Lipid rafts act as specialised domains for tetanus toxin binding and internalisation in neurons

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons.

Hepatocyte nuclear factor-4 alpha regulates the human apolipoprotein AV gene: Identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway

The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4 (HNF-4 ) as a novel regulator of human apoAV gene. Inhibition of HNF-4 expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4 directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4 consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor- coactivator-1 was capable of stimulating the HNF-4 -dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4 . Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4 gene revealed a species-distinct regulation of apoAV by HNF-4 , which resembles that of a subset of HNF-4 target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4 and underscore the role of HNF-4 in regulating triglyceride metabolism.

Spontaneous Cdc42 polarization independent of GDI-mediated extraction and actin-based trafficking.

The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.

Patterns of nucleotide diversity at the regions encompassing the Drosophila insulin-like peptide (dilp) genes: demography vs positive selection in Drosophila melanogaster.

In Drosophila, the insulin-signaling pathway controls some life history traits, such as fertility and lifespan, and it is considered to be the main metabolic pathway involved in establishing adult body size. Several observations concerning variation in body size in the Drosophila genus are suggestive of its adaptive character. Genes encoding proteins in this pathway are, therefore, good candidates to have experienced adaptive changes and to reveal the footprint of positive selection. The Drosophila insulin-like peptides (DILPs) are the ligands that trigger the insulin-signaling cascade. In Drosophila melanogaster, there are several peptides that are structurally similar to the single mammalian insulin peptide. The footprint of recent adaptive changes on nucleotide variation can be unveiled through the analysis of polymorphism and divergence. With this aim, we have surveyed nucleotide sequence variation at the dilp1-7 genes in a natural population of D. melanogaster. The comparison of polymorphism in D. melanogaster and divergence from D. simulans at different functional classes of the dilp genes provided no evidence of adaptive protein evolution after the split of the D. melanogaster and D. simulans lineages. However, our survey of polymorphism at the dilp gene regions of D. melanogaster has provided some evidence for the action of positive selection at or near these genes. The regions encompassing the dilp1-4 genes and the dilp6 gene stand out as likely affected by recent adaptive events.

Photocontrolled DNA Binding of a Receptor-Targeted Organometallic Ruthenium(II) Complex

A photoactivated ruthenium(II) arene complex has been conjugated to two receptor-binding peptides, a dicarba analogue of octreotide and the Arg-Gly-Asp (RGD) tripeptide. These peptides can act as"tumor-targeting devices" since their receptors are overexpressed on the membranes of tumor cells. Both ruthenium-peptide conjugates are stable in aqueous solution in the dark, but upon irradiation with visible light, the pyridyl-derivatized peptides were selectively photodissociated from the ruthenium complex, as inferred by UV-vis and NMR spectroscopy. Importantly, the reactive aqua species generated from the conjugates, [(η6-p-cym)Ru(bpm)(H2O)]2+, reacted with the model DNA nucleobase 9-ethylguanine as well as with guanines of two DNA sequences, 5′dCATGGCT and 5′dAGCCATG. Interestingly, when irradiation was performed in the presence of the oligonucleotides, a new ruthenium adduct involving both guanines was formed as a consequence of the photodriven loss of p-cymene from the two monofunctional adducts. The release of the arene ligand and the formation of a ruthenated product with a multidentate binding mode might have important implications for the biological activity of such photoactivated ruthenium(II) arene complexes. Finally, photoreactions with the peptide-oligonucleotide hybrid, Phac-His-Gly-Met-linker-p5′dCATGGCT, also led to arene release and to guanine adducts, including a GG chelate. The lack of interaction with the peptide fragment confirms the preference of such organometallic ruthenium(II) complexes for guanine over other potential biological ligands, such as histidine or methionine amino acids.

Differential Dimerization of Variants Linked to Enhanced S-Cone Sensitivity Syndrome (ESCS) Located in the NR2E3 Ligand-Binding Domain.

