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Identification of the integrin-binding site on coagulation factor VIIa required for proangiogenic PAR2 signaling.
2018
The tissue factor (TF) pathway serves both hemostasis and cell signaling, but how cells control these divergent functions of TF remains incompletely understood. TF is the receptor and scaffold of coagulation proteases cleaving protease-activated receptor 2 (PAR2) that plays pivotal roles in angiogenesis and tumor development. Here we demonstrate that coagulation factor VIIa (FVIIa) elicits TF cytoplasmic domain-dependent proangiogenic cell signaling independent of the alternative PAR2 activator matriptase. We identify a Lys-Gly-Glu (KGE) integrin-binding motif in the FVIIa protease domain that is required for association of the TF-FVIIa complex with the active conformer of integrin β1. A po…
Alkaline phosphatase dual-binding sites for collagen dictate cell migration and microvessel assembly in vitro
2020
Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro- or microvasculature) play a role in the formation of microvessel-like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor-beta 1, and interleukin-8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibro…
[Au(9-methylcaffein-8-ylidene) 2 ] + /DNA Tel23 System: Solution, Computational, and Biological Studies
2017
International audience; Physicochemical methods have been used to investigate interactions occurring in solution between the dicarbene gold(I) complex [Au(9‐methylcaffein‐8‐ylidene)2]BF4 (AuNHC) and a human telomeric DNA sequence, namely Tel23. Circular dichroism measurements allow identification of the conformational changes experienced by Tel23 upon interaction with AuNHC, and the respective binding stoichiometries and constants were determined. Computational studies provide a good link between previous crystallographic results of the same system and the present solution data, offering an exhaustive description of the inherent noncovalent metallodrug–DNA interactions. Remarkably, we found…
AlkAniline-Seq: Profiling of m7 G and m3 C RNA Modifications at Single Nucleotide Resolution.
2018
RNA modifications play essential roles in gene expression regulation. Only seven out of >150 known RNA modifications are detectable transcriptome-wide by deep sequencing. Here we describe a new principle of RNAseq library preparation, which relies on a chemistry based positive enrichment of reads in the resulting libraries, and therefore leads to unprecedented signal-to-noise ratios. The proposed approach eschews conventional RNA sequencing chemistry and rather exploits the generation of abasic sites and subsequent aniline cleavage. The newly generated 5'-phosphates are used as unique entry for ligation of an adapter in library preparation. This positive selection, embodied in the AlkAnilin…
The Binding Mechanism of Epolactaene to Hsp60 Unveiled by in Silico Modelling
2016
Molecular Dynamics (MD) simulations and DFT/MM calculations were performed in order to rationalize available experimental results and to provide structural details on the binding mechanism of Epolactaene (EPO) to the 60 KDa Heat Shock Protein (Hsp60). The available crystal structure of Hsp60 represents the last step of the chaperone folding cycle, while the Hsp60-EPO complex was obtained by using a homology model of Hsp60, in order to simulate a state related to the beginning of the folding cycle (Rs1). The results of MD simulations point out that EPO shows the highest binding affinity for the empty ATP binding site. The presence of ATP opens a channel that allows the entrance of both EPO d…
Alkyne-Functionalized Coumarin Compound for Analytic and Preparative 4-Thiouridine Labeling
2017
Bioconjugation of RNA is a dynamic field recently reinvigorated by a surge in research on post-transcriptional modification. This work focuses on the bioconjugation of 4-thiouridine, a nucleoside that occurs as a post-transcriptional modification in bacterial RNA and is used as a metabolic label and for cross-linking purposes in eukaryotic RNA. A newly designed coumarin compound named 4-bromomethyl-7-propargyloxycoumarin (PBC) is introduced, which exhibits remarkable selectivity for 4-thiouridine. Bearing a terminal alkyne group, it is conductive to secondary bioconjugation via “click chemistry”, thereby offering a wide range of preparative and analytical options. We applied PBC to quantita…
An Intronic cis-Regulatory Element Is Crucial for the Alpha Tubulin Pl-Tuba1a Gene Activation in the Ciliary Band and Animal Pole Neurogenic Domains …
2017
In sea urchin development, structures derived from neurogenic territory control the swimming and feeding responses of the pluteus as well as the process of metamorphosis. We have previously isolated an alpha tubulin family member of Paracentrotus lividus (Pl-Tuba1a, formerly known as Pl-Talpha2) that is specifically expressed in the ciliary band and animal pole neurogenic domains of the sea urchin embryo. In order to identify cis-regulatory elements controlling its spatio-temporal expression, we conducted gene transfer experiments, transgene deletions and site specific mutagenesis. Thus, a genomic region of about 2.6 Kb of Pl-Tuba1a, containing four Interspecifically Conserved Regions (ICRs…
Adaptation of gene loci to heterochromatin in the course of Drosophila evolution is associated with insulator proteins.
2020
AbstractPericentromeric heterochromatin is generally composed of repetitive DNA forming a transcriptionally repressive environment. Dozens of genes were embedded into pericentromeric heterochromatin during evolution of Drosophilidae lineage while retaining activity. However, factors that contribute to insusceptibility of gene loci to transcriptional silencing remain unknown. Here, we find that the promoter region of genes that can be embedded in both euchromatin and heterochromatin exhibits a conserved structure throughout the Drosophila phylogeny and carries motifs for binding of certain chromatin remodeling factors, including insulator proteins. Using ChIP-seq data, we demonstrate that ev…
iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins
2016
DNA adenine methyltransferase identification (DamID) has emerged as an alternative method to profile protein-DNA interactions; however, critical issues limit its widespread applicability. Here, we present iDamIDseq, a protocol that improves specificity and sensitivity by inverting the steps DpnI-DpnII and adding steps that involve a phosphatase and exonuclease. To determine genome-wide protein-DNA interactions efficiently, we present the analysis tool iDEAR (iDamIDseq Enrichment Analysis with R). The combination of DamID and iDEAR permits the establishment of consistent profiles for transcription factors, even in transient assays, as we exemplify using the small teleost medaka (Oryzias lati…
Q-nexus: a comprehensive and efficient analysis pipeline designed for ChIP-nexus
2016
Background: ChIP-nexus, an extension of the ChIP-exo protocol, can be used to map the borders of protein-bound DNA sequences at nucleotide resolution, requires less input DNA and enables selective PCR duplicate removal using random barcodes. However, the use of random barcodes requires additional preprocessing of the mapping data, which complicates the computational analysis. To date, only a very limited number of software packages are available for the analysis of ChIP-exo data, which have not yet been systematically tested and compared on ChIP-nexus data. Results: Here, we present a comprehensive software package for ChIP-nexus data that exploits the random barcodes for selective removal …