Altered cofactor binding affects stability and activity of human UDP-galactose 4′-epimerase: Implications for type III galactosemia

Deficiency of UDP-galactose 4'-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and no ability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD(+). p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two a-helices that contain residues that interact with NAD(+). These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation.

FhCaBP3: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the ß-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.

The effect of hypobaric hypoxia on misonidazole binding in normal and tumour-bearing mice

The effect of hypobaric hypoxia on the in vivo binding of misonidazole was investigated in normal mice and mice bearing T50/80 or CA NT mammary carcinomas. After the intraperitoneal injection of radiolabelled misonidazole, mice were randomised to breathe either room air or air at 0.5 atmospheres. The distribution of misonidazole in liver, kidney, heart, spleen and tumour tissue, 24 h later, was studied by scintillation counting and by autoradiography. Significantly higher misonidazole binding occurred in the livers (x2.5), kidneys (x2.4), spleens (x2.9) and hearts (x1.8) of hypoxic mice compared to controls. Hypobaric hypoxia was associated with a greater than four-fold increase in misonidazole binding within T50/80 tumours. However, significantly higher binding was not demonstrated within CA NT tumours after exposure of tumour-bearing animals to hypoxic conditions. In autoradiographs of hypoxic liver, labelling was intense in regions near to hepatic veins but sparse in areas surrounding portal tracts. This pattern was striking and consistent. In hypoxic kidney, labelling was most intense over tubular cells, less intense over glomeruli and sparse in the renal medulla. It is likely that the hepatic and renal cortical distributions of misonidazole binding reflect local oxygen gradients.

Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis

Introduction: Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic autoimmune diseases with variable clinical outcomes. We investigated whether the synovial fluid (SF) proteome could distinguish a subset of patients in whom disease extends to affect a large number of joints.

Methods: SF samples from 57 patients were obtained around time of initial diagnosis of JIA, labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression verified by immunochemical methods. Protein glycosylation status was confirmed by hydrophilic interaction liquid chromatography.

Results: A truncated isoform of vitamin D binding protein (VDBP) is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p < 0.05). Furthermore, sialylated forms of immunopurified synovial VDBP were significantly reduced in extended oligoarticular patients (p < 0.005).

Conclusion: Reduced conversion of VDBP to a macrophage activation factor may be used to stratify patients to determine risk of disease extension in JIA patients.

Single-stranded DNA-binding protein hSSB1 is critical for genomic stability

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein-protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.

The M17 leucine aminopeptidase of the malaria parasite Plasmodium falciparum:importance of active site metal ions in the binding of substrates and inhibitors

The M17 leucine aminopeptidase of the intraerythrocytic stages of the malaria parasite Plasmodium falciparum (PfLAP) plays a role in releasing amino acids from host hemoglobin that are used for parasite protein synthesis, growth, and development. This enzyme represents a target at which new antimalarials could be designed since metalloaminopeptidase inhibitors prevent the growth of the parasites in vitro and in vivo. A study on the metal ion binding characteristics of recombinant P. falciparum M17 leucine aminopeptidase (rPfLAP) shows that the active site of this exopeptidase contains two metal-binding sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The enzyme retains activity when the metal ion is removed from site 1, while removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by the addition of divalent metal cations. The metal ion at site 1 is readily exchangeable with several divalent metal ions and displays a preference in the order of preference Zn(2+) > Mn(2+) > Co(2+) > Mg(2+). While it is likely that native PfLAP contains a Zn(2+) in site 2, the metal ion located in site 1 may be dependent on the type and concentration of metal ions in the cytosolic compartment of the parasite. Importantly, the type of metal ion present at site 1 influences not only the catalytic efficiency of the enzyme for peptide substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.

Secretory leucoprotease inhibitor binds to NF-kappaB binding sites in monocytes and inhibits p65 binding

Secretory leucoprotease inhibitor (SLPI) is a nonglycosylated protein produced by epithelial cells. In addition to its antiprotease activity, SLPI has been shown to exhibit antiinflammatory properties, including down-regulation of tumor necrosis factor alpha expression by lipopolysaccharide (LPS) in macrophages and inhibition of nuclear factor (NF)-kappaB activation in a rat model of acute lung injury. We have previously shown that SLPI can inhibit LPS-induced NF-kappaB activation in monocytic cells by inhibiting degradation of IkappaBalpha without affecting the LPS-induced phosphorylation and ubiquitination of IkappaBalpha. Here, we present evidence to show that upon incubation with peripheral blood monocytes (PBMs) and the U937 monocytic cell line, SLPI enters the cells, becoming rapidly localized to the cytoplasm and nucleus, and affects NF-kappaB activation by binding directly to NF-kappaB binding sites in a site-specific manner. SLPI can also prevent p65 interaction with the NF-kappaB consensus region at concentrations commensurate with the physiological nuclear levels of SLPI and p65. We also demonstrate the presence of SLPI in nuclear fractions of PBMs and alveolar macrophages from individuals with cystic fibrosis and community-acquired pneumonia. Therefore, SLPI inhibition of NF-kappaB activation is mediated, in part, by competitive binding to the NF-kappaB consensus-binding site.

