Gene Wijffels

Peritrophic matrix proteins.

The peritrophic matrix (or peritrophic membrane) lines the gut of most insects at one or more stages of the life cycle. It has important roles in the facilitation of the digestive processes in the gut and the protection of the insect from invasion by microorganisms and parasites. The traditional view of the peritrophic matrix as a relatively insert sieve, composed largely of proteins and glycosaminoglycans embedded in a chitinous matrix, is under revision as more is learned about the molecular characteristics of the peritrophic matrix proteins. This review summarizes emerging knowledge of the main protein constituents of the peritrophic matrix. The availability of the first sequences of integral peritrophic matrix proteins has coincided with the explosion of information in sequence databases. It is therefore possible to examine common structural themes in this family of proteins as well as in proteins of unknown location and function from a variety of other insects, nematodes and viruses. The review concludes with speculation about the biological functions of the proteins in this matrix.

A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

The interaction between DNA polymerases and sliding clamp proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. The clamp proteins also participate in a variety of dynamic and competing protein–protein interactions. However, clamp-protein binding sequences have not so far been identified in the eubacteria. Here we show from three lines of evidence, bioinformatics, yeast two-hybrid analysis, and inhibition of protein–protein interaction by modified peptides, that variants of a pentapeptide motif (consensus QL[SD]LF) are sufficient to enable interaction of a number of proteins with an archetypal eubacterial sliding clamp (the β subunit of Escherichia coli DNA polymerase III holoenzyme). Representatives of this motif are present in most sequenced members of the eubacterial DnaE, PolC, PolB, DinB, and UmuC families of DNA polymerases and the MutS1 mismatch repair protein family. The component tripeptide DLF inhibits the binding of the α (DnaE) subunit of E. coli DNA polymerase III to β at μM concentration, identifying key residues. Comparison of the eubacterial, eukaryotic, and archaeal sliding clamp binding motifs suggests that the basic interactions have been conserved across the evolutionary landscape.

Primary sequence heterogeneity and tissue expression of glutathione S-transferases of Fasciola hepatica.

Glutathione S-transferases (GSTs) from Fasciola hepatica have been purified by glutathione affinity chromatography. Two closely migrating species of Mr 26,000 and 26,500 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several species resolved by two-dimensional gel analysis, indicating substantial heterogeneity among the GSTs. N-terminal amino acid sequencing revealed one core sequence containing three polymorphisms, whereas the sequence of GST peptides implied a minimum of three different GSTs. The amino acid sequence data assigned the F. hepatica GSTs to the mu class of GSTs with high similarities to these proteins in other helminths and mammals. The native GSTs of F. hepatica appeared to behave as dimers as determined by molecular sieving chromatography. The observation that the GSTs of F. hepatica are heterogeneous in sequence and behave as dimers in the native state suggest that these isoenzymes may exhibit considerable functional heterogeneity which may be of importance to the parasite. Immunocytochemical studies suggest that the main source of GST in F. hepatica are the parenchymal cells and peripheral tissues of the parasite. Some extracellular GST is associated with the lamellae of the intestinal epithelium. The identification of an intestinal GST is unique among trematodes studied to date.

Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and β proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with β in vitro. A new β-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified β-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind β. A 10-amino-acid peptide containing the E. coli Hda β-binding motif was shown to compete with Hda for binding to β in an Hda-β interaction assay. These results establish that the interaction of Hda with β is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

Flexibility revealed by the 1.85 Å crystal structure of the β sliding-clamp subunit of Escherichia coli DNA polymerase III

The beta subunit of the Escherichia coli replicative DNA polymerase III holoenzyme is the sliding clamp that interacts with the alpha (polymerase) subunit to maintain the high processivity of the enzyme. The beta protein is a ring-shaped dimer of 40.6 kDa subunits whose structure has previously been determined at a resolution of 2.5 A [Kong et al. (1992), Cell, 69, 425-437]. Here, the construction of a new plasmid that directs overproduction of beta to very high levels and a simple procedure for large-scale purification of the protein are described. Crystals grown under slightly modified conditions diffracted to beyond 1.9 A at 100 K at a synchrotron source. The structure of the beta dimer solved at 1.85 A resolution shows some differences from that reported previously. In particular, it was possible at this resolution to identify residues that differed in position between the two subunits in the unit cell; side chains of these and some other residues were found to occupy alternate conformations. This suggests that these residues are likely to be relatively mobile in solution. Some implications of this flexibility for the function of beta are discussed.

