Host S-nitrosylation inhibits clostridial small molecule-activated glucosylating toxins

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins. Virulence is dependent on the autoactivation of a toxin cysteine protease, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP(6)). Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP(6)- and inositol pyrophosphate (InsP(7))-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP(6) enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.     

Engineering toxins for 21st century therapies

'Engineering Toxins for 21st Century Therapies' (9-10 September 2010) was part of the Royal Society International Seminar series held at the Kavli International Centre, UK. Participants were assembled from a range of disciplines (academic, industry, regulatory, public health) to discuss the future potential of toxin-based therapies. The meeting explored how the current structural and mechanistic knowledge of toxins could be used to engineer future toxin-based therapies. To date, significant progress has been made in the design of novel recombinant biologics based on domains of natural toxins, engineered to exhibit advantageous properties. The meeting concluded, firstly that future product development vitally required the appropriate combination of creativity and innovation that can come from the academic, biotechnology and pharma sectors. Second, that continued investigation into understanding the basic science of the toxins and their targets was essential in order to develop new opportunities for the existing products and to create new products with enhanced properties. Finally, it was concluded that the clinical potential for development of novel biologics based on toxin domains was evident.     

Structural characterization of angiotensin I-converting enzyme in complex with a selenium analogue of captopril

Human somatic angiotensin I-converting enzyme (ACE), a zinc-dependent dipeptidyl carboxypeptidase, is central to the regulation of the renin-angiotensin aldosterone system. It is a well-known target for combating hypertension and related cardiovascular diseases. In a recent study by Bhuyan and Mugesh [Org. Biomol. Chem. (2011) 9, 1356-1365], it was shown that the selenium analogues of captopril (a well-known clinical inhibitor of ACE) not only inhibit ACE, but also protect against peroxynitrite-mediated nitration of peptides and proteins. Here, we report the crystal structures of human testis ACE (tACE) and a homologue of ACE, known as AnCE, from Drosophila melanogaster in complex with the most promising selenium analogue of captopril (SeCap) determined at 2.4 and 2.35 ? resolution, respectively. The inhibitor binds at the active site of tACE and AnCE in an analogous fashion to that observed for captopril and provide the first examples of a protein-selenolate interaction. These new structures of tACE-SeCap and AnCE-SeCap inhibitor complexes presented here provide important information for further exploration of zinc coordinating selenium-based ACE inhibitor pharmacophores with significant antioxidant activity.     

Functional and structural analyses of N-acylsulfonamide-linked dinucleoside inhibitors of RNase A

Molecular probes are useful for both studying and controlling the functions of enzymes and other proteins. The most useful probes have high affinity for their target, along with small size and resistance to degradation. Here, we report on new surrogates for nucleic acids that fulfill these criteria. Isosteres in which phosphoryl [R-O-P(O(2)(-))-O-R'] groups are replaced with N-acylsulfonamidyl [R-C(O)-N(-)-S(O(2))-R'] or sulfonimidyl [R-S(O(2))-N(-)-S(O(2))-R'] groups increase the number of nonbridging oxygens from two (phosphoryl) to three (N-acylsulfonamidyl) or four (sulfonimidyl). Six such isosteres were found to be more potent inhibitors of catalysis by bovine pancreatic RNase A than are parent compounds containing phosphoryl groups. The atomic structures of two RNase A·N-acylsulfonamide complexes were determined at high resolution by X-ray crystallography. The N-acylsulfonamidyl groups were observed to form more hydrogen bonds with active site residues than did the phosphoryl groups in analogous complexes. These data encourage the further development and use of N-acylsulfonamides and sulfonimides as antagonists of nucleic acid-binding proteins.     

Clinical proteomics of the neglected human malarial parasite Plasmodium vivax

Recent reports highlight the severity and the morbidity of disease caused by the long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties in the laboratory-propagation of P. vivax, the biology of this parasite has not been adequately explored. While the proteome of P. falciparum, the causative agent of cerebral malaria, has been extensively explored from several sources, there is limited information on the proteome of P. vivax. We have, for the first time, examined the proteome of P. vivax isolated directly from patients without adaptation to laboratory conditions. We have identified 153 proteins from clinical P. vivax, majority of which do not show homology to any previously known gene products. We also report 29 new proteins that were found to be expressed in P. vivax for the first time. In addition, several proteins previously implicated as anti-malarial targets, were also found in our analysis. Most importantly, we found several unique proteins expressed by P. vivax.This study is an important step in providing insight into physiology of the parasite under clinical settings.     