An enantioselective imprinted receptor for Z-glutamate exhibiting a binding induced color change

Using 1-(4-styryl)-3-(3-nitrophenyl)urea as host monomer for the imprinting of Z-(D or L)-Glu, a polymeric receptor exhibiting strong enantioselectivity and a change in color intensity upon binding of the guest was obtained.

Local-field and excitonic effects in the optical response of alpha-alumina

This paper introduces key ingredients of the dielectric response of a-alumina that go beyond an independent-particle (IP) treatment of the valence-electron excitations. The optical-response functions were calculated from first-principles both at the Bethe-Salpeter and the random-phase approximation (RPA) levels. Excitonic effects obtained within the Bethe-Salpeter framework were found essential for reproducing the low-energy part of the experimental spectra (below 15 eV) and the bound exciton in particular. For higher energies, local-field effects introduced through the RPA modified considerably the IP results and provided a satisfactory account of the reflectivity spectra and of the position and shape of the dominant bulk plasmon resonance in the electron energy-loss spectra.

Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr ΔF508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ?F508 mutation is the most common. ?F508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ?F508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ?F508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ?F508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ?F508 macrophages.

Lipopolysaccharide-binding protein and CD14 are increased in the bronchoalveolar lavage fluid of smokers

Lipopolysaccharide-binding protein (LBP) and CD14 contribute to the recognition of pathogens by cells, which triggers the activation of defence responses. Smoking is a risk factor for the development of chronic obstructive pulmonary disease (COPD) and respiratory infections. The current authors theorised that levels of LBP and CD14 in the lungs of smokers would be higher than those in the lungs of never-smokers. These elevated levels could affect host responses upon infection. LBP, soluble CD14 (sCD14) and interleukin (IL)-8 were detected by ELISA. Nuclear factor (NF)- ?B, p38 and the inhibitor I?Ba were studied by immunoassays. Gene expression was assessed by RT-PCR. Bronchoalveolar lavage levels of LBP and CD14 were significantly higher in smokers and COPD patients than in never-smokers, whereas levels of both proteins were not significantly different between smokers and COPD patients. IL-6, IL-1ß5 and cigarette smoke condensate induced the expression of LBP and CD14 by airway epithelial cells. LBP and sCD14 inhibited the nontypeable Haemophilus influenzae (NTHi)-dependent secretion of IL-8 and the activation of NF-?B and p38 mitogen-activated protein kinase signalling pathways but they increased the internalisation of NTHi by airway epithelial cells. Thus, in the inflamed airways of smokers both proteins could contribute to inhibit bacteria-dependent cellular activation without compromising the internalisation of pathogens by airway cells. Copyright©ERS Journals Ltd 2009.

Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners

Adrenomedullin (AM) is an important regulatory peptide involved in both physiological and pathological states. We have previously demonstrated the existence of a specific AM-binding protein (AMBP-1) in human plasma. In the present study, we developed a nonradioactive ligand blotting assay, which, together with high pressure liquid chromatography/SDS-polyacrylamide gel electrophoresis purification techniques, allowed us to isolate AMBP-1 to homogeneity. The purified protein was identified as human complement factor H. We show that AM/factor H interaction interferes with the established methodology for quantification of circulating AM. Our data suggest that this routine procedure does not take into account the AM bound to its binding protein. In addition, we show that factor H affects AM in vitro functions. It enhances AM-mediated induction of cAMP in fibroblasts, augments the AM-mediated growth of a cancer cell line, and suppresses the bactericidal capability of AM on Escherichia coli. Reciprocally, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. In summary, we report on a potentially new regulatory mechanism of AM biology, the influence of factor H on radioimmunoassay quantification of AM, and the possible involvement of AM as a regulator of the complement cascade.

Locating binding poses in protein-ligand systems using reconnaissance metadynamics

A molecular dynamics-based protocol is proposed for finding and scoring protein-ligand binding poses. This protocol uses the recently developed reconnaissance metadynamics method, which employs a self-learning algorithm to construct a bias that pushes the system away from the kinetic traps where it would otherwise remain. The exploration of phase space with this algorithm is shown to be roughly six to eight times faster than unbiased molecular dynamics and is only limited by the time taken to diffuse about the surface of the protein. We apply this method to the well-studied trypsin-benzamidine system and show that we are able to refind all the poses obtained from a reference EADock blind docking calculation. These poses can be scored based on the length of time the system remains trapped in the pose. Alternatively, one can perform dimensionality reduction on the output trajectory and obtain a map of phase space that can be used in more expensive free-energy calculations.

Binding of Calcium and Carbonate to Polyacrylates

Polyacrylate molecules can be used to slow the growth of calcium carbonate. However, little is known about the mechanism by which the molecules impede the growth rate. A recent computational study (Bulo et al. Macromolecules 2007, 40, 3437) used metadynamics to investigate the binding of calcium to polyacrylate chains and has thrown some light on the coiling and precipitation of these polymers. We extend these simulations to examine the binding of calcium and carbonate to polyacrylate chains. We show that calcium complexed with both carbonate and polyacrylate is a very stable species. The free energies of calcium-carbonate-polyacrylate complexes, with different polymer configurations, are calculated, and differences in the free energy of the binding of carbonate are shown to be due to differences in the amount of steric hindrance about the calcium, which prevents the approach of the carbonate ion.