Structure determination of T cell protein-tyrosine phosphatase

Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co-crystallize TC-PTP with the same set of inhibitors. This seems to be due to a multimerization process where residues 130-132, the DDQ loop, from one molecule is inserted into the active site of the neighboring molecule, resulting in a continuous string of interacting TC-PTP molecules. Importantly, despite the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme.     

A cell-free system for light-dependent nuclear import of phytochrome

Translocation from the cytosol to the nucleus is an essential step in phytochrome (phy) signal transduction. In the case of phytochrome A (phyA), this step occurs with the help of FHY1 (far-red-elongated hypocotyl 1), a specific transport protein. To investigate the components involved in phyA transport, we used a cell-free system that facilitates the controlled addition of transport factors. For this purpose, we isolated nuclei from the unicellular green algae Acetabularia acetabulum . These nuclei are up to 100 μm in diameter and allow easy detection of imported proteins. Experiments with isolated nuclei of Acetabularia showed that FHY1 is sufficient for phyA transport. The reconstituted system demonstrates all the characteristics of phytochrome transport in Arabidopsis thaliana . In addition, FHY1 was also actively exported from the nucleus, consistent with its role as a shuttle protein in plants. Therefore, we believe that isolated Acetabularia nuclei may be used as a general tool to study nuclear transport of plant proteins.     

RIG‐I detects infection with live Listeria by sensing secreted bacterial nucleic acids

Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill‐defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG‐I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG‐I‐dependent IL‐1β‐production and inflammasome activation. The signalling molecule CARD9 contributed to IL‐1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG‐I provides a mechanistic explanation for efficient induction of immunity by live bacteria.     

Seasonal Changes in the Content of Urotensin II in the Urophysis of the Goby,Gillichthys mirabilis

A seasonal study of urotensin II content of the urophysis of the goby, Gillichthys mirabilis. was conducted from March 1979 to June 1980 in relation to certain internal and environmental changes. Urotensin II content (lowest in November–January) is inversely correlated with female gonadosomatic index and to some extent with rainfall (and hence dilution of the environmental salinity). In addition, there appears to be a direct correlation between UII content and daylength and temperature.     

The Drosophila L1CAM homolog Neuroglian signals through distinct pathways to control different aspects of mushroom body axon development

The spatiotemporal integration of adhesion and signaling during neuritogenesis is an important prerequisite for the establishment of neuronal networks in the developing brain. In this study, we describe the role of the L1-type CAM Neuroglian protein (NRG) in different steps of Drosophila mushroom body (MB) neuron axonogenesis. Selective axon bundling in the peduncle requires both the extracellular and the intracellular domain of NRG. We uncover a novel role for the ZO-1 homolog Polychaetoid (PYD) in axon branching and in sister branch outgrowth and guidance downstream of the neuron-specific isoform NRG-180. Furthermore, genetic analyses show that the role of NRG in different aspects of MB axonal development not only involves PYD, but also TRIO, SEMA-1A and RAC1.     

N-Arylmethyl substituted iminoribitol derivatives as inhibitors of a purine specific nucleoside hydrolase

A key enzyme within the purine salvage pathway of parasites, nucleoside hydrolase, is proposed as a good target for new antiparasitic drugs. We have developed N-arylmethyl-iminoribitol derivatives as a novel class of inhibitors against a purine specific nucleoside hydrolase from Trypanosoma vivax. Several of our inhibitors exhibited low nanomolar activity, with 1,4-dideoxy-1,4-imino-N-(8-quinolinyl)methyl-d-ribitol (UAMC-00115, K(i) 10.8nM), N-(9-deaza-adenin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (K(i) 4.1nM), and N-(9-deazahypoxanthin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (K(i) 4.4nM) being the three most active compounds. Docking studies of the most active inhibitors revealed several important interactions with the enzyme. Among these interactions are aromatic stacking of the nucleobase mimic with two Trp-residues, and hydrogen bonds between the hydroxyl groups of the inhibitors and amino acid residues in the active site. During the course of these docking studies we also identified a strong interaction between the Asp40 residue from the enzyme and the inhibitor. This is an interaction which has not previously been considered as being important.     

From microbial gene essentiality to novel antimicrobial drug targets

Background

Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials.

Result

Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept.

Conclusion

Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-958) contains supplementary material, which is available to authorized users.