Abstract :

BMP-1/tolloid-like proteinases belong to the astacin family of human metalloproteinases, together with meprins and ovastacin. They represent promising targets to treat or prevent a wide range of diseases such as fibrotic disorders or cancer. However, the study of their pathophysiological roles is still impaired by the lack of well-characterized inhibitors and the questions that remain regarding their selectivity and in vivo efficiency. As a first step towards the identification of suitable tools to be used in functional studies, we have undertaken a systematic comparison of seven molecules known to affect the proteolytic activity of human astacins including three hydroxamates (FG-2575, UK383,367, S33A), the protein sizzled, a new phosphinic inhibitor (RXP-1001) and broad-spectrum protease inhibitors (GM6001, actinonin). Their efficacy in vitro, their cellular toxicity and efficacy in cell cultures were thoroughly characterized. We found that these molecules display very different potency and selectivity profiles, with hydroxamate FG-2575 and the protein sizzled being very powerful and selective inhibitors of BMP-1, whereas phosphinic peptide RXP-1001 behaves as a broad-spectrum inhibitor of astacins. Their use should therefore be carefully considered in agreement with the aim of the study to avoid result misinterpretation.          

DEC  2018     

DOI : 10.1002/2211-5463.12540          

Pubmed : 30524951

Abstract :

Mice from the Orkney archipelago exhibit an important diversity regarding molar shape. While on some islands mice display a usual dental pattern, teeth from other islands display additional cusplets and unusual phenotypes that may constitute case studies for evaluating the potential functional relevance of dental changes. We developed a multifaceted approach combining 2D and 3D geometric morphometrics, dental topography, dental wear, biomechanics, estimations of masticatory muscles force, and in vivo bite force on wild-derived lab descendants exemplifying the two extreme dental morphologies. The two strains differed in the geometry of the upper and lower tooth rows, and in the topography of the upper row only. Surprisingly, the most unusual tooth morphology appeared as the least complex because tooth simplification overwhelmed the signal provided by the occurrence of additional cusplets. No difference in bite force nor muscle force was evidenced, showing that the important change in dental morphology was accommodated without major changes in the rest of the masticatory apparatus. The evolution of unusual dental phenotypes was possibly fueled by drift and inbreeding in small and isolated populations on remote islands of the archipelago. No functional counter-selection impeded this diversification, since the unusual dental phenotypes did not disrupt occlusion and mastication.      

DEC  2018     

DOI : 10.1007/s11692-018-9459-6 

Abstract :

Chronic inflammation and fibrosis characterize Duchenne muscular dystrophy (DMD). We show that pro-inflammatory macrophages are associated with fibrosis in mouse and human DMD muscle. DMD-derived Ly6C(pos) macrophages exhibit a profibrotic activity by sustaining fibroblast production of collagen I. This is mediated by the high production of latent-TGF-beta 1 due to the higher expression of LTBP4, for which polymorphisms are associated with the progression of fibrosis in DMD patients. Skewing macrophage phenotype via AMPK activation decreases ltbp4 expression by Ly6C(pos) macrophages, blunts the production of latent-TGF-beta 1, and eventually reduces fibrosis and improves DMD muscle force. Moreover, fibro-adipogenic progenitors are the main providers of TGF-beta-activating enzymes in mouse and human DMD, leading to collagen production by fibroblasts. In vivo pharmacological inhibition of TGF-beta-activating enzymes improves the dystrophic phenotype. Thus, an AMPK-LTBP4 axis in inflammatory macrophages controls the production of TGF-beta 1, which is further activated by and acts on fibroblastic cells, leading to fibrosis in DMD.             

