Neil V. McFerran

The 67 kDa laminin receptor: structure, function and role in disease

The 67LR (67 kDa laminin receptor) is a cell-surface receptor with high affinity for its primary ligand. Its role as a laminin receptor makes it an important molecule both in cell adhesion to the basement membrane and in signalling transduction following this binding event. The protein also plays critical roles in the metastasis of tumour cells. Isolation of the protein from either normal or cancerous cells results in a product with an approx. molecular mass of 67 kDa. This protein is believed to be derived from a smaller precursor, the 37LRP (37 kDa laminin receptor precursor). However, the precise mechanism by which cytoplasmic 37LRP becomes cell-membrane-embedded 67LR is unclear. The process may involve post-translational fatty acylation of the protein combined with either homo- or hetero-dimerization, possibly with a galectin-3-epitope-containing partner. Furthermore, it has become clear that acting as a receptor for laminin is not the only function of this protein. 67LR also acts as a receptor for viruses, such as Sindbis virus and dengue virus, and is involved with internalization of the prion protein. Interestingly, unmodified 37LRP is a ribosomal component and homologues of this protein are found in all five kingdoms. In addition, it appears to be strongly associated with histones in the eukaryotic cell nucleus, although the precise role of these interactions is not clear. Here we review the current understanding of the structure and function of this molecule, as well as highlighting areas requiring further research.

Sequence analysis of a porcine enterovirus serotype 1 isolate: relationships with other picornaviruses

The majority of the genomic sequence of a porcine enterovirus serotype 1 (PEV-1) isolate was determined. The genome was found to contain a large open reading frame which encoded a leader protein prior to the capsid protein region. This showed no sequence identity to other picornavirus leader regions and the sequence data suggested that it does not possess proteolytic activity. The 2A protease was small and showed considerable sequence identity to the aphthoviruses and to equine rhinovirus serotype 2. The 2A/2B junction possessed the typical cleavage site (NPG/P) exhibited by these viruses. The other proteins shared less than 40% sequence identity with equivalent proteins from other picornavirus genera. Phylogenetic analyses of the P1 and 3D sequences indicated that this virus forms a distinct branch of the family Picornaviridae. On the basis of results presented in this paper PEV-1 has been assigned to a new picornavirus genus. The phylogeny of the virus in relation to other picornaviruses is discussed.

Development and implementation of split-GFP-based bimolecular fluorescence complementation (BiFC) assays in yeast

BiFC (bimolecular fluorescence complementation) is a tool for investigating interactions between proteins. Non-fluorescent fragments of, for example, GFP (green fluorescent protein) are fused to the interacting partners. The interaction brings the fragments together, which then fold, reassemble and fluoresce. This process can be carried out in living cells and provides information both on the interaction and its subcellular location. We have developed a split-GFP-based BiFC assay for use in the budding yeast Saccharomyces cerevisiae in which the modifications are carried out at the genomic level, thus resulting in the tagged yeast proteins being expressed at wild-type levels. The system is capable of detecting interactions in all subcellular compartments tested (the cytoplasm, mitochondria and nucleus) and makes a valuable addition to techniques for the investigation of protein-protein interactions in this model organism.

Microtubules as antiparasitic drug targets

Background: Parasitic diseases including malaria, leishmaniasis and schistosomiasis take a terrible toll of human life, health and productivity, especially in tropical and subtropical regions, and are also highly significant in animal health worldwide. Antiparasitic drugs are the mainstays of control of most of these diseases, but in many cases current therapies are inadequate and in some the situation is deteriorating because of drug resistance. Microtubules, as essential components of almost all eukaryotic cells, are proven drug targets in many helminth diseases and show promise as targets for the development of new antiprotozoal drugs. Objective: This article reviews the chemistry of the microtubule inhibitors in current use and under investigation as antiparasitic agents, their activities against the major parasites and their mechanisms of action. New directions in both inhibitor chemistry and biological evaluation are discussed. Conclusions: The most promising immediate avenues for discovery and design appear to lie in development of novel benzimidazoles for helminth parasites and compounds based on antimitotic herbicides for protozoal parasites. New understanding from functional genomics, structural biology and microtubular imaging will help accelerate the development of completely novel antiparasitic drugs targeting microtubules.

