Targeting the Nogo-A signalling pathway to promote recovery following acute CNS injury.

Functional recovery following acute CNS injury in humans, such as spinal cord injury and stroke, is exceptionally limited, leaving the affected individual with life-long neurological deficits such as loss of limb movement and sensation leading to a compromised quality of life. As yet, there is no effective treatment on the market for such injuries. This lack of functional recovery can at least in part be attributed to the restriction of axonal regeneration and neuroplasticity by several CNS myelin proteins that have been shown to be potent inhibitors of neurite outgrowth in vitro, namely myelin-associated glycoprotein (MAG), Nogo-A and oligodendrocyte myelin glycoprotein (OMgp). Nogo-A contains multiple neurite outgrowth inhibitory domains exposed on the surface of myelinating oligodendrocytes located within its amino-terminal region (amino-Nogo-A) and C-terminal region (Nogo-66). Although structurally dissimilar; Nogo-66, MAG and OMgp exert their inhibitory effects by binding the GPI-linked neuronal Nogo-66 receptor (NgR) that transduces the inhibitory signal to the cell interior via transmembrane co-receptors LINGO-1 and p75(NTR)or TROY. Although the receptor(s) for amino-Nogo-A are unknown, amino-Nogo-A and NgR ligands mutually activate the small GTPase RhoA. Consistent with their neurite outgrowth inhibitory function, approaches counter-acting Nogo-A using function-blocking antibodies, NgR using peptide antagonists and receptor bodies or RhoA using deactivating enzymes have been shown to significantly enhance axonal regeneration and neuroplasticity leading to improved functional recovery in animal models of acute CNS injury. These in vivo findings thus provide a sound basis for the development of an effective treatment for acute CNS injuries in humans.

Characterization of two novel proteins, NgRH1 and NgRH2, structurally and biochemically homologous to the Nogo-66 receptor.

Nogo-66 receptor (NgR) has recently been identified as the neuronal receptor of the myelin-associated proteins Nogo-A, oligodendrocyte protein (OMgp) and myelin-associated glycoprotein (MAG), and mediates inhibition of axonal regeneration both in vitro and in vivo. Through database searches, we have identified two novel proteins (NgRH1 and NgRH2) that turned out to be homologous in their primary structures, biochemical properties and expression patterns to NgR. Like NgR, the homologues contain eight leucine-rich repeats (LRR) flanked by a leucine-rich repeat C-terminus (LRRCT) and a leucine-rich repeat N-terminus (LRRNT), and also have a C-terminal GPI signal sequence. Northern blot analysis showed predominant expression of NgRH1 and NgRH2 mRNA in the brain. In situ hybridization and immunohistochemistry on rat brain slices revealed neuronal expression of the genes. NgRH1 and NgRH2 were detected on the cell surface of recombinant cell lines as N-glycosylated GPI anchored proteins and, consistent with other GPI anchored proteins, were localized within the lipid rafts of cellular membranes. In addition, an N-terminal proteolytic fragment of NgR comprising the majority of the ectodomain was found to be constitutively secreted from cells. Our data indicate that NgR, NgRH1 and NgRH2 constitute a novel receptor protein family, which may play related roles within the CNS.

Antimonite regulation of the ATPase activity of ArsA, the catalytic subunit of the arsenical pump.

The ArsA ATPase is the catalytic subunit of the pump protein, coupling the hydrolysis of ATP to the movement of arsenicals and antimonials through the membrane-spanning ArsB protein. Previously, we have shown the binding and hydrolysis of MgATP to ArsA to be a multi-step process in which the rate-limiting step is an isomerization between different conformational forms of ArsA. This isomerization occurs after product release, at the end of the ATPase reaction, and involves the return of the ArsA to its original conformation, which can then bind MgATP. ArsA possesses an allosteric site for antimonite [Sb(III)], the binding of which elevates the steady-state ATPase activity. We have used a transient kinetics approach to investigate the kinetics of ternary complex formation that lead to an enhancement in the ATPase activity. These studies revealed that ArsA exists in at least two conformational forms that differ in their ligand binding affinities, and that ATP favours one form and Sb(III) the other. Ternary complex formation is rate-limited by a slow transition between these conformational forms, leading to a lag in attaining maximal steady-state activity. Sb(III) enhances the steady-state ATPase activity by inducing rapid product release, allowing ArsA to adopt a conformation that can bind MgATP for the next catalytic cycle. In the presence of Sb(III), ArsA avoids the rate-limiting isomerization at the end of the ATPase reaction and ATP hydrolysis becomes rate-limiting for the reaction. The binding of Sb(III) probably results in more effective pumping of the substrates from the cell by enhancing the rate of efflux.

