Biomedical subjects

Richard James

Publications and source records attributed to Richard James.

At least 19 recordsLinked to original sources

PubMed2007

Differential effect of pioglitazone (PGZ) and rosiglitazone (RGZ) on postprandial glucose and lipid metabolism in patients with type 2 diabetes mellitus: a prospective, randomized crossover study.

BACKGROUND: Postprandial metabolism is impaired in patients with type 2 diabetes (T2Dm). Two thiazolidinediones pioglitazone (PGZ) and rosiglitazone (RGZ) have similar effects on glycaemic control but differ in their effects on fasting lipids. This study investigated the effects of RGZ and PGZ on postprandial metabolism in a prospective, randomized crossover trial. METHODS: Seventeen patients with T2Dm were randomized to RGZ or PGZ for 12 weeks, with an 8-week wash-out period. Fasting blood samples were taken for glucose (FPG), insulin, HbA(1c), lipids, apolipoproteins (apo), lipoprotein (LPL) and hepatic lipase (HL), and cholesterol ester transfer protein (CETP) activity. A standardized breakfast was served and postprandial glucose, insulin, and lipid subfraction profiles were determined. RESULTS: RGZ and PGZ treatment resulted in a similar improvement in FPG, HbA(1c) and homeostasis model assessment. Fasting and postprandial triglyceride (TG) levels were significantly lower following PGZ therapy (fasting: -0.35 vs 0.44 mmol/L; p < 0.04; postprandial AUC-TG: -195.6 vs 127.9 mmol/L/min; p < 0.02) associated with changes in VLDL-2-TG (-0.10 vs 0.21 mmol/L; p = 0.23) and VLDL-3-TG (0.0 vs 0.34 mmol/L; p < 0.04). Fasting cholesterol increased with RGZ compared to PGZ (0.06 vs 0.59 mmol/L; p < 0.04), particularly in VLDL-2-C (-0.30 vs 0.59 mmol/L; p < 0.03) and VLDL-3-C (-0.85 vs 2.11 mmol/L; p < 0.02). Postprandial VLDL lipid and protein content increased after RGZ and decreased after PGZ. Fasting apoB, apoA-I, apoC-II/C-III-ratio, and LPL activity did not differ. CETP activity decreased after RGZ and increased after PGZ (-6.2 vs 4.2 p/mol/mL/min; p < 0.002). CONCLUSIONS: Both the glitazones had similar effects on glucose metabolism. The additional beneficial effect of PGZ on lipid metabolism may be related to its effects on insulin-independent VLDL production and CETP activity.

Blood Glucose↗

PubMed2006

Internally quenched peptides for the study of lysostaphin: An antimicrobial protease that kills Staphylococcus aureus.

Lysostaphin (EC. 3.4.24.75) is a protein secreted by Staphylococcus simulans biovar staphylolyticus and has been shown to be active against methicillin resistant S. aureus (MRSA). The design and synthesis of three internally quenched substrates for lysostaphin based on the peptidoglycan crossbridges of S. aureus, and their use in fluorescence resonance energy transfer (FRET) assays is reported. These substrates enabled the gathering of information about the endopeptidase activity of lysostaphin and the effect that mutations have on its enzymatic ability. Significant problems with the inner filter effect and substrate aggregation were encountered; their minimisation and the subsequent estimation of the kinetic parameters for the interaction of lysostaphin with the substrates is described, as well as a comparison of substrates incorporating two FRET pairs: Abz-EDDnp and DABCYL-EDANS. In addition to this, the points of cleavage caused by lysostaphin in Abz-pentaglycine-EDDnp have been determined by HPLC and mass spectrometry analysis to be between glycines 2 and 3(approximately 60%) and glycines 3 and 4 (approximately 40%).

Fluorescence↗

PubMed2006

Competitive recruitment of the periplasmic translocation portal TolB by a natively disordered domain of colicin E9.