NR2E3 encodes the photoreceptor-specific nuclear hormone receptor that acts as a repressor of cone-specific gene expression in rod photoreceptors, and as an activator of several rod-specific genes. Recessive variants located in the ligand-binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive- (S-) cone syndrome (ESCS), a retinal degeneration characterized by an excess of S-cones and non-functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease-causing LBD missense variants by bioluminescence resonance energy transfer (BRET(2) ) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q, and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P, and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev-erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.

Microscopic-macroscopic approach for binding energies with the Wigner-Kirkwood method. II. Deformed nuclei

The binding energies of deformed even-even nuclei have been analyzed within the framework of a recently proposed microscopic-macroscopic model. We have used the semiclassical Wigner-Kirkwood ̄h expansion up to fourth order, instead of the usual Strutinsky averaging scheme, to compute the shell corrections in a deformed Woods-Saxon potential including the spin-orbit contribution. For a large set of 561 even-even nuclei with Z 8 and N 8, we find an rms deviation from the experiment of 610 keV in binding energies, comparable to the one found for the same set of nuclei using the finite range droplet model of Moller and Nix (656 keV). As applications of our model, we explore its predictive power near the proton and neutron drip lines as well as in the superheavy mass region. Next, we systematically explore the fourth-order Wigner-Kirkwood corrections to the smooth part of the energy. It is found that the ratio of the fourth-order to the second-order corrections behaves in a very regular manner as a function of the asymmetry parameter I=(N−Z)/A. This allows us to absorb the fourth-order corrections into the second-order contributions to the binding energy, which enables us us to simplify and speed up the calculation of deformed nuclei.

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties.

The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.

Sterol regulatory element binding protein-1a transactivates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene promoter

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB)catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key modulator of glycolysis-gluconeogenesis. To gain insight into the molecular mechanism behind hormonal and nutritional regulation of PFKFB expression, we have cloned and characterized the proximal promoter region of the liver isoform of PFKFB (PFKFB1) from gilthead sea bream (Sparus aurata). Transient transfection of HepG2 cells with deleted gene promoter constructs and electrophoretic mobility shift assays allowed us to identify a sterol regulatory element (SRE) to which SRE binding protein-1a (SREBP-1a)binds and transactivates PFKFB1 gene transcription. Mutating the SRE box abolished SREBP-1a binding and transactivation. The in vivo binding of SREBP-1a to the SRE box in the S. aurata PFKFB1 promoter was confirmed by chromatin immunoprecipitation assays. There is a great deal of evidence for a postprandial rise of PFKB1 mRNA levels in fish and rats. Consistently, starved-to-fed transition and treatment with glucose or insulin increased SREBP-1 immunodetectable levels, SREBP-1 association to PFKFB1 promoter, and PFKFB1 mRNA levels in the piscine liver. Our findings demonstrate involvement of SREBP-1a in the transcriptional activation of PFKFB1, and we conclude that SREBP-1a may exert a key role mediating postprandial activation of PFKFB1 transcription.

Allelic diversity and population structure in Vibrio cholerae O139 Bengal based on nucleotide sequence analysis

Comparative analysis of gene fragments of six housekeeping loci, distributed around the two chromosomes of Vibrio cholerae, has been carried out for a collection of 29 V. cholerae O139 Bengal strains isolated from India during the first epidemic period (1992 to 1993). A toxigenic O1 ElTor strain from the seventh pandemic and an environmental non-O1/non-O139 strain were also included in this study. All loci studied were polymorphic, with a small number of polymorphic sites in the sequenced fragments. The genetic diversity determined for our O139 population is concordant with a previous multilocus enzyme electrophoresis study in which we analyzed the same V. cholerae O139 strains. In both studies we have found a higher genetic diversity than reported previously in other molecular studies. The results of the present work showed that O139 strains clustered in several lineages of the dendrogram generated from the matrix of allelic mismatches between the different genotypes, a finding which does not support the hypothesis previously reported that the O139 serogroup is a unique clone. The statistical analysis performed in the V. cholerae O139 isolates suggested a clonal population structure. Moreover, the application of the Sawyer's test and split decomposition to detect intragenic recombination in the sequenced gene fragments did not indicate the existence of recombination in our O139 population.

