Heterozygous mutations of OTX2 cause severe ocular malformations

Major malformations of the human eye, including microphthalmia and anophthalmia, are examples of phenotypes that recur in families yet often show no clear Mendelian inheritance pattern. Defining loci by mapping is therefore rarely feasible. Using a candidate-gene approach, we have identified heterozygous coding-region changes in the homeobox gene OTX2 in eight families with ocular malformations. The expression pattern of OTX2 in human embryos is consistent with the eye phenotypes observed in the patients, which range from bilateral anophthalmia to retinal defects resembling Leber congenital amaurosis and pigmentary retinopathy. Magnetic resonance imaging scans revealed defects of the optic nerve, optic chiasm, and, in some cases, brain. In two families, the mutations appear to have occurred de novo in severely affected offspring, and, in two other families, the mutations have been inherited from a gonosomal mosaic parent. Data from these four families support a simple model in which OTX2 heterozygous loss-of-function mutations cause ocular malformations. Four additional families display complex inheritance patterns, suggesting that OTX2 mutations alone may not lead to consistent phenotypes. The high incidence of mosaicism and the reduced penetrance have implications for genetic counseling.     

Towards the identification of type II secretion signals in a nonacylated variant of pullulanase from Klebsiella oxytoca

Pullulanase (PulA) from the gram-negative bacterium Klebsiella oxytoca is a 116-kDa surface-anchored lipoprotein of the isoamylase family that allows growth on branched maltodextrin polymers. PulA is specifically secreted via a type II secretion system. PelBsp-PulA, a nonacylated variant of PulA made by replacing the lipoprotein signal peptide (sp) with the signal peptide of pectate lyase PelB from Erwinia chrysanthemi, was efficiently secreted into the medium. Two 80-amino-acid regions of PulA, designated A and B, were previously shown to promote secretion of beta-lactamase (BlaM) and endoglucanase CelZ fused to the C terminus. We show that A and B fused to the PelB signal peptide can also promote secretion of BlaM and CelZ but not that of nuclease NucB or several other reporter proteins. However, the deletion of most of region A or all of region B, either individually or together, had only a minor effect on PelBsp-PulA secretion. Four independent linker insertions between amino acids 234 and 324 in PelBsp-PulA abolished secretion. This part of PulA, region C, could contain part of the PulA secretion signal or be important for its correct presentation. Deletion of region C abolished PelBsp-PulA secretion without dramatically affecting its stability. PelBsp-PulA-NucB chimeras were secreted only if the PulA-NucB fusion point was located downstream from region C. The data show that at least three regions of PulA contain information that influences its secretion, depending on their context, and that some reporter proteins might contribute to the secretion of chimeras of which they are a part.     

Identification of a novel pharmacophore for peptide toxins interacting with K+ channels

KappaM-conotoxin RIIIK blocks TSha1 K+ channels from trout with high affinity by interacting with the ion channel pore. As opposed to many other peptides targeting K+ channels, kappaM-RIIIK does not possess a functional dyad. In this study we combine thermodynamic mutant cycle analysis and docking calculations to derive the binding mode of kappaM-conotoxin RIIIK to the TSha1 channel. The final model reveals a novel pharmacophore, where no positively charged side chain occludes the channel pore. Instead the positive-charged residues of the toxin form a basic ring; kappaM-RIIIK is anchored to the K+ channel via electrostatic interactions of this basic ring with the loop and pore helix residues of the channel. The channel amino acid Glu-354 is likely to be a fundamental determinant of the selectivity of kappaM-RIIIK for the TSha1 channel. The Cgamma-OH of Hyp-15 is in contact with the carbonyls of the selectivity filter, disturbing the charge distribution pattern necessary for the coordination of K+ ions. This novel, experimentally based pharmacophore model proves the existence of diverse binding modes of peptidic toxins to K+ channels and underlines the role of intermolecular electrostatic interactions involving channel loop side chains in determining the selectivity of toxins for specific K+ channel types.     