The natively disordered N-terminal 83-aa translocation (T) domain of E group nuclease colicins recruits OmpF to a colicin-receptor complex in the outer membrane (OM) as well as TolB in the periplasm of Escherichia coli, the latter triggering translocation of the toxin across the OM. We have identified the 16-residue TolB binding epitope in the natively disordered T-domain of the nuclease colicin E9 (ColE9) and solved the crystal structure of the complex. ColE9 folds into a distorted hairpin within a canyon of the six-bladed beta-propeller of TolB, using two tryptophans to bolt the toxin to the canyon floor and numerous intramolecular hydrogen bonds to stabilize the bound conformation. This mode of binding enables colicin side chains to hydrogen-bond TolB residues in and around the channel that runs through the beta-propeller and that constitutes the binding site of peptidoglycan-associated lipoprotein (Pal). Pal is a globular binding partner of TolB, and their association is known to be important for OM integrity. The structure is therefore consistent with translocation models wherein the colicin disrupts the TolB-Pal complex causing local instability of the OM as a prelude to toxin import. Intriguingly, Ca(2+) ions, which bind within the beta-propeller channel and switch the surface electrostatics from negative to positive, are needed for the negatively charged T-domain to bind TolB with an affinity equivalent to that of Pal and competitively displace it. Our study demonstrates that natively disordered proteins can compete with globular proteins for binding to folded scaffolds but that this can require cofactors such as metal ions to offset unfavorable interactions.

Binding Sites↗

PubMed2006

Global structural rearrangement of the cell penetrating ribonuclease colicin E3 on interaction with phospholipid membranes.

Nuclease type colicins and related bacteriocins possess the unprecedented ability to translocate an enzymatic polypeptide chain across the Gram-negative cell envelope. Here we use the rRNase domain of the cytotoxic ribonuclease colicin E3 to examine the structural changes on its interaction with the membrane. Using phospholipid vesicles as model membranes we show that anionic membranes destabilize the nuclease domain of the rRNase type colicin E3. Intrinsic tryptophan fluorescence and circular dichroism show that vesicles consisting of pure DOPA act as a powerful protein denaturant toward the rRNase domain, although this interaction can be entirely prevented by the addition of salt. Binding of E3 rRNase to DOPA vesicles is an endothermic process (DeltaH=24 kcal mol-1), reflecting unfolding of the protein. Consistent with this, binding of a highly destabilized mutant of the E3 rRNase to DOPA vesicles is exothermic. With mixed vesicles containing anionic and neutral phospholipids at a ratio of 1:3, set to mimic the charge of the Escherichia coli inner membrane, destabilization of E3 rRNase is lessened, although the melting temperature of the protein at pH 7.0 is greatly reduced from 50 degrees C to 30 degrees C. The interaction of E3 rRNase with 1:3 DOPA:DOPC vesicles is also highly dependent on both ionic strength and temperature. We discuss these results in terms of the likely interaction of the E3 rRNase and the related E9 DNase domains with the E. coli inner membrane and their subsequent translocation to the cell cytoplasm.

Anions↗

PubMed2005

Cell entry mechanism of enzymatic bacterial colicins: porin recruitment and the thermodynamics of receptor binding.

Binding of enzymatic E colicins to the vitamin B12 receptor, BtuB, is the first stage in a cascade of events that culminate in the translocation of the cytotoxic nuclease into the Escherichia coli cytoplasm and release of its tightly bound immunity protein. A dogma of colicin biology is that the toxin coiled-coil connecting its functional domains must unfold or unfurl to span the periplasm, with recent reports claiming this reaction is initiated by receptor binding. We report isothermal titration calorimetry data of BtuB binding the endonuclease toxin ColE9 and a disulfide form (ColE9S-S) where unfolding of the coiled-coil is prevented and, as a consequence, the toxin is biologically inactive. Contrary to expectation, the thermodynamics of receptor binding, characterized by large negative values for TDeltaS, are identical for the two colicins, arguing against any form of BtuB-induced unfolding. We go on to delineate key features of the "colicin translocon" that assembles at the cell surface after BtuB binding by using a complex of histidine-tagged Im9 bound to ColE9S-S. First, we show that the porin OmpF is recruited directly to the BtuB.colicin complex to form the translocon. Second, recruitment is through the natively unfolded region of the colicin translocation domain, with this domain likely having two contact points for OmpF. Finally, the immunity protein is not released during its assembly. Our study demonstrates that although colicin unfolding is undoubtedly a prerequisite for E. coli cell death, it must occur after assembly of the translocon.

Bacterial Outer Membrane Proteins↗

PubMed2005

Clusters in an intrinsically disordered protein create a protein-binding site: the TolB-binding region of colicin E9.