NOV  2018     

DOI: 10.1016/j.celrep.2018.10.077    

Pubmed : 30463013

Abstract :

Chromatin acetylation, a critical regulator of synaptic plasticity and memory processes, is thought to be altered in neurodegenerative diseases. Here, we demonstrate that spatial memory and plasticity (LTD, dendritic spine formation) deficits can be restored in a mouse model of tauopathy following treatment with CSP-TTK21, a small-molecule activator of CBP/p300 histone acetyltransferases (HAT). At the transcriptional level, CSP-TTK21 re-established half of the hippocampal transcriptome in learning mice, likely through increased expression of neuronal activity genes and memory enhancers. At the epigenomic level, the hippocampus of tauopathic mice showed a significant decrease in H2B but not H3K27 acetylation levels, both marks co-localizing at TSS and CBP enhancers. Importantly, CSP-TTK21 treatment increased H2B acetylation levels at decreased peaks, CBP enhancers, and TSS, including genes associated with plasticity and neuronal functions, overall providing a 95% rescue of the H2B acetylome in tauopathic mice. This study is the first to provide in vivo proof-of-concept evidence that CBP/p300 HAT activation efficiently reverses epigenetic, transcriptional, synaptic plasticity, and behavioral deficits associated with Alzheimer's disease lesions in mice. 

NOV 2018     

DOI : 10.15252/emmm.201708587     

Pubmed : 30275019

Abstract :

Eukaryotic-like serine/threonine kinases (eSTKs) with extracellular PASTA repeats are key membrane regulators of bacterial cell division. How PASTA repeats govern eSTK activation and function remains elusive. Using evolution- and structural-guided approaches combined with cell imaging, we disentangle the role of each PASTA repeat of the eSTK StkP from Streptococcus pneumoniae. While the three membrane-proximal PASTA repeats behave as interchangeable modules required for the activation of StkP independently of cell wall binding, they also control the septal cell wall thickness. In contrast, the fourth and membrane-distal PASTA repeat directs StkP localization at the division septum and encompasses a specific motif that is critical for final cell separation through interaction with the cell wall hydrolase LytB. We propose a model in which the extracellular four-PASTA domain of StkP plays a dual function in interconnecting the phosphorylation of StkP endogenous targets along with septal cell wall remodelling to allow cell division of the pneumococcus. 

FEB 2018     

DOI : 10.1038/s41564-017-0069-3

Pubmed : 29203882

Abstract :

Most living organisms show circadian rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. To study peripheral clocks in vivo, we developed an original model, the Rev-Luc mouse to follow noninvasively and longitudinally Rev-Luc oscillations in peripheral clocks using in vivo bioluminescence imaging. We found in vitro and in vivo a robust diurnal rhythm of Rev-Luc, mainly in liver, intestine, kidney and adipose tissues. We further confirmed in vivo that Rev-Luc peripheral tissues are food-entrainable oscillators, not affected by age or sex. These data strongly support the relevance of the Rev-Luc model for circadian studies, especially to investigate in vivo the establishment and the entrainment of the rhythm throughout ontogenesis. We then showed that Rev-Luc expression develops dynamically and gradually, both in amplitude and in phase, during fetal and postnatal development. We also demonstrate for the first time that the immature peripheral circadian system of offspring in utero is mainly entrained by maternal cues from feeding regimen. The prenatal entrainment will also differentially determine the Rev-Luc expression in pups before weaning underlining the importance of the maternal chrononutrition on the circadian system entrainment of the offspring.           

NOV  2018     

DOI : 10.1007/s00018-018-2845-5      

Pubmed : 29804258

Abstract :

Prominent changes in the gut microbiota (referred to as "dysbiosis") play a key role in the development of allergic disorders, but the underlying mechanisms remain unknown. Study of the delayedtype hypersensitivity (DTH) response in mice contributed to our knowledge of the pathophysiology of human allergic contact dermatitis. Here we report a negative regulatory role of the RIG-I-like receptor adaptor mitochondrial antiviral signaling (MAVS) on DTH bymodulating gut bacterial ecology. Cohousing and fecal transplantation experiments revealed that the dysbiotic microbiota of Mavs(-/-) mice conferred a proallergic phenotype that is communicable to wild-type mice. DTH sensitization coincided with increased intestinal permeability and bacterial translocation within lymphoid organs that enhanced DTH severity. Collectively, we unveiled an unexpected impact of RIG-I-like signaling on the gut microbiota with consequences on allergic skin disease outcome. Primarily, these data indicate that manipulating the gut microbiota may help in the development of therapeutic strategies for the treatment of human allergic skin pathologies.             