Characterisation of two calmodulin-like proteins from the liver fluke, Fasciola hepatica.

Abstract Calmodulin is a calcium ion-sensing signalling protein found in eukaryotics. Two calmodulin-like gene sequences were identified in an EST library from adult liver flukes. One codes for a protein (FhCaM1) homologous to mammalian calmodulins (98% identity), whereas the other protein (FhCaM2) has only 41% identity. These genes were cloned into expression vectors and the recombinant proteins were expressed in Escherichia coli. Gel shift assays showed that both proteins bind to calcium, magnesium and zinc ions. Homology models have been built for both proteins. As expected, FhCaM1 has a highly similar structure to other calmodulins. Although FhCaM2 has a similar fold, its surface charge is higher than FhCaM1. One of the potential metal ion-binding sites has lower metal-ion co-ordination capability, while another has an adjacent lysine residue, both of which may decrease the metal-binding affinity. These differences may reflect a specialised role for FhCaM2 in the liver fluke.

Liver fluke β-tubulin isotype 2 binds albendazole and is thus a probable target of this drug.

Albendazole is a benzimidazole drug which can be used to treat liver fluke (Fasciola hepatica) infections. Its mode of action is believed to be the inhibition of microtubule formation through binding to β-tubulin. However, F. hepatica expresses at least six different isotypes of β-tubulin, and this has confused, rather than clarified, understanding of the molecular mechanisms of benzimidazole drugs in this organism. Recombinant F. hepatica β-tubulin proteins were expressed in, and purified from, Escherichia coli. These proteins were then used in pull-down assays in which albendazole was covalently linked to Sepharose. β-Tubulin isotype 2 was pulled down in this assay, and this interaction could be reduced by adding competing albendazole. Molecular modelling of β-tubulin isotypes suggests that changes in the side change conformations of residue 200 in the putative albendazole binding site may be important in determining whether, or not, a particular isotype will bind to the drug. These results, together with previous work demonstrating that albendazole causes disruption of microtubules in the liver fluke, strongly suggest that β-tubulin isotype 2 is one of the targets of this drug.

Detection and localisation of protein-protein interactions in Saccharomyces cerevisiae using a split-GFP method

An alternative method for monitoring protein-protein interactions in Saccharomyces cerevisiae has been developed. It relies on the ability of two fragments of enhanced green fluorescent protein (EGFP) to reassemble and fluoresce when fused to interacting proteins. Since this fluorescence can be detected in living cells, simultaneous detection and localisation of interacting pairs is possible. DNA sequences encoding N- and C-terminal EGFP fragments flanked by sequences from the genes of interest were transformed into S. cerevisiae JPY5 cells and homologous recombination into the genome verified by PCR. The system was evaluated by testing known interacting proteins: labelling of the phosphofructokinase subunits, Pfk1p and Pfk2p, with N- and C-terminal EGFP fragments, respectively, resulted in green fluorescence in the cytoplasm. The system works in other cellular compartments: labelling of Idh1p and Idh2p (mitochondrial matrix), Sdh3p and Sdh4p (mitochondrial membrane) and Pap2p and Mtr4p (nucleus) all resulted in fluorescence in the appropriate cellular compartment.

The isolated perfused liver response to a 'second hit' of portal endotoxin during severe acute pancreatitis.

