Molecular Mechanism of Action of Antimalarial Benzoisothiazolones: Species-Selective Inhibitors of the Plasmodium spp. MEP Pathway enzyme, IspD

The methylerythritol phosphate (MEP) pathway is an essential metabolic pathway found in malaria parasites, but absent in mammals, making it a highly attractive target for the discovery of novel and selective antimalarial therapies. Using high-throughput screening, we have identified 2-phenyl benzo[d]isothiazol-3(2H)-ones as species-selective inhibitors of Plasmodium spp. 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD), the third catalytic enzyme of the MEP pathway. 2-Phenyl benzo[d]isothiazol-3(2H)-ones display nanomolar inhibitory activity against P. falciparum and P. vivax IspD and prevent the growth of P. falciparum in culture, with EC50 values below 400 nM. In silico modeling, along with enzymatic, genetic and crystallographic studies, have established a mechanism-of-action involving initial non-covalent recognition of inhibitors at the IspD binding site, followed by disulfide bond formation through attack of an active site cysteine residue on the benzo[d]isothiazol-3(2H)-one core. The species-selective inhibitory activity of these small molecules against Plasmodium spp. IspD and cultured parasites suggests they have potential as lead compounds in the pursuit of novel drugs to treat malaria

Protein Complex Evolution Does Not Involve Extensive Network Rewiring

The formation of proteins into stable protein complexes plays a fundamental role in the operation of the cell. The study of the degree of evolutionary conservation of protein complexes between species and the evolution of protein-protein interactions has been hampered by lack of comprehensive coverage of the high-throughput (HTP) technologies that measure the interactome. We show that new high-throughput datasets on protein co-purification in yeast have a substantially lower false negative rate than previous datasets when compared to known complexes. These datasets are therefore more suitable to estimate the conservation of protein complex membership than hitherto possible. We perform comparative genomics between curated protein complexes from human and the HTP data in Saccharomyces cerevisiae to study the evolution of co-complex memberships. This analysis revealed that out of the 5,960 protein pairs that are part of the same complex in human, 2,216 are absent because both proteins lack an ortholog in S. cerevisiae, while for 1,828 the co-complex membership is disrupted because one of the two proteins lacks an ortholog. For the remaining 1,916 protein pairs, only 10% were never co-purified in the large-scale experiments. This implies a conservation level of co-complex membership of 90% when the genes coding for the protein pairs that participate in the same protein complex are also conserved. We conclude that the evolutionary dynamics of protein complexes are, by and large, not the result of network rewiring (i.e. acquisition or loss of co-complex memberships), but mainly due to genomic acquisition or loss of genes coding for subunits. We thus reveal evidence for the tight interrelation of genomic and network evolution

Genome-Wide Profiling of H3K56 Acetylation and Transcription Factor Binding Sites in Human Adipocytes

The growing epidemic of obesity and metabolic diseases calls for a better understanding of adipocyte biology. The regulation of transcription in adipocytes is particularly important, as it is a target for several therapeutic approaches. Transcriptional outcomes are influenced by both histone modifications and transcription factor binding. Although the epigenetic states and binding sites of several important transcription factors have been profiled in the mouse 3T3-L1 cell line, such data are lacking in human adipocytes. In this study, we identified H3K56 acetylation sites in human adipocytes derived from mesenchymal stem cells. H3K56 is acetylated by CBP and p300, and deacetylated by SIRT1, all are proteins with important roles in diabetes and insulin signaling. We found that while almost half of the genome shows signs of H3K56 acetylation, the highest level of H3K56 acetylation is associated with transcription factors and proteins in the adipokine signaling and Type II Diabetes pathways. In order to discover the transcription factors that recruit acetyltransferases and deacetylases to sites of H3K56 acetylation, we analyzed DNA sequences near H3K56 acetylated regions and found that the E2F recognition sequence was enriched. Using chromatin immunoprecipitation followed by high-throughput sequencing, we confirmed that genes bound by E2F4, as well as those by HSF-1 and C/EBPα, have higher than expected levels of H3K56 acetylation, and that the transcription factor binding sites and acetylation sites are often adjacent but rarely overlap. We also discovered a significant difference between bound targets of C/EBPα in 3T3-L1 and human adipocytes, highlighting the need to construct species-specific epigenetic and transcription factor binding site maps. This is the first genome-wide profile of H3K56 acetylation, E2F4, C/EBPα and HSF-1 binding in human adipocytes, and will serve as an important resource for better understanding adipocyte transcriptional regulation.Singapore. Agency for Science, Technology and Research (National Science Scholarship )Massachusetts Institute of Technology (Eugene Bell Career Development Chair)National Science Foundation (U.S.) (Award No. DBI-0821391)Pfizer Inc

Structure-Function Analysis of Human TYW2 Enzyme Required for the Biosynthesis of a Highly Modified Wybutosine (yW) Base in Phenylalanine-tRNA

