Backbone and sidechain methyl Ile (δ1), Leu and Val chemical shift assignments of RDE-4 (1–243), an RNA interference initiation protein in C. elegans

The RNAi pathway of several organisms requires presence of double stranded RNA binding proteins for functioning of Dicer in gene regulation. In C. elegans, a double stranded RNA binding protein, RDE-4 (385 aa, 44 kDa) recognizes long exogenous dsRNA and initiates the RNAi pathway. We have achieved complete backbone and stereospecific methyl sidechain Ile (δ1), Leu and Val chemical shifts of first 243 amino acids of RDE-4, namely RDE-4ΔC.     

Design and synthesis of novel furoquinoline based inhibitors of multiple targets in the PI3K/Akt-mTOR pathway

We herein report the design and synthesis of furoquinoline based novel molecules (16-36) and their in vitro multiple targeted inhibitory potency against PI3K/Akt phosphorylation and mTOR using cell based and cell-free kinase assay. In particular, compound 23 in addition to PI3K-mTOR inhibitory potency, it has shown potent inhibition of hypoxia-induced accumulation of HIF-1alpha protein in U251-HRE cell line. The inhibitory activities of compound 23 were confirmed by Western blot analysis, using human non-small cell lung carcinoma H-460 cell line and glioblastoma U251 cell lines.     

<Emphasis Type="Italic">Arabidopsis HOG1</Emphasis> gene and its petunia homolog <Emphasis Type="Italic">PETCBP</Emphasis> act as key regulators of yield parameters

Plant hormones influence the key parameters that contribute to crop yield, including biomass, branching and seed number. We tested manipulation of cytokinin signaling as an avenue for influencing these growth parameters. Here we report a full-length cDNA coding for a cytokinin binding protein, Petunia cytokinin binding protein (PETCBP) from Petunia hybrida cv. Mitchell. PETCBP encodes for a protein that exhibits high sequence similarity to S-adenosyl-L: -homocysteine hydrolase (SAHH). Transgenic petunia plants expressing this gene in antisense orientation displayed profuse branching, delayed flowering and delayed shoot bud induction from leaf explants in vitro. Homologs were also isolated from Arabidopsis thaliana homology-dependent gene silencing 1 (HOG1) and Orzya sativa (OsCBP). Arabidopsis HOG1 showed high affinity cytokinin binding activity and modified plant architecture similar to PETCBP. Transgenic Arabidopsis plants overexpressing HOG1 showed early flowering with a significantly reduced plant biomass and number of leaves. In contrast, profuse branching, delayed flowering, increased leaf size and higher seed yield were the major phenotypes observed in the antisense suppression lines. These results suggest that genetic manipulation of this cytokinin binding protein or its orthologs could be used for improving crop biomass and seed yield.     

Preliminary computational modeling of nitric oxide synthase 3 interactions with caveolin-1： influence of exon 7 Glu298Asp polymorphism

The significance of endothelial nitric oxide synthase 3 （NOS3） activity has been recognized for many years, however it was only recently that the complicated regulation of this constitutively expressed enzyme in endothelial cells was identified. A critical component of the NOS3 regulatory cyde in endothelial cells is its intracellnlar localization to caveolae. The caveolar coordination of NOS3, more specifically its interaction with caveolin-1 （Cav-1）, plays a major role in normal endothelial NOS3 activity and vascular bioavailability of nitric oxide. We have recently shown that the presence of NOS3 exon 7 Glu298Asp polymorphism caused diminished shear-dependent NOS activation, was less extensively associated with caveolae, and had a decreased degree of interaction with Cav-1. Here, we carried out preliminary investigations to identify possible mechanisms of the genotype-dependent endothelial cell responses we observed in our previous investigations. Through this approach we tested the hypothesis that computer simulations could provide insights regarding the contribution of this single nucleotide polymorphism to regulation of the NOS3 isoform. We observed that in the Glu/Asp and Asp/Asp mutant genotypes, the amount of NOS3 associated with Cav-1 was significantly lower. Additionally, we have shown, using a theoretical computational model, that mutation of an amino acid at position 298 might affect the protein-protein interactions and localization of the NOS3 protein. These alterations might also affect the protein function and explain the enhanced disease risk associated with the presence of Glu298Asp polymorphism in the NOS3 protein.     