The 61-kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins and kills them by hydrolyzing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies [Collins et al. (2002) J. Mol. Biol. 318, 787-904; MacDonald et al. (2004), J. Biomol. NMR 30, 81-96] have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is intrinsically disordered and contains clusters of interacting side chains. To further define the properties of this region of colicin E9, we have investigated the effects on the dynamical and TolB-binding properties of three mutations of colicin E9 that inactivate it as a toxin. The mutations were contained in a fusion protein consisting of residues 1-61 of colicin E9 connected to the N terminus of the E9 DNase by an eight-residue linking sequence. The NMR data reveals that the mutations cause major alterations to the properties of some of the clusters, consistent with some form of association between them and other more distant parts of the amino acid sequence, particularly toward the N terminus of the protein. However, (15)N T(2) measurements indicates that residues 5-13 of the fusion protein bound to the 43-kDa TolB remain as flexible as they are in the free protein. The NMR data point to considerable dynamic ordering within the intrinsically disordered translocation domain of the colicin that is important for creating the TolB-binding site. Furthermore, amino acid sequence considerations suggest that the clusters of amino acids occur because of the size and polarities of the side chains forming them influenced by the propensities of the residues within the clusters and those immediately surrounding them in sequence space to form beta turns.

Amino Acid Sequence↗

PubMed2005

Free vascularised iliac crest graft: an audit of 26 consecutive cases.

Over an 18 month period 26 vascularised myo-osseus, and one myo-osseus-cutaneous iliac crest flaps were used to reconstruct 18 mandibular and eight maxillary defects. During the operation one flap failed to reperfuse and was removed. Postoperatively three patients were returned to theatre for appraisal and reanastomoses, which in two cases involved thrombolysis. In all three salvage was deemed successful but in all there was partial muscle necrosis. There were 10 cases of anaesthesia of the skin supplied by the lateral cutaneous nerve of the thigh, three incisional hernias, one wound dehiscence in the thigh, two oronasal fistulas, one wound dehiscence in the neck, one infection by methicillin resistant Staphylococcus aureus in the thigh, and two long-term and one medium-term disturbances of gait. Complications were graded as severe (4%), intermediate (27%), and minor (46%).

Adult↗

PubMed2005

Insulin resistance is associated with liver fibrosis in non-diabetic chronic hepatitis C patients.

BACKGROUND/AIMS: Liver steatosis is a frequent finding in chronic hepatitis C. An association has been suggested between steatosis and fibrosis progression rate, but the pathogenetic mechanisms linking fatty infiltration and collagen deposition are unknown. METHODS: We measured the levels of insulin resistance (as HOMA score) and leptin in 221 non-diabetic chronic hepatitis C patients, to assess their impact on liver steatosis and fibrosis, relative to other factors, using a multivariable logistic regression. RESULTS: When all 221 patients were considered, steatosis was associated with excessive alcohol intake, genotype 3, and serum HCV RNA level, whereas fibrosis was associated with HOMA score and age. In 152 patients infected with genotype non-3, steatosis was associated with alcohol abuse and HCV RNA level, and fibrosis with HOMA score and age. In the 69 patients with genotype 3, steatosis and fibrosis were associated with each other. The association between fibrosis and HOMA score held also when 22 obese patients were excluded from the analysis. Levels of insulin resistance were not correlated with the presence of steatosis. CONCLUSIONS: Thus, insulin resistance (but not leptin) may play a role in fibrogenesis in chronic hepatitis C patients infected with genotype non-3.

Adult↗

PubMed2005

T-cell-stimulating protein A elicits immune responses during meningococcal carriage and human disease.

In recognition of the need for immunological memory-inducing components for future Neisseria meningitidis group B vaccines, we previously searched the proteome of N. meningitidis and identified T-cell-stimulating protein A (TspA). This study was designed to confirm the immunogencity of TspA and to examine the subset of T-helper cell responses to the protein in patients and nasopharyngeal carriers. The tspA gene was reconstructed, cloned, and expressed in Escherichia coli, and the recombinant TspA (rTspA) protein was affinity purified. T-cell proliferative responses to rTspA were detected in the peripheral blood mononuclear cells (PBMCs) of convalescent patients and carriers, confirming that TspA-specific T-cell responses were stimulated by invasive disease and nasopharyngeal colonization. Following stimulation of PBMCs with meningococcal lysate, increased frequencies of both Th1 and Th2 cells were observed, indicating that, as during carriage, invasive meningococcal disease induced an unbiased T-helper subset response. A similar unbiased T-helper response was also detected against rTspA in the PBMCs of convalescent patients. The response of PBMCs from the carriers to TspA stimulation, however, was very weak, and the frequencies of cytokine-positive CD4 cells were not significantly greater than the frequencies in unstimulated control cultures. All of the patients and carriers responded with serum antimeningococcal immunoglobulin G (IgG) antibodies, while four of six samples from patients and 5 of 14 samples from carriers contained detectable anti-rTspA IgG antibodies. Taken together, the results of this study confirmed the immunogenicity of TspA in humans during natural meningococcal infection, and therefore, TspA is worthy of further investigation as a possible T-cell stimulating component of future vaccines.