OCT   2018     

DOI : 10.1073/pnas.1722372115         

Pubmed : 30249647

Abstract :

LAM is a rare low-grade metastasizing lung neoplasm. Inhibitors of mTOR improve clinical outcome of LAM patients by preventing loss of lung function. Nevertheless, other cell targets may be of interest for drug development. Therefore, we explored the potential role of EDN1(endothelin) in LAM. We report an increased endothelin blood level in LAM patients as well as EDN1 overexpression and EDN1 receptor downregulation in LAM-derived primary cells and in TSC2(NEG) cells mutated in TSC2. We evidenced EDN pathway dysregulation based on EDN1, EDNRA, EDNRB and ARRB1 mRNA expression in LAM-derived primary cells. We showed overexpression of EDN1 and ARRB1 mRNAs in TSC2(NEG) cells; these cells lost their ability to respond to stimulation by endothelin. We analyzed the effects of endothelin receptor antagonists alone or in combination with rapamycin, an mTOR inhibitor, on proliferation and migration of LAM cells. Rapamycin treatment ofTSC2(NEG) cells significantly reduced cell proliferation or migration, while none of the tested inhibitors of EDN receptors impaired these functions. We showed that TSC2(NEG) cells have acquired a transformed phenotype as showed by their ability to grow as spheroids in semi-solid medium and that unlike endothelin receptors antagonists, rapamycin reduced anchorage-independent cell growth and prevented expansion of TSC2(NEG) spheroids.             

OCT   2018     

DOI : 10.1038/s41598-018-32795-3    

Pubmed : 30279475

Abstract :

Procollagen C-proteinase enhancer-1 (PCPE-1) is a secreted protein that specifically accelerates proteolytic release of the C-propeptides from fibrillar procollagens, a crucial step in fibril assembly. As such, it is a potential therapeutic target to improve tissue repair and prevent fibrosis, a major cause of mortality worldwide. Here we present the crystal structure of the active CUB1CUB2 fragment of PCPE-1 bound to the C-propeptide trimer of procollagen III (CPIII). This shows that the two CUB domains bind to two different chains of CPIII and that the N-terminal region of one CPIII chain, close to the proteolytic cleavage site, lies in the cleft between CUB1 and CUB2. This suggests that enhancing activity involves unraveling of this chain from the rest of the trimer, thus facilitating the action of the proteinase involved. Support for this hypothesis comes from site-directed mutagenesis, enzyme assays, binding studies, and molecular modeling.     

OCT   2018     

DOI : 10.1016/j.str.2018.06.011           

Pubmed : 30078642

Abstract :

The nucleoplasm and nucleolus are the two main territories of the nucleus. While specific functions are associated with each of these territories (such as mRNA synthesis in the nucleoplasm and ribosomal rRNA synthesis in the nucleolus), some proteins are known to be located in both. Here, we investigated the molecular function of REBELOTE (RBL), an Arabidopsis thaliana protein previously characterized as a regulator of floral meristem termination. We show that RBL displays a dual localization, in the nucleolus and nucleoplasm. Moreover, we used direct and global approaches to demonstrate that RBL interacts with nucleic acid-binding proteins. It binds to the NOC proteins SWA2, AtNOC2 and AtNOC3 in both the nucleolus and nucleoplasm, and also to OBE1 and VFP3/ENAP1. Taking into account the identities of these RBL interactors, we hypothesize that RBL acts both in ribosomal biogenesis and in the regulation of gene expression.            

