Crystal structure of human kynurenine aminotransferase II, a drug target for the treatment of schizophrenia

Kynurenic acid is an endogenous neuroactive compound whose unbalancing is involved in the pathogenesis and progression of several neurological diseases. Kynurenic acid synthesis in the human brain is sustained by the catalytic activity of two kynurenine aminotransferases, hKAT I and hKAT II. A wealth of pharmacological data highlight hKAT II as a sensible target for the treatment of neuropathological conditions characterized by a kynurenic acid excess, such as schizophrenia and cognitive impairment. We have solved the structure of human KAT II by means of the single-wavelength anomalous dispersion method at 2.3-A resolution. Although closely resembling the classical aminotransferase fold, the hKAT II architecture displays unique features. Structural comparison with a prototypical aspartate aminotransferase reveals a novel antiparallel strand-loop-strand motif that forms an unprecedented intersubunit beta-sheet in the functional hKAT II dimer. Moreover, the N-terminal regions of hKAT II and aspartate aminotransferase appear to have converged to highly similar although 2-fold symmetry-related conformations, which fulfill the same functional role. A detailed structural comparison of hKAT I and hKAT II reveals a larger and more aliphatic character to the active site of hKAT II due to the absence of the aromatic cage involved in ligand binding in hKAT I. The observed structural differences could be exploited for the rational design of highly selective hKAT II inhibitors.     

Lower expression of TLR2 and SOCS-3 is associated with Schistosoma haematobium infection and with lower risk for allergic reactivity in children living in a rural area in Ghana

Background

Helminth infections are prevalent in rural areas of developing countries and have in some studies been negatively associated with allergic disorders and atopy. In this context little is known of the molecular mechanisms of modulation involved. We have characterized the innate immune responses, at the molecular level, in children according to their helminth infection status and their atopic reactivity to allergens.

Methodology/Principal Findings

The mRNA expression of several genes of the innate immune system that have been associated with microbial exposure and allergy was examined in 120 school children in a rural area in Ghana. Helminth infections were common and atopy rare in the study area. The analysis of gene expression in ex vivo whole blood samples reflected the levels of corresponding proteins. Using this approach in a population of school children in whom the presence of Schistosoma haematobium infection was associated with protection from atopic reactivity, we found that the level of TLR2 and SOCS-3, genes associated with atopy in the children, were significantly downregulated by presence of S. haematobium infection.

Conclusions

S. haematobium infections modulate the expression of genes of the innate immune system (TLR2 and SOCS-3); these are genes that are associated with increased allergic inflammatory processes, providing a molecular link between the negative association of this infection and atopy in rural children in Ghana.     

A molecular phylogeny of the Sylvia cantillans complex: cryptic species within the Mediterranean basin

The subalpine warbler Sylvia cantillans is formally considered a polytypic species, with four subspecies, European S. c. cantillans, albistriata, moltonii (recently resumed name: subalpina) and North African S. c. inornata. They are very similar in external morphology but clearly differ in their vocalizations. We evaluated their uncertain taxonomic status reconstructing the phylogenetic and phylogeographic relationships among populations sampled across major biogeographical areas in the European species’ range, using nucleotide sequences of the mitochondrial cytochrome b gene (mtDNA cyt b). A variety of phylogenetic analyses concordantly led to identify four major groups, only partially corresponding to the three European nominal subspecies. Phylogenetic trees showed a monophyletic group including all moltonii individuals, well diverged from all other taxa. Populations taxonomically assigned to cantillans were polyphyletic being split into two distinct clades (western and southern cantillans), with monophyletic albistriata closely related to southern cantillans. Individuals of moltonii and southern cantillans sampled in sites of sympatry in central Italy were assigned to their respective groups, with perfect concordance between phenotypic and genetic identifications. All findings indicate that moltonii should be ranked as a distinct species. Former subspecies cantillans is polyphyletic, but additional data are needed to define the taxonomic status of its two clades. Albistriata is phylogenetically related to southern cantillans and should be provisionally kept as a subspecies of S. cantillans. The cantillans complex thus provides an interesting case-study illustrating geographical structuring across small geographical ranges, and it exemplifies speciation through differentiation in allopatry leading to reproductive isolation after a secondary contact.     

The Italian External Quality Assessment scheme for fragile x syndrome: the results of a 5-year survey

The Italian External Quality Assessment scheme for fragile X syndrome started in 2001 as an activity funded by the National Health System and coordinated by the National Institute of Public Health. The aim of this work is to present the data of 5 years (2001--2004 and 2006) of survey. The External Quality Assessment scheme was designed to cover the following points: (a) genotyping and (b) interpretation and reporting of results. Overall, the scheme covered about 65% of all Italian public laboratories. The average reporting of results was 91.6%, with an overall success rate of 76%. The rate of diagnostic errors observed was on average 5%. Inaccuracy in sizing of CGG repeats of normal and premutated alleles was reported. During the survey the proportion of laboratories using a Southern blotting, polymerase chain reaction, and ABI sizing kit in combination rose from 36.8% to 70.6%. The reports from laboratories showed incompleteness and considerable variations in expected outcomes. For this reason, in 2004 a model for written reports was introduced. In conclusion, these data underscore the need to participate in External Quality Assessment schemes as an educational resource to ensure quality in molecular genetic testing.     