Antigens, Bacterial↗

PubMed2005

Rapid detection of colicin E9-induced DNA damage using Escherichia coli cells carrying SOS promoter-lux fusions.

ColE9 is a plasmid-encoded protein antibiotic produced by Escherichia coli and closely related species that kills E. coli cells expressing the BtuB receptor. The 15-kDa cytotoxic DNase domain of colicin E9 preferentially nicks double-stranded DNA at thymine bases and shares a common active-site structural motif with a variety of other nucleases, including the H-N-H homing endonucleases and the apoptotic CAD proteins of eukaryotes. Studies of the mechanism by which the DNase domain of ColE9 reaches the cytoplasm of E. coli cells are limited by the lack of a rapid, sensitive assay for the DNA damage that results. Here, we report the development of an SOS promoter-lux fusion reporter system for monitoring DNA damage in colicin-treated cells and illustrate the value of this reporter system in experiments that probe the mechanism and time required for the DNase domain of colicin E9 to reach the cytoplasm.

Colicins↗

PubMed2005

Interactions of TolB with the translocation domain of colicin E9 require an extended TolB box.

The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill Escherichia coli cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46. The affinity of the TolB box mutants for TolB was determined by surface plasmon resonance to confirm that the loss of biological activity in all except one (N44A) of the extended TolB box mutants correlates with a reduced affinity of binding to TolB. We used a PCR mutagenesis protocol to isolate residues that restored activity to the inactive ColE9 D35A, S37A, and W39A mutants. A serine residue at position 35, a threonine residue at position 37, and phenylalanine or tyrosine residues at position 39 restored biological activity of the mutant ColE9. The average area predicted to be buried upon folding (AABUF) was correlated with the activity of the variants at positions 35, 37, and 39 of the TolB box. All active variants had AABUF profiles that were similar to the wild-type residues at those positions and provided information on the size, stereochemistry, and potential folding pattern of the residues of the TolB Box.

Amino Acid Sequence↗

PubMed2004

Social networks in the guppy (Poecilia reticulata).

Social network theory is used to elicit details of the social structure of a population of free-ranging guppies, Poecilia reticulata. They were found to have a complex and highly structured social network, which exhibited characteristics consistent with the 'small world' phenomenon. Stable partner associations between individuals were observed, a finding that fulfils the basic prerequisite for the evolution of reciprocal altruism. The findings are discussed in relation to the ecology and evolution of the wild population, highlighting the potential application of network theory to social associations in animals.

Animals↗

PubMed2004

Identification of the catalytic motif of the microbial ribosome inactivating cytotoxin colicin E3.

Colicin E3 is a cytotoxic ribonuclease that specifically cleaves 16S rRNA at the ribosomal A-site to abolish protein synthesis in sensitive Escherichia coli cells. We have performed extensive mutagenesis of the 96-residue colicin E3 cytotoxic domain (E3 rRNase), assayed mutant colicins for in vivo cytotoxicity, and tested the corresponding E3 rRNase domains for their ability to inactivate ribosome function in vitro. From 21 alanine mutants, we identified five positions where mutation resulted in a colicin with no measurable cytotoxicity (Y52, D55, H58, E62, and Y64) and four positions (R40, R42, E60, and R90) where mutation caused a significant reduction in cytotoxicity. Mutations that were found to have large in vivo and in vitro effects were tested for structural integrity through circular dichroism and fluorescence spectroscopy using purified rRNase domains. Our data indicate that H58 and E62 likely act as the acid-base pair during catalysis with other residues likely involved in transition state stabilization. Both the Y52 and Y64 mutants were found to be highly destabilized and this is the likely origin of the loss of their cytotoxicity. The identification of important active site residues and sequence alignments of known rRNase homologs has allowed us to identify other proteins containing the putative rRNase active site motif. Proteins that contained this active site motif included three hemagglutinin-type adhesins and we speculate that these have evolved to deliver a cytotoxic rRNase into eukaryotic cells during pathogenesis.