Membrane topology influences N-glycosylation of the prion protein.

The glycosylation state of the glycosyl-phosphatidylinositol (GPI) anchored cellular prion protein (PrPC) can influence the formation of the disease form of the protein responsible for the neurodegenerative spongiform encephalopathies. We have investigated the role of membrane topology in the N-glycosylation of PrP by expressing a C-terminal transmembrane anchored form, PrP-CTM, an N-terminal transmembrane anchored form, PrP-NTM, a double-anchored form, PrP-DA, and a truncated form, PrPDeltaGPI, in human neuroblastoma SH-SY5Y cells. Wild-type PrP, PrP- CTM and PrP-DA were membrane anchored and present on the cell surface as glycosylated forms. In contrast, PrP-NTM, although membrane anchored and localized at the cell surface, was not N-glycosylated. PrPDeltaGPI was secreted from the cells into the medium in a hydrophilic form that was unglycosylated. The 4-fold slower rate at which PrPDeltaGPI was trafficked through the cell compared with wild-type PrP was due to the absence of the GPI anchor not the lack of N-glycans. Retention of PrPDeltaGPI in the endoplasmic reticulum did not lead to its glycosylation. These results indicate that C-terminal membrane anchorage is required for N-glycosylation of PrP.

The structure and function of drug pumps.

Resistance to drugs has emerged in biological systems as diverse as cancer cells undergoing chemotherapy and microbial pathogens undergoing treatment with antimicrobials. This medical problem is escalating and there is an urgent need for the development of new classes of drugs. In the case of pathogenic bacteria, we are rapidly approaching a scenario where there will be no effective antibiotics in the armoury of drugs available for treating the infectious diseases that these bacteria cause, returning us to the pre-antibiotic era when infectious diseases were rife because they were untreatable. One of the most frequently employed resistance strategies in both prokaryotes and eukaryotes is the transmembrane-protein-catalysed extrusion of drugs from the cell, with these proteins acting like bilge pumps, reducing the intracellular drug concentration to subtoxic levels. There is currently much scientific interest in understanding how these pumps operate, so that we might design transport inhibitors that would block them, allowing a renaissance for drugs that are no longer effective owing to their efflux.

A kinetic model for the action of a resistance efflux pump.

ArsA is the catalytic subunit of the arsenical pump, coupling ATP hydrolysis to the efflux of arsenicals through the ArsB membrane protein. It is a paradigm for understanding the structure-function of the nucleotide binding domains (NBD) of medically important efflux pumps, such as P-glycoprotein, because it has two sequence-related, interacting NBD, for which the structure is known. On the basis of a rigorous analysis of the pre-steady-state kinetics of nucleotide binding and hydrolysis, we propose a model in which ArsA alternates between two mutually exclusive conformations as follows: the ArsA(1) conformation in which the A1 site is closed but the A2 site open; and the ArsA(2) conformation, in which the A1 and A2 sites are open and closed, respectively. Antimonite elicits its effects by sequestering ArsA in the ArsA(1) conformation, which catalyzes rapid ATP hydrolysis at the A2 site to drive ArsA between conformations that have high (nucleotide-bound ArsA) and low affinity (nucleotide-free ArsA) for Sb(III). ArsA potentially utilizes this process to sequester Sb(III) from the medium and eject it into the channel of ArsB.

The quarternary molecular architecture of TetA, a secondary tetracycline transporter from Escherichia coli.

TetA, a tetracycline cation/proton antiporter, was expressed in Escherichia coli with a C-terminal tag of six histidines, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two-dimensional crystals were obtained after reconstitution of purified protein with lipids. Electron microscopy of negatively stained crystals revealed a trigonal symmetry, from which we infer that this secondary transporter has a trimeric structure. An overall molecular envelope can be described by a triangle of side approximately 100 A enclosing a central stain-filled depression. These dimensions are consistent with those obtained from projection views of single, isolated TetA particles that also display a trimeric architecture, confirming that the threefold symmetry is not simply a consequence of crystal-packing interactions. These data represent the first direct view of the quarternary arrangement of any antibiotic efflux pump. They are fully consistent with biochemical data on TetA, which indicate that it functions as a multimer and that the monomer consists of two domains, one of which plays the major part in oligomerization interactions.

Structure-function relationships in an anion-translocating ATPase.

