Cyclosporin A Associated Helicase-Like Protein Facilitates the Association of Hepatitis C Virus RNA Polymerase with Its Cellular Cyclophilin B

BACKGROUND: Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology

Global Analysis of Dynamical Decision-Making Models through Local Computation around the Hidden Saddle

Bistable dynamical switches are frequently encountered in mathematical modeling of biological systems because binary decisions are at the core of many cellular processes. Bistable switches present two stable steady-states, each of them corresponding to a distinct decision. In response to a transient signal, the system can flip back and forth between these two stable steady-states, switching between both decisions. Understanding which parameters and states affect this switch between stable states may shed light on the mechanisms underlying the decision-making process. Yet, answering such a question involves analyzing the global dynamical (i.e., transient) behavior of a nonlinear, possibly high dimensional model. In this paper, we show how a local analysis at a particular equilibrium point of bistable systems is highly relevant to understand the global properties of the switching system. The local analysis is performed at the saddle point, an often disregarded equilibrium point of bistable models but which is shown to be a key ruler of the decision-making process. Results are illustrated on three previously published models of biological switches: two models of apoptosis, the programmed cell death and one model of long-term potentiation, a phenomenon underlying synaptic plasticity

Nucleosome-coupled expression differences in closely-related species

<p>Abstract</p> <p>Background</p> <p>Genome-wide nucleosome occupancy is negatively related to the average level of transcription factor motif binding based on studies in yeast and several other model organisms. The degree to which nucleosome-motif interactions relate to phenotypic changes across species is, however, unknown.</p> <p>Results</p> <p>We address this challenge by generating nucleosome positioning and cell cycle expression data for <it>Saccharomyces bayanus </it>and show that differences in nucleosome occupancy reflect cell cycle expression divergence between two yeast species, <it>S. bayanus </it>and <it>S. cerevisiae</it>. Specifically, genes with nucleosome-depleted MBP1 motifs upstream of their coding sequence show periodic expression during the cell cycle, whereas genes with nucleosome-shielded motifs do not. In addition, conserved cell cycle regulatory motifs across these two species are more nucleosome-depleted compared to those that are not conserved, suggesting that the degree of conservation of regulatory sites varies, and is reflected by nucleosome occupancy patterns. Finally, many changes in cell cycle gene expression patterns across species can be correlated to changes in nucleosome occupancy on motifs (rather than to the presence or absence of motifs).</p> <p>Conclusions</p> <p>Our observations suggest that alteration of nucleosome occupancy is a previously uncharacterized feature related to the divergence of cell cycle expression between species.</p

Endoreplication Controls Cell Fate Maintenance

Cell-fate specification is typically thought to precede and determine cell-cycle regulation during differentiation. Here we show that endoreplication, also known as endoreduplication, a specialized cell-cycle variant often associated with cell differentiation but also frequently occurring in malignant cells, plays a role in maintaining cell fate. For our study we have used Arabidopsis trichomes as a model system and have manipulated endoreplication levels via mutants of cell-cycle regulators and overexpression of cell-cycle inhibitors under a trichome-specific promoter. Strikingly, a reduction of endoreplication resulted in reduced trichome numbers and caused trichomes to lose their identity. Live observations of young Arabidopsis leaves revealed that dedifferentiating trichomes re-entered mitosis and were re-integrated into the epidermal pavement-cell layer, acquiring the typical characteristics of the surrounding epidermal cells. Conversely, when we promoted endoreplication in glabrous patterning mutants, trichome fate could be restored, demonstrating that endoreplication is an important determinant of cell identity. Our data lead to a new model of cell-fate control and tissue integrity during development by revealing a cell-fate quality control system at the tissue level

Prediction of Human Disease Genes by Human-Mouse Conserved Coexpression Analysis

One of the most limiting aspects of biological research in the post-genomic era is the capability to integrate massive datasets on gene structure and function for producing useful biological knowledge. In this report we have applied an integrative approach to address the problem of identifying likely candidate genes within loci associated with human genetic diseases. Despite the recent progress in sequencing technologies, approaching this problem from an experimental perspective still represents a very demanding task, because the critical region may typically contain hundreds of positional candidates. We found that by concentrating only on genes sharing similar expression profiles in both human and mouse, massive microarray datasets can be used to reliably identify disease-relevant relationships among genes. Moreover, we found that integrating the coexpression criterion with systematic phenome analysis allows efficient identification of disease genes in large genomic regions. Using this approach on 850 OMIM loci characterized by unknown molecular basis, we propose high-probability candidates for 81 genetic diseases