Posttranscriptional modifications are critical for structure and function of tRNAs. Wybutosine (yW) and its derivatives are hyper-modified guanosines found at the position 37 of eukaryotic and archaeal tRNAPhe. TYW2 is an enzyme that catalyzes α-amino-α-carboxypropyl transfer activity at the third step of yW biogenesis. Using complementation of a ΔTYW2 strain, we demonstrate here that human TYW2 (hTYW2) is active in yeast and can synthesize the yW of yeast tRNAPhe. Structure-guided analysis identified several conserved residues in hTYW2 that interact with S-adenosyl-methionine (AdoMet), and mutation studies revealed that K225 and E265 are critical residues for the enzymatic activity. We previously reported that the human TYW2 is overexpressed in breast cancer. However, no difference in the tRNAPhe modification status was observed in either normal mouse tissue or a mouse tumor model that overexpresses Tyw2, indicating that hTYW2 may have a role in tumorigenesis unrelated to yW biogenesis

An exceptional horizontal gene transfer in plastids: gene replacement by a distant bacterial paralog and evidence that haptophyte and cryptophyte plastids are sisters

BACKGROUND: Horizontal gene transfer (HGT) to the plant mitochondrial genome has recently been shown to occur at a surprisingly high rate; however, little evidence has been found for HGT to the plastid genome, despite extensive sequencing. In this study, we analyzed all genes from sequenced plastid genomes to unearth any neglected cases of HGT and to obtain a measure of the overall extent of HGT to the plastid. RESULTS: Although several genes gave strongly supported conflicting trees under certain conditions, we are confident of HGT in only a single case beyond the rubisco HGT already reported. Most of the conflicts involved near neighbors connected by long branches (e.g. red algae and their secondary hosts), where phylogenetic methods are prone to mislead. However, three genes – clpP, ycf2, and rpl36 – provided strong support for taxa moving far from their organismal position. Further taxon sampling of clpP and ycf2 resulted in rejection of HGT due to long-branch attraction and a serious error in the published plastid genome sequence of Oenothera elata, respectively. A single new case, a bacterial rpl36 gene transferred into the ancestor of the cryptophyte and haptophyte plastids, appears to be a true HGT event. Interestingly, this rpl36 gene is a distantly related paralog of the rpl36 type found in other plastids and most eubacteria. Moreover, the transferred gene has physically replaced the native rpl36 gene, yet flanking genes and intergenic regions show no sign of HGT. This suggests that gene replacement somehow occurred by recombination at the very ends of rpl36, without the level and length of similarity normally expected to support recombination. CONCLUSION: The rpl36 HGT discovered in this study is of considerable interest in terms of both molecular mechanism and phylogeny. The plastid acquisition of a bacterial rpl36 gene via HGT provides the first strong evidence for a sister-group relationship between haptophyte and cryptophyte plastids to the exclusion of heterokont and alveolate plastids. Moreover, the bacterial gene has replaced the native plastid rpl36 gene by an uncertain mechanism that appears inconsistent with existing models for the recombinational basis of gene conversion

Strongly exchange-coupled triplet pairs in an organic semiconductor

From biological complexes to devices based on organic semiconductors, spin interactions play a key role in the function of molecular systems. For instance, triplet-pair reactions impact operation of organic light-emitting diodes as well as photovoltaic devices. Conventional models for triplet pairs assume they interact only weakly. Here, using electron spin resonance, we observe long-lived, strongly-interacting triplet pairs in an organic semiconductor, generated via singlet fission. Using coherent spin-manipulation of these two-triplet states, we identify exchange-coupled (spin-2) quintet complexes co-existing with weakly coupled (spin-1) triplets. We measure strongly coupled pairs with a lifetime approaching 3 µs and a spin coherence time approaching 1 µs, at 10 K. Our results pave the way for the utilization of high-spin systems in organic semiconductors.Gates-Cambridge Trust, Winton Programme for the Physics of Sustainability, Freie Universität Berlin within the Excellence Initiative of the German Research Foundation, Engineering and Physical Sciences Research Council (Grant ID: EP/G060738/1)This is the author accepted manuscript. The final version is available from Nature Publishing Group at http://dx.doi.org/10.1038/nphys3908

Transcriptional Homeostasis of a Mangrove Species, Ceriops tagal, in Saline Environments, as Revealed by Microarray Analysis