Sterol regulatory element binding protein-1a transactivates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene promoter

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB)catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key modulator of glycolysis-gluconeogenesis. To gain insight into the molecular mechanism behind hormonal and nutritional regulation of PFKFB expression, we have cloned and characterized the proximal promoter region of the liver isoform of PFKFB (PFKFB1) from gilthead sea bream (Sparus aurata). Transient transfection of HepG2 cells with deleted gene promoter constructs and electrophoretic mobility shift assays allowed us to identify a sterol regulatory element (SRE) to which SRE binding protein-1a (SREBP-1a)binds and transactivates PFKFB1 gene transcription. Mutating the SRE box abolished SREBP-1a binding and transactivation. The in vivo binding of SREBP-1a to the SRE box in the S. aurata PFKFB1 promoter was confirmed by chromatin immunoprecipitation assays. There is a great deal of evidence for a postprandial rise of PFKB1 mRNA levels in fish and rats. Consistently, starved-to-fed transition and treatment with glucose or insulin increased SREBP-1 immunodetectable levels, SREBP-1 association to PFKFB1 promoter, and PFKFB1 mRNA levels in the piscine liver. Our findings demonstrate involvement of SREBP-1a in the transcriptional activation of PFKFB1, and we conclude that SREBP-1a may exert a key role mediating postprandial activation of PFKFB1 transcription.

On the origin of the selectivity of plasmidic H-NS towards horizontally acquired DNA: Linking H-NS oligomerization and cooperative DNA binding

The nucleoid-associated protein H-NS is a global modulator of the expression of genes associated with adaptation to environmental changes. A variant of H-NS expressed in the R27 plasmid was previously shown to selectively modulate the expression of horizontally acquired genes, with minimal effects on core genes that are repressed by the chromosomal form of H-NS. Both H-NS proteins are formed by an oligomerization domain and a DNA-binding domain, which are connected by a linker that is highly flexible in the absence of DNA. We studied DNA binding by means of oligomer-forming chimeric proteins in which domains of the chromosomal and plasmidic variants are exchanged, as well as in monomeric truncated forms containing the DNA-binding domain and variable portions of the linker. Point mutations in the linker were also examined in full-length and truncated H-NS constructs. These experiments show that the linker region contributes to DNA binding affinity and that it is a main component of the distinct DNA binding properties of chromosomal and plasmidic H-NS. We propose that interactions between the linker and DNA limit the flexibility of the connection between H- NS oligomerization and DNA binding and provide an allosteric indirect readout mechanism to detect long- range distortions of DNA, thus enabling discrimination between core and horizontally acquired DNA.

Distinct OGT-Binding Sites Promote HCF-1 Cleavage.

Human HCF-1 (also referred to as HCFC-1) is a transcriptional co-regulator that undergoes a complex maturation process involving extensive O-GlcNAcylation and site-specific proteolysis. HCF-1 proteolysis results in two active, noncovalently associated HCF-1N and HCF-1C subunits that regulate distinct phases of the cell-division cycle. HCF-1 O-GlcNAcylation and site-specific proteolysis are both catalyzed by O-GlcNAc transferase (OGT), which thus displays an unusual dual enzymatic activity. OGT cleaves HCF-1 at six highly conserved 26 amino acid repeat sequences called HCF-1PRO repeats. Here we characterize the substrate requirements for OGT cleavage of HCF-1. We show that the HCF-1PRO-repeat cleavage signal possesses particular OGT-binding properties. The glutamate residue at the cleavage site that is intimately involved in the cleavage reaction specifically inhibits association with OGT and its bound cofactor UDP-GlcNAc. Further, we identify a novel OGT-binding sequence nearby the first HCF-1PRO-repeat cleavage signal that enhances cleavage. These results demonstrate that distinct OGT-binding sites in HCF-1 promote proteolysis, and provide novel insights into the mechanism of this unusual protease activity.