Alpha-conotoxin BuIA, a novel peptide from Conus bullatus, distinguishes among neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. Alpha subunits, together with beta 2 and/or beta 4 subunits, form ligand-binding sites at alpha/beta subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the alpha-conotoxin signal sequence and 3'-untranslated sequence region, we have cloned a novel gene from the fish-eating snail, Conus bullatus; the gene codes for a previously unreported alpha-conotoxin with unusual 4/4 spacing of amino acids in the two disulfide loops. Chemical synthesis of the predicted mature toxin was performed. The resulting peptide, alpha-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for alpha 3- and chimeric alpha 6-containing nAChRs; BuIA blocks alpha 6/alpha 3 beta 2 nAChRs with a 40,000-fold lower IC(50) than alpha 4 beta 2 nAChRs. The kinetics of toxin unblock are dependent on the beta subunit. nAChRs with a beta 4 subunit have very slow off-times, compared with the corresponding beta 2 subunit-containing nAChR. In each instance, rat alpha x beta 4 may be distinguished from rat alpha x beta 2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse alpha 3 beta 2 to mouse alpha 3 beta 4, and human alpha 3 beta2 to human alpha 3 beta 4, indicating that the beta subunit dependence extends across species. Thus, alpha-conotoxin BuIA also represents a novel probe for distinguishing between beta 2- and beta 4-containing nAChRs.     

The truncated oxygen-avid hemoglobin from Bacillus subtilis: X-ray structure and ligand binding properties

The group II truncated hemoglobin from Bacillus subtilis has been cloned, expressed, purified, and characterized. B. subtilis truncated hemoglobin is a monomeric protein endowed with an unusually high oxygen affinity (in the nanomolar range) such that the apparent thermodynamic binding constant for O2 exceeds that for CO by 1 order of magnitude. The kinetic basis of the high oxygen affinity resides mainly in the very slow rate of ligand release. The extremely stable ferrous oxygenated adduct is resistant to oxidation, which can be achieved only with oxidant in large excess, e.g. ferricyanide in 50-fold molar excess. The three-dimensional crystal structure of the cyano-Met derivative was determined at 2.15 A resolution. Although the overall fold resembles that of other truncated hemoglobins, the distal heme pocket displays a unique array of hydrophilic side chains in the topological positions that dominate the steric interaction with iron-bound ligands. In fact, the Tyr-B10, Thr-E7, and Gln-E11 oxygens on one side of the heme pocket and the Trp-G8 indole NE1 nitrogen on the other form a novel pattern of the "ligand-inclusive hydrogen bond network" described for mycobacterial HbO. On the proximal side, the histidine residue is in an unstrained conformation, and the iron-His bond is unusually short (1.91 A).     

Secretions of interleukin-1beta and tumor necrosis factor alpha by whole fetal membranes depend on initial interactions of amnion or choriodecidua with lipopolysaccharides or group B streptococci

The present study evaluated the secretions of interleukin (IL)-1beta and tumor necrosis factor (TNF) alpha by fetal membranes stimulated with group B streptococci (GBS) and lipopolysaccharide (LPS). The aim was to evaluate the initial response of full-thickness membranes to the microbial insult using an in vitro experimental model that allowed testing of the individual contributions of amnion and choriodecidua to stimulation. Full-thickness membranes were obtained after delivery by elective cesarean section from women at 37-40 wk of gestation without evidence of active labor. The membranes were mounted in Transwell devices, physically separating the upper and lower chambers. The LPS (500 ng/ml) or GBS (1 x 10(6) colony-forming units/ml) was added to either the amniotic or choriodecidual surface, and accumulation of IL-1beta and TNFalpha were measured in both compartments using a specific ELISA. Fetal membranes followed different patterns of secretion of proinflammatory cytokines that depended on the side to which the stimulus was added or the nature of the stimulus itself. The TNFalpha was secreted by amnion and choriodecidua in the presence of LPS or GBS, and stimulation with GBS induced a greater synthesis of IL-1beta than did stimulation with LPS. Choriodecidual tissue was more responsive than amniotic tissue, and this response tended to be higher even when the stimulation was only on the amniotic side. However, the amnion plays an active role in recognizing LPS or GBS, contributing a significant amount of TNFalpha. Thus, cooperative and bidirectional communications occur between amnion and choriodecidua in response to bacterial products, which include intermembranous cytokine traffic and signaling between tissues.     