Combining ChIP-chip and Expression Profiling to Model the MoCRZ1 Mediated Circuit for Ca2+/Calcineurin Signaling in the Rice Blast Fungus

Significant progress has been made in defining the central signaling networks in many organisms, but collectively we know little about the downstream targets of these networks and the genes they regulate. To reconstruct the regulatory circuit of calcineurin signal transduction via MoCRZ1, a Magnaporthe oryzae C2H2 transcription factor activated by calcineurin dephosphorylation, we used a combined approach of chromatin immunoprecipitation - chip (ChIP-chip), coupled with microarray expression studies. One hundred forty genes were identified as being both a direct target of MoCRZ1 and having expression concurrently differentially regulated in a calcium/calcineurin/MoCRZ1 dependent manner. Highly represented were genes involved in calcium signaling, small molecule transport, ion homeostasis, cell wall synthesis/maintenance, and fungal virulence. Of particular note, genes involved in vesicle mediated secretion necessary for establishing host associations, were also found. MoCRZ1 itself was a target, suggesting a previously unreported autoregulation control point. The data also implicated a previously unreported feedback regulation mechanism of calcineurin activity. We propose that calcium/calcineurin regulated signal transduction circuits controlling development and pathogenicity manifest through multiple layers of regulation. We present results from the ChIP-chip and expression analysis along with a refined model of calcium/calcineurin signaling in this important plant pathogen

Multi-level engineering facilitates the production of phenylpropanoid compounds in tomato

Phenylpropanoids comprise an important class of plant secondary metabolites. A number of transcription factors have been used to upregulate-specific branches of phenylpropanoid metabolism, but by far the most effective has been the fruit-specific expression of AtMYB12 in tomato, which resulted in as much as 10% of fruit dry weight accumulating as flavonols and hydroxycinnamates. We show that AtMYB12 not only increases the demand of flavonoid biosynthesis but also increases the supply of carbon from primary metabolism, energy and reducing power, which may fuel the shikimate and phenylalanine biosynthetic pathways to supply more aromatic amino acids for secondary metabolism. AtMYB12 directly binds promoters of genes encoding enzymes of primary metabolism. The enhanced supply of precursors, energy and reducing power achieved by AtMYB12 expression can be harnessed to engineer high levels of novel phenylpropanoids in tomato fruit, offering an effective production system for bioactives and other high value ingredients

Mutant K-ras oncogene regulates steroidogenesis of normal human adrenocortical cells by the RAF-MEK-MAPK pathway

The result of our previous study has shown that the K-ras mutant (pK568MRSV) transfected human adrenocortical cells can significantly increase cortisol production and independently cause cell transformation. The aim of this study is to investigate the effect of the active K-ras oncogene on the cortisol production in normal human adrenocortical cells. First we used isopropyl thiogalactoside to induce the inducible mutant K-ras expression plasmid, pK568MRSV, in the stable transfected human adrenocortical cells. The result showed that the increase of RasGTP levels in transfected cells was time-dependent after isopropyl thiogalactoside induction. Additionally, results from Western blot analysis revealed significant elevation in phosphorylation of c-Raf-1 and Mitogen-activated protein kinase. We also detected the levels of mRNA encoding Cholesterol side-chain cleavage enzyme (P450SCC), 17α-Hydroxylase/17,20-lyase (P450c17) and 3β-Hydroxysteroid dehydrogenase (3βHSD) were increased in human adrenocortical cells transfected with mutant K-ras after IPTG treatment. The increase of mRNA amount in P450scc P450c17 and 3βHSD and the elevation of cortisol level were inhibited with a pretreatment of PD098059, a specific extracellular signal-regulated kinase inhibitor. In our previous report, we proved that lovastatin, a pharmacological inhibitor of p21ras function, also reversed the increase of cortisol level in mutant K-ras stably transfected human adrenocortical cells. Taken together, these findings proved that the active mutant Ras enhanced not only cell proliferation but also steroidogenesis in steroidogenic phenotype cells by activating Raf-MEK-MAPK related signal transduction pathway. Therefore, we believe that K-ras mutants influence regulation of steroidogenesis in adrenocortical cells through RAF-MEK-MAPK pathway