Amino Acid Motifs↗

PubMed2004

Highly discriminating protein-protein interaction specificities in the context of a conserved binding energy hotspot.

We explore the thermodynamic basis for high affinity binding and specificity in conserved protein complexes using colicin endonuclease-immunity protein complexes as our model system. We investigated the ability of each colicin-specific immunity protein (Im2, Im7, Im8 and Im9) to bind the endonuclease (DNase) domains of colicins E2, E7 and E8 in vitro and compared these to the previously studied colicin E9. We find that high affinity binding (Kd < or = 10(-14) M) is a common feature of cognate colicin DNase-Im protein complexes as are non-cognate protein-protein associations, which are generally 10(6)-10(8)-fold weaker. Comparative alanine scanning of Im2 and Im9 residues involved in binding the E2 DNase revealed similar behaviour to that of the two proteins binding the E9 DNase; helix III forms a conserved binding energy hotspot with specificity residues from helix II only contributing favourably in a cognate interaction, a combination we have termed as "dual recognition". Significant differences are seen, however, in the number and side-chain chemistries of specificity sites that contribute to cognate binding. In Im2, Asp33 from helix II dominates colicin E2 specificity, whereas in Im9 several hydrophobic residues, including position 33 (leucine), help define its colicin specificity. A similar distribution of specificity sites was seen using phage display where, with Im2 as the template, a library of randomised sequences was generated in helix II and the library panned against either the E2 or E9 DNase. Position 33 was the dominant specificity site recovered in all E2 DNase-selected clones, whereas a number of Im9 specificity sites were recovered in E9 DNase-selected clones, including position 33. In order to probe the relationship between biological specificity and in vitro binding affinity we compared the degree of protection afforded to bacteria against colicin E9 toxicity by a set of immunity proteins whose affinities for the E9 DNase differed by up to ten orders of magnitude. This analysis indicated that the Kd required for complete biological protection is <10(-10)M and that the "affinity window" over which the selection of novel immunity protein specificities likely evolves is 10(-6)-10(-10)M. This comprehensive survey of colicin DNase-immunity protein complexes illustrates how high affinity protein-protein interactions can be very discriminating even though binding is dominated by a conserved hotspot, with single or multiple specificity sites modulating the overall binding free energy. We discuss these results in the context of other conserved protein complexes and suggest that they point to a generic specificity mechanism in divergently evolved protein-protein interactions.

Bacterial Proteins↗

PubMed2004

Destabilization of the colicin E9 Endonuclease domain by interaction with negatively charged phospholipids: implications for colicin translocation into bacteria.

We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers (Mosbahi, K., Lemaître, C., Keeble, A. H., Mobasheri, H., Morel, B., James, R., Moore, G. R., Lea, E. J., and Kleanthous, C. (2002) Nat. Struct. Biol. 9, 476-484). It was proposed that the E9 DNase mediates its own translocation across the cytoplasmic membrane and that the formation of ion channels is essential to this process. Here we describe changes to the structure and stability of the E9 DNase that accompany interaction of the protein with phospholipid vesicles. Formation of the protein-lipid complex at pH 7.5 resulted in a red-shift of the intrinsic protein fluorescence emission maximum (lambda(max)) from 333 to 346 nm. At pH 4.0, where the E9 DNase lacks tertiary structure but retains secondary structure, DOPG induced a blue-shift in lambda(max), from 354 to 342 nm. Changes in lambda(max) were specific for anionic phospholipid vesicles at both pHs, suggesting electrostatics play a role in this association. The effects of phospholipid were negated by Im9 binding, the high affinity, acidic, exosite inhibitor protein, but not by zinc, which binds at the active site. Fluorescence-quenching experiments further demonstrated that similar protein-phospholipid complexes are formed regardless of whether the E9 DNase is initially in its native conformation. Consistent with these observations, chemical and thermal denaturation data as well as proteolytic susceptibility experiments showed that association with negatively charged phospholipids destabilize the E9 DNase. We suggest that formation of a destabilizing protein-lipid complex pre-empts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane.

Acrylamide↗

PubMed2004

Population-based study of SR-BI genetic variation and lipid profile.