<div><h3>Background</h3><p>Differential responses to the environmental stresses at the level of transcription play a critical role in adaptation. Mangrove species compose a dominant community in intertidal zones and form dense forests at the sea-land interface, and although the anatomical and physiological features associated with their salt-tolerant lifestyles have been well characterized, little is known about the impact of transcriptional phenotypes on their adaptation to these saline environments.</p> <h3>Methodology and Principal findings</h3><p>We report the time-course transcript profiles in the roots of a true mangrove species, <em>Ceriops tagal</em>, as revealed by a series of microarray experiments. The expression of a total of 432 transcripts changed significantly in the roots of <em>C. tagal</em> under salt shock, of which 83 had a more than 2-fold change and were further assembled into 59 unigenes. Global transcription was stable at the early stage of salt stress and then was gradually dysregulated with the increased duration of the stress. Importantly, a pair-wise comparison of predicted homologous gene pairs revealed that the transcriptional regulations of most of the differentially expressed genes were highly divergent in <em>C. tagal</em> from that in salt-sensitive species, <em>Arabidopsis thaliana</em>.</p> <h3>Conclusions/Significance</h3><p>This work suggests that transcriptional homeostasis and specific transcriptional regulation are major events in the roots of <em>C. tagal</em> when subjected to salt shock, which could contribute to the establishment of adaptation to saline environments and, thus, facilitate the salt-tolerant lifestyle of this mangrove species. Furthermore, the candidate genes underlying the adaptation were identified through comparative analyses. This study provides a foundation for dissecting the genetic basis of the adaptation of mangroves to intertidal environments.</p> </div

Multilayered epithelium in a rat model and human Barrett's esophagus: Similar expression patterns of transcription factors and differentiation markers

<p>Abstract</p> <p>Background</p> <p>In rats, esophagogastroduodenal anastomosis (EGDA) without concomitant chemical carcinogen treatment leads to gastroesophageal reflux disease, multilayered epithelium (MLE, a presumed precursor in intestinal metaplasia), columnar-lined esophagus, dysplasia, and esophageal adenocarcinoma. Previously we have shown that columnar-lined esophagus in EGDA rats resembled human Barrett's esophagus (BE) in its morphology, mucin features and expression of differentiation markers (<it>Lab. Invest. 2004;84:753–765</it>). The purpose of this study was to compare the phenotype of rat MLE with human MLE, in order to gain insight into the nature of MLE and its potential role in the development of BE.</p> <p>Methods</p> <p>Serial sectioning was performed on tissue samples from 32 EGDA rats and 13 patients with established BE. Tissue sections were immunohistochemically stained for a variety of transcription factors and differentiation markers of esophageal squamous epithelium and intestinal columnar epithelium.</p> <p>Results</p> <p>We detected MLE in 56.3% (18/32) of EGDA rats, and in all human samples. As expected, both rat and human squamous epithelium, but not intestinal metaplasia, expressed squamous transcription factors and differentiation markers (p63, Sox2, CK14 and CK4) in all cases. Both rat and human intestinal metaplasia, but not squamous epithelium, expressed intestinal transcription factors and differentiation markers (Cdx2, GATA4, HNF1α, villin and Muc2) in all cases. Rat MLE shared expression patterns of Sox2, CK4, Cdx2, GATA4, villin and Muc2 with human MLE. However, p63 and CK14 were expressed in a higher proportion of rat MLE compared to humans.</p> <p>Conclusion</p> <p>These data indicate that rat MLE shares similar properties to human MLE in its expression pattern of these markers, not withstanding small differences, and support the concept that MLE may be a transitional stage in the metaplastic conversion of squamous to columnar epithelium in BE.</p

Toxicity Testing in the 21st Century: Defining New Risk Assessment Approaches Based on Perturbation of Intracellular Toxicity Pathways

The approaches to quantitatively assessing the health risks of chemical exposure have not changed appreciably in the past 50 to 80 years, the focus remaining on high-dose studies that measure adverse outcomes in homogeneous animal populations. This expensive, low-throughput approach relies on conservative extrapolations to relate animal studies to much lower-dose human exposures and is of questionable relevance to predicting risks to humans at their typical low exposures. It makes little use of a mechanistic understanding of the mode of action by which chemicals perturb biological processes in human cells and tissues. An alternative vision, proposed by the U.S. National Research Council (NRC) report Toxicity Testing in the 21st Century: A Vision and a Strategy, called for moving away from traditional high-dose animal studies to an approach based on perturbation of cellular responses using well-designed in vitro assays. Central to this vision are (a) “toxicity pathways” (the innate cellular pathways that may be perturbed by chemicals) and (b) the determination of chemical concentration ranges where those perturbations are likely to be excessive, thereby leading to adverse health effects if present for a prolonged duration in an intact organism. In this paper we briefly review the original NRC report and responses to that report over the past 3 years, and discuss how the change in testing might be achieved in the U.S. and in the European Union (EU). EU initiatives in developing alternatives to animal testing of cosmetic ingredients have run very much in parallel with the NRC report. Moving from current practice to the NRC vision would require using prototype toxicity pathways to develop case studies showing the new vision in action. In this vein, we also discuss how the proposed strategy for toxicity testing might be applied to the toxicity pathways associated with DNA damage and repair