Molecular recognition of human eosinophil-derived neurotoxin (RNase 2) by placental ribonuclease inhibitor

Placental ribonuclease inhibitor (RI) binds diverse mammalian RNases with dissociation constants that are in the femtomolar range. Previous studies on the complexes of RI with RNase A and angiogenin revealed that RI utilises largely distinctive interactions to achieve high affinity for these two ligands. Here we report a 2.0 angstroms resolution crystal structure of RI in complex with a third ligand, eosinophil-derived neurotoxin (EDN), and a mutational analysis based on this structure. The RI-EDN interface is more extensive than those of the other two complexes and contains a considerably larger set of interactions. Few of the contacts present in the RI-angiogenin complex are replicated; the correspondence to the RI-RNase A complex is somewhat greater, but still modest. The energetic contributions of various interface regions differ strikingly from those in the earlier complexes. These findings provide insight into the structural basis for the unusual combination of high avidity and relaxed stringency that RI displays.     

Neuroprotective effect of Azadirachta indica on cerebral post-ischemic reperfusion and hypoperfusion in rats

We assessed the effect of Azadirachta indica (A. indica), a plant that has been reported to possess antioxidant, anti-inflammatory and anxiolytic properties, on cerebral reperfusion injury and long term cerebral hypoperfusion. When blood flow to brain region that has undergone critical period of ischemia is re-established, additional injury is to be expected from the reperfusion. In the present study, bilateral common carotid artery (BCCA) occlusion for 30 min followed by 45 min reperfusion resulted in increase in lipid peroxidation, superoxide dismutase (SOD) activity and fall in total tissue sulfhydryl (T-SH) groups. A. indica pretreatment (500 mg/kg/day x 7 days) attenuated the reperfusion induced enhanced lipid peroxidation, SOD activity and prevented fall in T-SH groups. Moreover, A.indica per se increased brain ascorbic acid level, which was unchanged during reperfusion insult. Long-term cerebral hypoperfusion induced by permanent BCCA occlusion has been reported to cause behavioral and histopathological abnormalities. In the present study, as tested by open field paradigm and Morris' water maze, a propensity towards anxiety and disturbances of learning/memory were observed in animals subjected to hypoperfusion for 2 weeks. A. indica (500 mg/kg/day x 15 days) significantly reduced these hypoperfusion induced functional disturbances. Reactive changes in brain histology like gliosis, perivascular lymphocytic infiltration, recruitment of macrophages and cellular edema following long term hypoperfusion were also attenuated effectively by A. indica. We conclude that our study provides an experimental evidence for possible neuroprotective potentiality of A. indica.     

Clustering of diverse genomic data using information fusion

MOTIVATION: Genome sequencing projects and high-through-put technologies like DNA and Protein arrays have resulted in a very large amount of information-rich data. Microarray experimental data are a valuable, but limited source for inferring gene regulation mechanisms on a genomic scale. Additional information such as promoter sequences of genes/DNA binding motifs, gene ontologies, and location data, when combined with gene expression analysis can increase the statistical significance of the finding. This paper introduces a machine learning approach to information fusion for combining heterogeneous genomic data. The algorithm uses an unsupervised joint learning mechanism that identifies clusters of genes using the combined data. RESULTS: The correlation between gene expression time-series patterns obtained from different experimental conditions and the presence of several distinct and repeated motifs in their upstream sequences is examined here using publicly available yeast cell-cycle data. The results show that the combined learning approach taken here identifies correlated genes effectively. The algorithm provides an automated clustering method, but allows the user to specify apriori the influence of each data type on the final clustering using probabilities. AVAILABILITY: Software code is available by request from the first author. CONTACT: jkasturi@cse.psu.edu.     

Binding of non-natural 3'-nucleotides to ribonuclease A

2'-Fluoro-2'-deoxyuridine 3'-phosphate (dU(F)MP) and arabinouridine 3'-phosphate (araUMP) have non-natural furanose rings. dU(F)MP and araUMP were prepared by chemical synthesis and found to have three- to sevenfold higher affinity than uridine 3'-phosphate (3'-UMP) or 2'-deoxyuridine 3'-phosphate (dUMP) for ribonuclease A (RNase A). These differences probably arise (in part) from the phosphoryl groups of 3'-UMP, dU(F)MP, and araUMP (pK(a) = 5.9) being more anionic than that of dUMP (pK(a) = 6.3). The three-dimensional structures of the crystalline complexes of RNase A with dUMP, dU(F)MP and araUMP were determined at < 1.7 A resolution by X-ray diffraction analysis. In these three structures, the uracil nucleobases and phosphoryl groups bind to the enzyme in a nearly identical position. Unlike 3'-UMP and dU(F)MP, dUMP and araUMP bind with their furanose rings in the preferred pucker. In the RNase A.araUMP complex, the 2'-hydroxyl group is exposed to the solvent. All four 3'-nucleotides bind more tightly to wild-type RNase A than to its T45G variant, which lacks the residue that interacts most closely with the uracil nucleobase. These findings illuminate in atomic detail the interaction of RNase A and 3'-nucleotides, and indicate that non-natural furanose rings can serve as the basis for more potent inhibitors of catalysis by RNase A.