Inhibition of mitochondrial respiration by the anticancer agent 2-methoxyestradiol

2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have antiproliferative, antiangiogenic, and proapoptotic activity. Mechanistically, 2ME2 has been shown to downregulate hypoxia-inducible factor 1alpha (HIF1alpha) and to induce apoptosis in tumour cells by generating reactive oxygen species (ROS). In this study we report that 2ME2 inhibits mitochondrial respiration in both intact cells and submitochondrial particles, and that this effect is due to inhibition of complex I of the mitochondrial electron transport chain (ETC). The prevention by 2ME2 of hypoxia-induced stabilisation of HIF1alpha in HEK293 cells was found not to be due to an effect on HIF1alpha synthesis but rather to an effect on protein degradation. This is in agreement with our recent observation using other inhibitors of mitochondrial respiration which bring about rapid degradation of HIF1alpha in hypoxia due to increased availability of oxygen and reactivation of prolyl hydroxylases. The concentrations of 2ME2 that inhibited complex I also induced the generation of ROS. 2ME2 did not, however, cause generation of ROS in 143B rho(-) cells, which lack a functional mitochondrial ETC. We conclude that inhibition of mitochondrial respiration explains, at least in part, the effect of 2ME2 on hypoxia-dependent HIF1alpha stabilisation and cellular ROS production. Since these actions of 2ME2 occur at higher concentrations than those known to inhibit cell proliferation, it remains to be established whether they contribute to its therapeutic effect.     

Propeptide does not act as an intramolecular chaperone but facilitates protein disulfide isomerase-assisted folding of a conotoxin precursor

Conotoxins comprise a large and diverse group of peptide neurotoxins derived from Conus snail venoms; most contain multiple disulfide bonds. The conotoxin precursors consist of three distinct domains: the N-terminal signal sequence, an intervening propeptide region, and the C-terminal mature conotoxin. Formation of the native disulfide bonds during the oxidative folding of conotoxins is a prerequisite for their proper biological function, but in numerous in vitro folding experiments with mature conotoxins, a lack of specificity in formation of the native Cys-Cys connectivities is observed. The mechanisms that ensure that the native disulfide bonds are formed in venom ducts during biosynthesis remain unknown. To evaluate whether the propeptide could potentially function as an intramolecular chaperone, we studied the oxidative folding of a conotoxin precursor, pro-GI, belonging to the alpha-conotoxin family. Our results indicate that the propeptide sequence did not directly contribute to folding kinetics and thermodynamics. However, we found that the propeptide region of pro-GI played an important role when oxidative folding was catalyzed by protein disulfide isomerase (PDI). The PDI-assisted reaction was more efficient during the early folding in the context of the propeptide sequence (pro-GI), as compared to that of the mature conotoxin (alpha-GI). Taken together, our results suggest for the first time that the propeptide region may play a role in the PDI-catalyzed oxidative folding of conotoxin precursors.     

Role of cross-talk between IFN-alpha-induced monocyte-derived dendritic cells and NK cells in priming CD8+ T cell responses against human tumor antigens

In the present study we evaluated the role of IFN-alpha in the generation of dendritic cells (IFN-DCs) with priming activity on CD8(+) T lymphocytes directed against human tumor Ags. A 3-day treatment of monocytes, obtained as adherent PBMCs from HLA-A*0201(+) healthy donors, with IFN-alpha and GM-CSF led to the differentiation of DCs displaying a semimature phenotype, but promptly inducing CD8(+) T cell responses after one in vitro sensitization with peptides derived from melanoma (gp100(209-217) and MART-1/Melan-A(27-35)) and adenocarcinoma (CEA(605-613)) Ags. However, these features were lost when IFN-DCs were generated from immunosorted CD14(+) monocytes. The ability of adherent PBMCs to differentiate into IFN-DCs expressing higher levels of costimulatory molecules and exerting efficient T cell priming capacity was associated with the presence of contaminating NK cells, which underwent phenotypic and functional activation upon IFN-alpha treatment. NK cell boost appeared to be mediated by both direct and indirect (i.e., mediated by IFN-DCs) mechanisms. Experiments performed to prove the role of contaminating NK cells in DC differentiation showed that IFN-DCs generated in the absence of NK were phenotypically less mature and could not efficiently prime antitumor CD8(+) lymphocytes. Reciprocally, IFN-DCs raised from immunosorted CD14(+) monocytes regained their T cell priming activity when NK cells were added to the culture before IFN-alpha and GM-CSF treatment. Together, our data suggest that the ability of IFN-DCs to efficiently prime anti-tumor CD8(+) T lymphocytes relied mostly on the positive cross-talk occurring between DCs and NK cells upon stimulation with IFN-alpha.