The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli. This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells. The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit ArsB. The complex is similar in many ways to ATP-binding cassette ('ABC') transporters, which typically have two groups of six transmembrane-spanning helical segments and two nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 transmembrane-spanning segments. ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter. The 63 kDa ArsA protein is a substrate-activated ATPase. It has two homologous halves, A1 and A2, which are clearly the result of an ancestral gene duplication and fusion. Each half has a consensus NBD. The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses. Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA. The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme. Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step. Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.

cAMP signalling in pathogenic fungi: control of dimorphic switching and pathogenicity.

Morphological changes in pathogenic fungi often underlie the development of virulence and infection by these organisms. Our knowledge of the components of the cell signalling pathways controlling morphological switching has, to a large extent, come from studies of pseudohyphal growth of the model organism Saccharomyces cerevisiae, in which control is exerted via changes in the intracellular cAMP and mitogen-activated protein kinase cascades. There is evidence that pathogenic fungi also utilize these pathways to control dimorphic switching between saprobic and pathogenic forms and, as such, the elements of these pathways have potential as drug targets.

Mechanism of the ArsA ATPase.

The ArsAB ATPase confers metalloid resistance in Escherichia coli by pumping toxic anions out of the cells. This transport ATPase shares structural and perhaps mechanism features with ABC transporters. The ArsAB pump is composed of a membrane subunit that has two groups of six transmembrane segments, and the catalytic subunit, the ArsA ATPase. As is the case with many ABC transporters, ArsA has an internal repeat, each with an ATP binding domain, and is allosterically activated by substrates of the pump. The mechanism of allosteric activation of the ArsA ATPase has been elucidated at the molecular level. Binding of the activator produces a conformational change that forms a tight interface of the nucleotide binding domains. In the rate-limiting step in the overall reaction, the enzyme undergoes a slow conformational change. The allosteric activator accelerates catalysis by increasing the velocity of this rate-limiting step. We postulate that similar conformational changes may be rate-limiting in the mechanism of ABC transporters.

Structure and function of facilitative sugar transporters.

Sugar transporters from one group of the major facilitator superfamily of membrane transporters. A conserved common central pore structure lies at the heart of these transporters and diverse functionality is brought about by alterations to this pore or regions associated with it. Recent mutagenesis studies of sugar transporters within the framework of tenable models for the distantly related lactose permease argue that this model is a good paradigm for other members of the major facilitator superfamily.

The ATPase mechanism of ArsA, the catalytic subunit of the arsenite pump.

The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein. The single tryptophan-containing Trp159 ArsA was used to elucidate the elementary steps of the ATPase mechanism by fluorescence stopped-flow experiments. The binding and hydrolysis of MgATP is a multistep process with a minimal kinetic mechanism (Mechanism 1). A notable feature of the reaction is that MgATP binding induces a slow transient increase in fluorescence of ArsA, which is independent of the ATP concentration, indicative of the build-up of a pre-steady state intermediate. This finding, coupled with a phosphate burst, implies that the steady-state intermediate builds up subsequent to product release. We propose that the rate-limiting step is an isomerization between different conformational forms of ArsA. kcat is faster than the phosphate burst, indicating that both nucleotide binding sites of ArsA are catalytic. Consistent with this interpretation, approximately 2 mol of phosphate are released per mole of ArsA during the phosphate burst.

Intracellular retention of procollagen within the endoplasmic reticulum is mediated by prolyl 4-hydroxylase.

The correct folding and assembly of proteins within the endoplasmic reticulum (ER) are prerequisites for subsequent transport from this organelle to the Golgi apparatus. The mechanisms underlying the ability of the cell to recognize and retain unassembled or malfolded proteins generally require binding to molecular chaperones within the ER. One classic example of this process occurs during the biosynthesis of procollagen. Here partially folded intermediates are retained and prevented from secretion, leading to a build up of unfolded chains within the cell. The accumulation of these partially folded intermediates occurs during vitamin C deficiency due to incomplete proline hydroxylation, as vitamin C is an essential co-factor of the enzyme prolyl 4-hydroxylase. In this report we show that this retention is tightly regulated with little or no secretion occurring under conditions preventing proline hydroxylation. We studied the molecular mechanism underlying retention by determining which proteins associate with partially folded procollagen intermediates within the ER. By using a combination of cross-linking and sucrose gradient analysis, we show that the major protein binding to procollagen during its biosynthesis is prolyl 4-hydroxylase, and no binding to other ER resident proteins including Hsp47 was detected. This binding is regulated by the folding status rather than the extent of hydroxylation of the chains demonstrating that this enzyme can recognize and retain unfolded procollagen chains and can release these chains for further transport once they have folded correctly.