Coffee oil as a potential feedstock for biodiesel production

A preliminary evaluation of the feasibility of producing biodiesel using oil extracted from defective coffee beans was conducted as an alternative means of utilizing these beans instead of roasting for consumption of beverage with depreciated quality. Direct transesterifications of triglycerides from refined soybean oil (reference) and from oils extracted from healthy and defective coffee beans were performed. Type of alcohol employed and time were the reaction parameters studied. Sodium methoxide was used as alkaline catalyst. There was optimal phase separation after reactions using both soybean and healthy coffee beans oils when methanol was used. This was not observed when using the oil from defective beans which required further processing to obtain purified alkyl esters. Nevertheless, coffee oil was demonstrated to be a potential feedstock for biodiesel production, both from healthy and defective beans, since the corresponding oils were successfully converted to fatty acid methyl and ethyl esters.     

Synthesis, SAR and in vitro evaluation of new cyclic Arg-Gly-Asp pseudopentapeptides containing a s-cis peptide bond as integrin alphavbeta3 and alphavbeta5 ligands

The solid-phase synthesis of two diastereomeric cyclic pseudopeptides containing the Arg-Gly-Asp sequence and the dipeptide isostere 2-amino-3-oxotetrahydro-1H-pyrrolizine-7a(5H)-carboxylic acid (GPTM) is described. Competition binding assays to purified alphavbeta3 and alphavbeta5 integrins with respect to [125I]echistatin showed a high inhibitory activity for the (2S,7aS)-GPTM derivative. Effects of the structural constraint induced by the two enantiomeric scaffolds (2R,7aR)-GPTM and (2S,7aS)-GPTM on the conformation of Arg-Gly-Asp sequence have been computationally investigated using as a reference the recently solved X-ray structure of cyclo(Arg-Gly-Asp-d-Phe-[N-Me]Val) in complex with the extracellular fragment of the alphavbeta3 receptor. The computational method disclosed the key role played by a bridging water molecule on differentiating the two ligands by a diverse stabilization of the ligand-protein complex.     

Molecular interactions of ASPP1 and ASPP2 with the p53 protein family and the apoptotic promoters PUMA and Bax

The apoptosis stimulating p53 proteins, ASPP1 and ASPP2, are the first two common activators of the p53 protein family that selectively enable the latter to regulate specific apoptotic target genes, which facilitates yes yet unknown mechanisms for discrimination between cell cycle arrest and apoptosis. To better understand the interplay between ASPP- and p53-family of proteins we investigated the molecular interactions between them using biochemical methods and structure-based homology modelling. The data demonstrate that: (i) the binding of ASPP1 and ASPP2 to p53, p63 and p73 is direct; (ii) the C-termini of ASPP1 and ASPP2 interact with the DNA-binding domains of p53 protein family with dissociation constants, K_d, in the lower micro-molar range; (iii) the stoichiometry of binding is 1:1; (iv) the DNA-binding domains of p53 family members are sufficient for these protein–protein interactions; (v) EMSA titrations revealed that while tri-complex formation between ASPPs, p53 family of proteins and PUMA/Bax is mutually exclusive, ASPP2 (but not ASPP1) formed a complex with PUMA (but not Bax) and displaced p53 and p73. The structure-based homology modelling revealed subtle differences between ASPP2 and ASPP1 and together with the experimental data provide novel mechanistic insights.     

Thiol-modifying inhibitors for understanding squalene cyclase function.

The function of squalene-hopene cyclase from Alicyclobacillus acidocaldarius was studied by labelling critical cysteine residues of the enzyme, either native or inserted by site-directed mutagenesis, with different thiol-reacting molecules. The access of the substrate to the active centre cavity through a nonpolar channel that contains a narrow constriction harbouring a cysteine residue (C435) was probed by labelling experiments on both a C435S mutant, lacking C435 of the channel constriction, and a C25S/C50S/C455S/C537S mutant, bearing C435 as the only cysteine residue. Labelling experiments with tritiated 3-carboxy-4-nitrophenyl-dithio-1,1',2-trisnorsqualene (CNDT-squalene) showed that the cysteine residue at the channel constriction was covalently modified by the squalene-like inhibitor. Time-dependent inactivation of the C25S/C50S/C455S/C537S mutant by a number of squalene analogues and other agents with thiol-modifying activity suggested that modifying C435 caused the obstruction of the channel constriction thus blocking access of the substrate to the active site. The tryptic fragment comprising C435 of the quadruple mutant labelled with the most effective inhibitor had the expected altered molecular mass, as determined by LC-ESI-MS measurements. The arrangement of the substrate in the active site cavity was studied by using thiol reagents as probes in labelling experiments with the double mutant D376C/C435S in which D376, supposedly the substrate-protonating residue, was substituted by cysteine. The inhibitory effect was evaluated in terms of the reduced ability to cyclize oxidosqualene, as the mutant is unable to catalyse the reaction of squalene to hopene. Among the inhibitors tested, the substrate analogue squalene-maleimide proved to be a very effective time-dependent inhibitor.