DNA Area and NETosis Analysis (DANA): a High-Throughput Method to Quantify Neutrophil Extracellular Traps in Fluorescent Microscope Images

Background

Neutrophil extracellular traps (NETs), extracellular structures composed of decondensed chromatin and antimicrobial molecules, are released in a process called NETosis. NETs, which are part of normal host defense, have also been implicated in multiple human diseases. Unfortunately, methods for quantifying NETs have limitations which constrain the study of NETs in disease. Establishing optimal methods for NET quantification holds the potential to further elucidate the role of NETs in normal and pathologic processes.

Results

To better quantify NETs and NET-like structures, we created DNA Area and NETosis Analysis (DANA), a novel ImageJ/Java based program which provides a simple, semi-automated approach to quantify NET-like structures and DNA area. DANA can analyze many fluorescent microscope images at once and provides data on a per cell, per image, and per sample basis. Using fluorescent microscope images of Sytox-stained human neutrophils, DANA quantified a similar frequency of NET-like structures to the frequency determined by two different individuals counting by eye, and in a fraction of the time. As expected, DANA also detected increased DNA area and frequency of NET-like structures in neutrophils from subjects with rheumatoid arthritis as compared to control subjects. Using images of DAPI-stained murine neutrophils, DANA (installed by an individual with no programming background) gave similar frequencies of NET-like structures as the frequency of NETs determined by two individuals counting by eye. Further, DANA quantified more NETs in stimulated murine neutrophils compared to unstimulated, as expected.

Conclusions

DANA provides a means to quantify DNA decondensation and the frequency of NET-like structures using a variety of different fluorescent markers in a rapid, reliable, simple, high-throughput, and cost-effective manner making it optimal to assess NETosis in a variety of conditions.

     

Staphylococcus aureus Sortase A transpeptidase. Calcium promotes sorting signal binding by altering the mobility and structure of an active site loop

Many virulence factors in gram-positive bacteria are covalently anchored to the cell-wall peptidoglycan by sortase enzymes, a group of widely distributed cysteine transpeptidases. The Staphylococcus aureus Sortase A protein (SrtA) is the archetypal member of the Sortase family and is activated by Ca2+, an adaptation that may facilitate host colonization as elevated concentrations of this ion are encountered in human tissue. Here we show that a single Ca2+ ion bound to an ordered pocket on SrtA allosterically activates catalysis by modulating both the structure and dynamics of a large active site loop. Detailed nitrogen-15 relaxation measurements indicate that Ca2+ may facilitate the adaptive recognition of the substrate by inducing slow micro- to millisecond time-scale dynamics in the active site. Interestingly, relaxation compensated Carr-Purcell-Meiboom-Gill experiments suggest that the time scale of these motions is directly correlated with ion binding. The results of site-directed mutagenesis indicate that this motional coupling is mediated by the side chain of Glu-171, which is positioned within the beta6/beta7 loop and shown to contribute to Ca2+ binding. The available structural and dynamics data are compatible with a loop closure model of Ca2+ activation, in which the beta6/beta7 loop fluctuates between a binding competent closed form that is stabilized by Ca2+, and an open, highly flexible state that removes key substrate contacting residues from the active site.     

Functional characterisation of <Emphasis Type="Italic">romeharsha</Emphasis> and <Emphasis Type="Italic">clint1</Emphasis> reaffirms the link between plasma membrane homeostasis,cell size maintenance and tissue homeostasis in developing zebrafish epidermis