The variability of the Class B Type I Scavenger Receptor (SR-BI) gene in human populations and the relation of its variants to blood lipids was investigated in a random sample of 1756 untreated adult residents of Geneva, Switzerland, during 1999-2000. A three-step study approach yielded the following results: (1) resequencing the gene's exons and flanking regions in 95 subjects identified four common single nucleotide polymorphisms (SNPs with rare allele frequency >3%); (2) association study of the four common SNPs in subjects with extreme HDL-cholesterol (HDL-C) and LDL-C phenotypes (186 "atherogenic cases" and 185 "non-atherogenic controls") showed that the synonymous exon 8 C-T (allelic frequency 48%) polymorphism, A350A, was associated with atheroprotection in men (odds ratios (OR) = 0.36, 95% confidence intervals (CI) = 0.15-0.90, P < 0.03), but not in women (2.09, 0.79-5.49, P = 0.14); and (3) population clinical effects of A350A genotypes assessed in all 1756 subjects, showed that the case-control study findings reflected a protective HDL-C effect in men (CC: 1.17 mmol/L, CT: 1.22 mmol/L, and TT: 1.24 mmol/L, trend P = 0.0062) and a deleterious LDL-C effect in women (CC: 3.58 mmol/L, CT: 3.72 mmol/L, and TT: 3.79 mmol/L, trend P = 0.014). The allelic frequencies of the common SR-BI variants appear to be very similar in European and North American populations. The HDL-C effect increased with age. SR-BI A350A appears to have gender-specific and age-related effects on cholesterol transport lipoproteins.

Adult↗

PubMed2004

Presence of autoantibodies to apolipoprotein A-1 in patients with acute coronary syndrome further links autoimmunity to cardiovascular disease.

Anti-apolipoprotein A-1 (Apo A-1) autoantibodies were described in autoimmune disorders such as systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) and might be involved in the genesis of arterial and venous thrombotic events. To investigate the presence of these autoantibodies in patients with acute coronary syndrome (ACS) without other features of autoimmunity, we set up an enzyme-linked immunosorbent assay (ELISA) for anti-Apo A-1 antibodies. We used it to investigate their prevalence in ACS as compared to SLE and APS and correlated them to plasma Apo A-1 and serum amyloid A protein (SAA) concentrations. The prevalence of anti-Apo A-1 autoantibodies in the healthy control group was 1% (1/92), but was significantly higher in other groups: 21% (11/53) in ACS group (P=0.001), 13% (12/92) in SLE and/or APS group (P=0.005). Multiple linear regression revealed a significant correlation between plasma Apo A-1 (r=-0.72, P=0.013), plasma SAA concentration (r=0.76, P=0.0066) and anti-Apo A-1 IgG titre in ACS patients. The presence of anti-Apo A-1 autoantibodies in patients with ACS highlights an additional link between autoimmunity, inflammation and atherosclerosis.

Acute Disease↗

PubMed2004

Characterisation of a mobile protein-binding epitope in the translocation domain of colicin E9.

The 61 kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins, and kills them by hydrolysing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies (Collins et al., 2002 J. Mol. Biol. 318, 787-804) have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is largely unstructured and highly flexible. In order to further define the properties of this region we have studied a fusion protein containing residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight-residue linking sequence. 53 of the expected 58 backbone NH resonances for the first 61 residues and all of the expected 7 backbone NH resonances of the linking sequence were assigned with 3D (1)H-(13)C-(15)N NMR experiments, and the backbone dynamics of these regions investigated through measurement of (1)H-(15)N relaxation properties. Reduced spectral density mapping, extended Lipari-Szabo modelling, and fitting backbone R(2) relaxation rates to a polymer dynamics model identifies three clusters of interacting residues, each containing a tryptophan. Each of these clusters is perturbed by TolB binding to the intact colicin, showing that the significant region for TolB binding extends beyond the recognized five amino acids of the TolB box and demonstrating that the binding epitope for TolB involves a considerable degree of order within an otherwise disordered and flexible domain. Abbreviations : Im9, the immunity protein for colicin E9; E9 DNase, the endonuclease domain of colicin E9; HSQC, heteronuclear single quantum coherence; ppm, parts per million; DSS, 2,2-(dimethylsilyl)propanesulfonic acid; TSP, sodium 3-trimethylsilypropionate; T(1 - 61)-DNase fusion protein, residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight residue thrombin cleavage sequence.

Amino Acid Sequence↗