Background/Aim: During severe acute pancreatitis (AP), the liver may show an exaggerated response to the inflammatory products of gut injury transported in the portal vein. Our aim was to explore liver proinflammatory mediator production after a ‘second hit’ of portal lipopolysaccharide (LPS) during AP. Methods: Twenty-four rats underwent one of three ‘first-hit’ scenarios: (1) severe AP induced by intraductal glycodeoxycholic acid injection and intravenous caerulein infusion, (2) sham laparotomy, or (3) no first intervention. Eighteen hours later, all animals received a ‘second hit’ of portal LPS in an isolated liver perfusion system. Tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 concentrations were measured in portal and systemic serum, and in the perfusate 30 and 90 min after the ‘second hit’. Neutrophil activation by the perfusate was assayed using dihydrorhodamine-123 fluorescence. Results: We observed a six-fold increase in IL-6 concentration across the liver during AP. All livers produced TNF-α after the portal LPS challenge, but this was not exaggerated by AP. No differential neutrophil activation by the perfusate was seen. Conclusion: TNF-α, IL-1β, IL-6 and neutrophil activator production by the isolated perfused liver, in response to a ‘second hit’ of portal LPS, does not appear to be enhanced during AP.

A novel calmodulin-like protein from the liver fluke, Fasciola hepatica

An 18.2 kDa protein from the liver fluke, Fasciola hepatica has been identified and characterised. The protein shows strongest sequence similarity to egg antigen proteins from Schistosoma mansoni, Schistosoma japonicum and Clonorchis sinensis. The protein is predicted to adopt a calmodulin-like fold; it thus represents the third calmodulin-like protein to be characterised in F. hepatica and has been named FhCaM3. Compared to the classical calmodulin structure there are some variations. Most noticeably, the central, linker helix is disrupted by a cysteine residue. Alkaline native gel electrophoresis showed that FhCaM3 binds calcium ions. This binding event increases the ability of the protein to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, consistent with an increase in surface hydrophobicity as seen in other calmodulins. FhCaM3 binds to the calmodulin antagonists trifluoperazine and W7, but not to the myosin regulatory light chain binding compound praziquantel. Immunolocalisation demonstrated that the protein is found in eggs and vitelline cells. Given the critical role of calcium ions in egg formation and hatching this suggests that FhCaM3 may play a role in calcium signalling in these processes. Consequently the antagonism of FhCaM3 may, potentially, offer a method for inhibiting egg production and thus reducing the spread of infection.

Differential Preservation of Lipopolysaccharide-Induced Chemokine/Cytokine Expression during Experimental Pancreatitis-Associated Organ Failure in Rats Shows a Regulatory Expressed Phenotype

Background: Altered lipopolysaccharide (LPS)-responsiveness is a key feature of acute pancreatitis (AP)-associated multiple organ failure (AP-MOF) in rats and humans. Aim: To determine the differential expression of 16 cytokines and chemokines in response to delayed LPS administration in established experimental AP-MOF in rats. Methods: In a cubic factorial group design (12 groups, n = 6 rats/group), 0, 6 and 30 µg/kg Escherichia coli 0111:B4 LPS was administered intra-arterially, 18 h into experimental AP-MOF or sham laparotomy. AP was induced by intraductal glycodeoxycholic acid and intravenous caerulein. Central venous serum concentrations of 16 cytokines and chemokines were measured by Searchlight™ multiplex ELISA. Results: Four patterns were observed: (1) TNF-α, IL-1α, IL-1β, IL-6, IFN-γ, MCP-1, MIP-2α, MIP-3α, fractalkine and RANTES showed a diminished LPS response in AP versus sham (p < 0.001, ANOVA); (2) IL-2, IL-4 and GM-CSF levels were undetectable; (3) CINC-2α and GRO/KC showed little or no difference between AP and controls, and (4) IL-10 concentrations after 0 and 6 µg/kg, but not 30 µg/kg LPS injection were significantly higher in AP than controls (p < 0.001, ANOVA). Conclusion: Experimental AP-MOF in rats results in differential preservation of the cytokine and chemokine response to LPS challenge, with a predominantly regulatory expressed phenotype.