into cardiomyocytes and endothelial cells. Under such conditions, administration of transport inhibitors increases extracellular concentrations of adenosine, leading to vasodilatation. 8 However, increased adenine nucleotide breakdown and inhibition of adenosine kinase duri...

Proton-dependent oligopeptide transporters (POTs) are secondary active symporters that utilize the proton gradient to drive the inward translocation of di- and tripeptides. We have mutated two highly conserved membraneembedded glutamate residues (Glu20 and Glu388) in the E. coli POT YjdL to probe their possible functional roles, in particular if they were involved/implicated in recognition of the substrate N-terminus. The mutants (Glu20Asp, Glu20Gln, Glu388Asp, and Glu388Gln) were tested for substrate uptake, which indicated that both the negative charge and the side chain length were important for function. The IC50 values of dipeptides with lack of or varying N-terminus (Ac-Lys, Gly- Lys, β-Ala-Lys, and 4-GABA-Lys), showed that Gly-Lys and β-Ala-Lys ranged between ~0.1 to ~1.0 mM for wild type and Glu20 mutants. However, for Glu388Gln the IC50 increased to ~2.0 and > 10 mM for Gly-Lys and β-Ala-Lys, respectively, suggesting that Glu388, and not Glu20, is able to sense the position of the N-terminus and important for the interaction. Furthermore, uptake as a function of pH showed that the optimum at around pH 6.5 for wild type YjdL shifted to 7.0-7.5 for the Glu388Asp/Gln mutants while the Glu20Asp retained the wild type optimum. Uptake by the Glu20Gln on the other hand was completely unaffected by the bulk pH in the range tested, which indicated a possible role of Glu20 in proton translocation.

YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.

Membrane proteins represent a significant fraction of all genomes and play key roles in many aspects of biology, but their structural analysis has been hampered by difficulties in large-scale production and crystallisation. To overcome the first of these hurdles, we present here a systematic approach for expression and affinity-tagging which takes into account transmembrane topology. Using a set of bacterial transporters with known topologies, we tested the efficacy of a panel of conventional and Gateway recombinational cloning vectors designed for protein expression under the control of the tac promoter, and for the addition of differing N- and C-terminal affinity tags. For transporters in which both termini are cytoplasmic, C-terminal oligohistidine tagging by recombinational cloning typically yielded functional protein at levels equivalent to or greater than those achieved by conventional cloning. In contrast, it was not effective for examples of the substantial minority of proteins that have one or both termini located on the periplasmic side of the membrane, possibly because of impairment of membrane insertion by the tag and/or att-site-encoded sequences. However, fusion either of an oligohistidine tag to cytoplasmic (but not periplasmic) termini, or of a Strep-tag II peptide to periplasmic termini using conventional cloning vectors did not interfere with membrane insertion, enabling high-level expression of such proteins. In conjunction with use of a C-terminal Lumio fluorescence tag, which we found to be compatible with both periplasmic and cytoplasmic locations, these findings offer a system for strategic planning of construct design for high throughput expression of membrane proteins for structural genomics projects.

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

No abstract

Newcastle disease virus (NDV) is an infectious agent of a large variety of birds, including chicken, which poses a real threat to the agriculture industry. Matrix (M) proteins of NDV and many other viruses perform critical functions during viral assembly and budding from the host cell. M-proteins are well conserved and therefore are potential targets for antiviral therapies. To validate this, we expressed the NDV M-protein in its native form in Saccharomyces cerevisiae and in inclusion bodies in Escherichia coli. Proper refolding of the recombinant protein produced in E. coli was verified using circular dichroism and infrared spectroscopies and electron microscopy. Immunization of chickens with the NDV M-protein elicited significant serum antibody titers. However, the antibodies conferred little protection against the ND following lethal viral challenges. We conclude that the M-protein is not exposed on the surface of the host cell or the virus at any stage during its life cycle. We discuss how the conserved M-protein can further be exploited as an antiviral drug target.

Escherichia coli glutamate/aspartate-proton symporter GltP is a member of the Dicarboxylate/Amino Acid:Cation Symporter family of secondary active transport proteins. A range of computational, chemical, biochemical and biophysical methods characterised evolutionary relationships, structural features, substrate binding affinities and transport kinetics of wild-type and mutant forms of GltP. Sequence alignments and phylogenetic analysis revealed close homologies of GltP with human glutamate transporters involved in neurotransmission, neutral amino acid transporters and with the archaeal aspartate transporter GltPh. Topology predictions and comparisons with the crystal structure of GltPh were consistent with eight transmembrane-spanning α-helices and two hairpin re-entrant loops in GltP. Amplified expression of recombinant GltP with C-terminal affinity tags was achieved at 10% of total membrane protein in E. coli and purification to homogeneity with a yield of 0.8 mg/litre. Binding of substrates to GltP in native inner membranes and to purified protein solubilised in detergent was observed and quantified using solid-state NMR and fluorescence spectroscopy, respectively. A homology model of GltP docked with L-glutamate identified a putative binding site and residues predicted to interact with substrate. Sequence alignments identified further highly conserved residues predicted to have essential roles in GltP function. Residues were investigated by measuring transport activities, kinetics and response to thiol-specific reagents in 42 site-specific mutants compared with cysteine-less GltP (C256A) having an apparent affinity of initial rate transport (K) for H-L-glutamate of 22.6 ± 5.5 μM in energised E. coli cells. This confirmed GltP residues involved in substrate binding and transport, especially in transmembrane helices VII and VIII.