Biochemical analysis of recombinant glutathione S-transferase of Fasciola hepatica

Four cDNAs encoding GST (rGST1, rGST7, rGST47 and rGST51) of Fasciola hepatica were expressed in Escherichia coli and the rGST proteins purified for biochemical analyses. The rGST proteins are 95% pure as indicated by Coomassie staining of proteins separated by SDS-PAGE. Molecular sieving by HPLC infers that, like the native protein, the rGST proteins form homodimers under non-denaturing conditions. The rGST proteins are recognised by antisera raised to the native GST of F. hepatica. All four rGST proteins from F. hepatica actively conjugate glutathione to the universal substrate, 1-chloro-2,4-dinitrobenzene. The activity of the rGSTs was also measured for substrates which have been shown to have partial specificity for the Alpha, Mu or Pi classes of mammalian GSTs (trans-4-phenyl-3-buten-2-one, ethacrynic acid), for substrates known to be products of lipid peroxidation (trans-2-nonenal, trans,trans-2,4-decadienal) and for epoxy-3-(p-nitrophenoxy)-propane (EPNP), a known substrate for the theta class of GST. No rGST were active with EPNP. rGST47 and 51 showed activity with the other four substrates. rGST7 was active with three substrates whereas rGST1 showed relatively low activity with all substrates except trans,trans-2,4-decadienal. The sensitivity of the rGST activity to inhibition by the GST inhibitors triphenyltin chloride and bromosulphophthalein also varied among the rGSTs with rGST1 showing a 800-fold difference in sensitivity between the inhibitors. These results show that F. hepatica expresses a family of GST isoenzymes which exhibit unique substrate and inhibitor profiles.

Binding inhibitors of the bacterial sliding clamp by design

The bacterial replisome is a target for the development of new antibiotics to combat drug resistant strains. The β(2) sliding clamp is an essential component of the replicative machinery, providing a platform for recruitment and function of other replisomal components and ensuring polymerase processivity during DNA replication and repair. A single binding region of the clamp is utilized by its binding partners, which all contain conserved binding motifs. The C-terminal Leu and Phe residues of these motifs are integral to the binding interaction. We acquired three-dimensional structural information on the binding site in β(2) by a study of the binding of modified peptides. Development of a three-dimensional pharmacophore based on the C-terminal dipeptide of the motif enabled identification of compounds that on further development inhibited α-β(2) interaction at low micromolar concentrations. We report the crystal structure of the complex containing one of these inhibitors, a biphenyl oxime, bound to β(2), as a starting point for further inhibitor design.

Neuropeptide profiling of the bovine hypothalamus: Thermal stabilization is an effective tool in inhibiting post-mortem degradation

The hypothalamus is the central regulatory region of the brain that links the nervous system to the endocrine system via the pituitary gland. It synthesizes and secretes neuropeptide hormones, which in turn act to stimulate or inhibit the secretion of pituitary hormones. We have undertaken a detailed MS investigation of the peptides present in the bovine hypothalamus by adapting a novel heat stabilization methodology, which improved peptide discovery to direct our studies into the molecular mechanisms involved in bovine reproduction. The untreated samples contained large numbers of protein degradation products that interfered with the analysis of the neuropeptides. In the thermally stabilized samples, we were able to identify many more neuropeptides that are known to be expressed in the bovine hypothalamus. Furthermore, we have characterized a range of post‐translational modifications that indicate the presence of processed intact mature neuropeptides in the stabilized tissue samples, whereas we detected many trimmed or truncated peptides resulting from post‐mortem degradation in the untreated tissue samples. Altogether, using an optimized workflow, we were able to identify 140 candidate neuropeptides. We also nominate six new candidate neuropeptides derived from proSAAS, secretogranin‐2 and proTRH.

Performance Evaluation of Algorithms for the Classification of Metabolic 1H NMR Fingerprints

Nontargeted metabolite fingerprinting is increasingly applied to biomedical classification. The choice of classification algorithm may have a considerable impact on outcome. In this study, employing nested cross-validation for assessing predictive performance, six binary classification algorithms in combination with different strategies for data-driven feature selection were systematically compared on five data sets of urine, serum, plasma, and milk one-dimensional fingerprints obtained by proton nuclear magnetic resonance (NMR) spectroscopy. Support Vector Machines and Random Forests combined with t-score-based feature filtering performed well on most data sets, whereas the performance of the other tested methods varied between data sets.

The Plasmid RK2 Replication Initiator Protein (TrfA) Binds to the Sliding Clamp β Subunit of DNA Polymerase III: Implication for the Toxicity of a Peptide Derived from the Amino-Terminal Portion of 33-Kilodalton TrfA

The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. It encodes the essential replication initiation protein TrfA, which binds to the host initiation protein, DnaA, at the plasmid origin of replication (oriV). There are two versions of the TrfA protein, 44 and 33 kDa, resulting from alternate in-frame translational starts. We have shown that the smaller protein, TrfA-33, and its 64-residue amino-terminal peptide (designated T1) physically interact with the Escherichia coli beta sliding clamp (beta(2)). This interaction appears to be mediated through a QLSLF peptide motif located near the amino-terminal end of TrfA-33 and T1, which is identical to the previously described eubacterial clamp-binding consensus motif. T1 forms a stable complex with beta(2) and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and beta(2) and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1. The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the beta-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent over-initiation in E. coli through its interaction with the initiator protein, DnaA, and beta(2). Our results support a model in which T1 toxicity is caused by T1 binding to beta(2), especially when T1 is overexpressed, preventing beta(2) from interacting with host replication proteins such as Hda during the early events of chromosome replication.