Differential expression of an hsp70 gene during transition from the mycelial to the infective yeast form of the human pathogenic fungus Paracoccidioides brasiliensis.

We have isolated and characterized cDNA and genomic clones that encode a 70 kDa heat shock protein (Hsp70) from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. The gene encodes a 649-amino-acid protein showing high identity with other members of the hsp70 gene family. The hsp70 gene is induced during both heat shock of yeast cells at 42 degrees C and the mycelial to yeast transition. A differential expression of this gene can be observed between mycelial and yeast forms, with a much higher level of expression in the yeast. We found two introns of 178 and 72 nucleotides in the P. brasiliensis hsp70 gene. Splicing of these introns is regulated during the heat shock process and possibly during infection. In order to analyse the differential accumulation of unspliced mRNA following cellular differentiation and/or heat shock, reverse transcriptase-polymerase chain reaction (RT-PCR) experiments were carried out. The temperature-induced mycelial to yeast transition results in the transient accumulation of unspliced hsp70 mRNA transcripts. Yeast cells, after adaptation at 36 degrees C, seem to be more proficient at splicing, at least with respect to hsp70 mRNA because, during a severe heat shock (42 degrees C), the unspliced form of this mRNA does not accumulate. The mycelial to yeast differentiation will have the adaptational effect of increasing the resistance of the organism to environmental stress, which may be necessary for parasite survival in the mammalian host.

Analysis of amino and carboxy terminal GLUT-4 targeting motifs in 3T3-L1 adipocytes using an endosomal ablation technique.

The targeting of the insulin-responsive glucose transporter, GLUT-4, to an intracellular compartment in adipocytes and muscle is one of the key features responsible for the unique insulin sensitivity of this transporter. Through expression of epitope-tagged GLUT-4 mutants in 3T3-L1 adipocytes, two motifs have been identified as playing a central role in GLUT-4 targeting: FQQI in the amino terminus and a di-leucine motif in the carboxy terminus. The goal of this study was to explore the role of these targeting motifs in the intracellular sorting of GLUT-4 using the Tf-HRP ablation technique. This technique provides a quantitative assessment of the amount of GLUT-4 located in recycling endosomes. In basal adipocytes, we find that approximately 40% of GLUT-4 is ablated following Tf-HRP loading. In contrast, here we demonstrate that the intracellular pool of a mutant in which F5 was mutated to A5 is localized to the recycling endosomal pathway, suggesting that the amino terminal FQQI motif functions in trafficking GLUT-4 from early endosomes. In contrast, GLUT-4 in which L489L490 was mutated to A489A490 was localized predominantly to a nonablated compartment. These data imply a role for the di-leucine motif in sorting from a separate intracellular compartment, such as the TGN. Our findings are discussed within the context of a revised multicompartment model for GLUT-4 trafficking in adipocytes, in which mutations in either the FQQI or LL motifs result in the altered subcellular trafficking of GLUT-4 between multiple intracellular compartments.

Sugar transporters from bacteria, parasites and mammals: structure-activity relationships.

Sugar transport across the plasma membrane is one of the most important transport processes. The cloning and expression of cDNAs from a superfamily of related sugar transporters that all adopt a 12-membrane-spanning-domain structure has opened new avenues of investigation, including presteady-state kinetic analysis. Structure-function analyses of mammalian and bacterial sugar transporters, and comparisons of these transporters with those of parasitic trypanosomatids, indicate that different environmental pressures have tailored the evolution of the various members of the sugar-transporter superfamily. Subtle distinctions in the function of these proteins can be related to particular amino acid residue substitutions.

Asparagine 394 in putative helix 11 of the galactose-H+ symport protein (GalP) from Escherichia coli is associated with the internal binding site for cytochalasin B and sugar.