In vertebrates, early developing epidermis is a bilayered epithelium consisting of an outer periderm and the underlying basal epidermis. It eventually develops into a multi-layered epithelium. The mechanisms that control the architecture and homeostasis of early developing bilayered epidermis have remained poorly understood. Recently, we have shown that the function of Myosin Vb, an actin based molecular motor, is essential in peridermal cells for maintenance of plasma membrane homeostasis. Furthermore, our analyses of the goosepimples/myosin Vb mutant unravelled a direct link between plasma membrane homeostasis, cell size maintenance and tissue homeostasis in the developing epidermis. However, it remained unclear whether this link is specific to myosin Vb mutant or this is a general principle. Here we have identified two more genetic conditions, romeharsha mutant and clint1 knockdown, in which membrane homeostasis is perturbed, as evident by increased endocytosis and accumulation of lysosomes. As a consequence, peridermal cells exhibit smaller size and increased proliferation. We further show that decreasing endocytosis in romeharsha mutant and clint1 morphants rescues or mitigates the effect on cell size, cell proliferation and morphological phenotype. Our data confirms generality of the principle by reaffirming the causal link between plasma membrane homeostasis, cell size maintenance and tissue homeostasis.     

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization,phase separation,and RNA splicing

TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD). Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS) in vitro, fluidizes liquid–liquid phase separated nuclear TDP‐43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization. These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS. In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation.     

Identification of chikungunya virus interacting proteins in mammalian cells

Identification and characterization of virus host interactions is an essential step for the development of novel antiviral strategies. Very few studies have been targeted towards identification of chikungunya virus (CHIKV) interacting host proteins. In current study, virus overlay protein binding assay (VOPBA) and matrix-assisted laser desorption/ionization time of flight analysis (MALDI TOF/TOF) were employed for the identification of CHIKV binding proteins in mammalian cells. HSP70 and actin were identified as virus binding proteins in HEK-293T and Vero-E6 cells, whereas STAT-2 was identified as an additional protein in Vero-E6 cells. Pre-incubation with anti-HSP70 antibody and miRNA silencing of HSP70 significantly reduced the CHIKV production in HEK-293T and Vero-E6 cells at early time points. These results suggest that CHIKV exploits the housekeeping molecules such as actin, HSP70 and STAT-2 to establish infection in the mammalian cells.     

DNA damage-induced expression of p53 suppresses mitotic checkpoint kinase hMps1: the lack of this suppression in p53MUT cells contributes to apoptosis

DNA damage induced by the topoisomerase I inhibitor irinotecan (CPT-11) triggers in p53(WT) colorectal carcinoma cells a long term cell cycle arrest and in p53MUT cells a transient arrest followed by apoptosis (Magrini, R., Bhonde, M. R., Hanski, M. L., Notter, M., Scherübl, H., Boland, C. R., Zeitz, M., and Hanski, C. (2002) Int. J. Cancer 101, 23-31; Bhonde, M. R., Hanski, M. L., Notter, M., Gillissen, B. F., Daniel, P. T., Zeitz, M., and Hanski, C. (2006) Oncogene 25, 165-175). The mechanism of the p53-independent apoptosis still remains largely unclear. Here we used five p53WT and five p53MUT established colon carcinoma cell lines to identify gene expression alterations associated with apoptosis in p53MUT cells after treatment with SN-38, the irinotecan metabolite. After treatment, 16 mitosis-related genes were found to be expressed at least 2-fold stronger in the apoptosis-executing p53MUT cells than in the cell cycle-arrested p53WT cells by oligonucleotide microarray analysis. One of the genes whose strong post-treatment expression was associated with apoptosis was the mitotic checkpoint kinase hMps1 (human ortholog of the yeast monopolar spindle 1 kinase). hMps1 mRNA and protein expression were suppressed by the treatment-induced and by the exogenous adenovirus-coded p53 protein. The direct suppression of hMps1 on RNA level or inhibition of its activity by a dominant-negative hMps1 partly suppressed apoptosis. Together, these data indicate that the high expression of mitotic genes in p53MUT cells after SN-38 treatment contributes to DNA damage-induced apoptosis, whereas their suppression in p53WT cells acts as a safeguard mechanism preventing mitosis initiation and the subsequent apoptosis. hMps1 kinase is one of the mitotic checkpoint proteins whose expression after DNA damage in p53MUT cells activates the checkpoint and contributes to apoptosis.