The galactose-H+ symport protein (GalP) of Escherichia coli is very similar to the human glucose transport protein, GLUT1, and both contain a highly conserved Asn residue in predicted helix 11 that is different in a cytochalasin B-resistant member of this sugar transport family (XylE). The role of the Asn394 residue (which is predicted to be in putative trans-membrane alpha-helix 11) in the structure/activity relationship of the D-galactose-H+ symporter (GalP) was therefore assessed by measuring the interaction of sugar substrates and the inhibitory antibiotics, cytochalasin B, and forskolin with the wild-type and Asn394 --> Gln mutant proteins. Steady-state fluorescence quenching experiments show that the mutant protein binds cytochalasin B with a Kd 37-53-fold higher than the wild type. This low affinity binding was not detected with equilibrium binding or photolabeling experiments. In contrast, the mutant protein binds forskolin with a Kd similar to that of the wild type and is photolabeled by 3-125I-4-azido-phenethylamido-7-O-succinyl-desacetyl-forskolin. The mutant protein displays an increased amount of steady-state fluorescence quenching with the binding of forskolin, suggesting that the substitution of the Asn residue has altered the environment of a tryptophan, probably Trp395, in a conformationally active region of the protein. Time-resolved fluorescence measurements on the mutant protein provided association and dissociation rate constants (k2 and k-2), describing the initial interaction of cytochalasin B to the inward-facing binding site (Ti), that are decreased (9-fold) and increased (4.9-fold) compared with the wild type. This yielded a dissociation constant (K2) for cytochalasin B to the inward-facing binding site 44-fold higher than that of the wild type. The binding of forskolin gave values for k2 and k-2 3.9- and 3.6-fold lower, respectively, yielding a K2 value for Ti similar to that of the wild type. The low overall affinity (high Kd) of the mutant protein for cytochalasin B is due mainly to a disruption in binding to the Ti conformation. It is proposed that Asn394 forms either a direct binding interaction with cytochalasin B or is part of the immediate environment of the binding site and that Asn394 is in the immediate environment, but not part, of the forskolin binding site. The ability of the mutant protein to catalyze energized transport is only mildly impaired with 4.8- and 2.1-fold reduction in Vmax/Km values for D-galactose and D-glucose, respectively. In stark contrast, the overall Kd describing binding of D-galactose and D-glucose to the inward-facing conformation of the mutant and their subsequent translocation across the membrane is substantially increased (64-fold for D-galactose and 163.3-fold for D-glucose). These data indicate that Asn394 is associated with both the cytochalasin B and internal sugar binding sites. This conclusion is also supported by data showing that the sugar specificity of the mutant protein has been altered for D-xylose. This work powerfully illustrates how comparisons of the aligned amino acid sequences of homologous membrane proteins of unknown structure and characterization of their phenotypes can be used to map substrate and ligand binding sites.

Kinetic studies on the binding of 1,N6-etheno-NAD+ to glutamate dehydrogenase from Clostridium symbiosum.

The mechanism of the binding of reduced coenzyme (NAD+) to clostridial glutamate dehydrogenase (GDH) was determined by transient kinetics. The fluorescent 1,N6-ethenoadenine analogue of NAD+ (epsilonNAD+) was used as a probe of nucleotide binary and ternary complex formation because the binding of NAD+ is optically silent. The kinetics of epsilonNAD+ binding were consistent with a 3-step binding process. The enzyme was found to oscillate between two conformational forms, termed E1 and E2, in the presence and absence of L-glutamate. However, L-glutamate shifted the equilibrium from 96.8% to 99% of the enzyme in the E1 form. The rapid-equilibrium binding of epsilonNAD+ to the E2 form was rate limited by a slow isomerisation of the ternary complex as the binary complex became saturated with epsilonNAD+. The L-glutamate binary complex had a greater affinity for the coenzyme (Kd = 11 microM) than the free enzyme (Km = 39 microM), indicative of a positive interaction of the substrate and coenzyme binding sites. Steady-state studies were also indicative of a positive interaction in the formation of the catalytic complex, with this complex having a Kd for epsilonNAD+ of 6.8 microM. Consequently, there is stabilization of successive complexes on the reaction pathway.

Design of kallidin-releasing tissue kallikrein inhibitors based on the specificities of the enzyme's binding subsites.

The tissue kallikrein inhibitors reported in the present work were derived by selectively replacing residues in Nalpha-substituted arginine- or phenylalanine-pNA (where pNA is p-nitroanilide), and in peptide substrates for these enzymes. Phenylacetyl-Arg-pNA was found to be an efficient inhibitor of human tissue kallikrein (Ki 0.4 microM) and was neither a substrate nor an inhibitor of plasma kallikrein. The peptide inhibitors having phenylalanine as the P1 residue behaved as specific inhibitors for kallidin-releasing tissue kallikreins, while plasma kallikrein showed high affinity for inhibitors containing (p-nitro)phenylalanine at the same position. The Ki value of the most potent inhibitor developed, Abz-Phe-Arg-Arg-Pro-Arg-EDDnp [where Abz is o-aminobenzoyl and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine], was 0.08 microM for human tissue kallikrein. Progress curve analyses of the inhibition of human tissue kallikrein by benzoyl-Arg-pNA and phenylacetyl-Phe-Ser-Arg-EDDnp indicated a single-step mechanism for reversible formation of the enzyme-inhibitor complex.