OCT  2018     

DOI : 10.1002/2211-5463.12504          

Pubmed : 30338215

Abstract :

Studying intracellular dynamics in neurons is crucial to better understand how brain circuits communicate and adapt to environmental changes. In neurons, axonal secretory vesicles underlie various functions from growth during development to plasticity in the mature brain. Similarly, transport of mitochondria, the power plant of the cell, regulates both axonal development and synaptic homeostasis. However, because of their submicrometric size and rapid velocities, studying the kinetics of these organelles in projecting axons in vivo is technically challenging. In parallel, primary neuronal cultures are adapted to study axonal transport but they lack the physiological organization of neuronal networks, which in turn may bias observations. We previously developed a microfluidic platform to reconstruct a physiologically-relevant and functional corticostriatal network in vitro that is compatible with high-resolution videorecording of axonal trafficking. Here, using this system we report progressive changes in axonal transport kinetics of both dense core vesicles and mitochondria that correlate with network development and maturation. Interestingly, axonal flow of both types of organelles change in opposite directions, with rates increasing for vesicles and decreasing for mitochondria. Overall, our observations highlight the need for a better spatiotemporal control for the study of intracellular dynamics in order to avoid misinterpretations and improve reproducibility. 

SEP 2018     

DOI : 10.1038/s41598-018-31759-x    

Pubmed : 30194421

Abstract :

CLP rats presented hypotension, lactic acidosis, liver cytolysis, and upregulation of proinflammatory cytokines mRNA as compared to controls. Liver mitochondria showed a decrease in ATP synthesis and oxygen consumption at 24 h following CLP. A marked uncoupling of oxidative phosphorylation appeared 36 h following CLP and was associated with a decrease in cytochrome c oxidase activity and content and ATP synthase subunit β content (slip mechanism) and an increase in mitochondrial oligomycin-insensitive respiration, but no change in mitochondrial inner membrane permeability (no leak). Upregulation of UCP2 mRNA resulted in a decrease in mitochondrial ROS generation 24 h after the onset of CLP, whereas ROS over-generation associated with slip at cytochrome c oxidase observed at 36 h was concomitant with a decrease in UCP2 mRNA expression.

SEP 2018     

DOI : 10.1186/s40635-018-0197-y   

Pubmed : 30187255

Abstract :

Lentiviral vectors have emerged as an efficient, safe therapeutic tool for gene therapy based on hematopoietic stem cells (HSCs) or T cells. However, the monitoring of transduced cells in preclinical models remains challenging because of the inefficient transduction of murine primary T cells with lentiviral vectors, in contrast to gammaretroviral vectors. The use of this later in preclinical proof of concept is not considered as relevant when a lentiviral vector will be used in a clinical trial. Hence, there is an urgent need to develop an efficient transduction protocol for murine cells with lentiviral vectors. Here, we describe an optimized protocol in which a nontoxic transduction enhancer (Lentiboost) enables the efficient transduction of primary murine T cells with lentiviral vectors. The optimized protocol combines low toxicity and high transduction efficiency. We achieved a high-level transduction of murine CD4(+) and CDS+ T cells with a VSV-G-pseudotyped lentiviral vector with no changes in the phenotypes of transduced T cells, which were stable and long-lived in culture. This enhancer also increased the transduction of murine HSCs. Hence, use of this new transduction enhancer overcomes the limitations of lentiviral vectors in preclinical experiments and should facilitate the translation of strategies based on lentiviral vectors from the bench to the clinic.          

SEP 2018     

DOI : 10.1016/j.omtm.2018.08.002    

Pubmed : 30191160

Abstract :

Increasing evidence suggests that dysregulation of lipid metabolism is associated with neurodegeneration in retinal diseases such as age-related macular degeneration and in brain disorders such as Alzheimer's and Parkinson's diseases. Lipid storage organelles (lipid droplets, LDs), accumulate in many cell types in response to stress, and it is now clear that LDs function not only as lipid stores but also as dynamic regulators of the stress response. However, whether these LDs are always protective or can also be deleterious to the cell is unknown. Here, we investigated the consequences of LD accumulation on retinal cell homeostasis under physiological and stress conditions in Drosophila and in mice. In wild-type Drosophila, we show that dFatp is required and sufficient for expansion of LD size in retinal pigment cells (RPCs) and that LDs in RPCs are required for photoreceptor survival during aging. Similarly, in mice, LD accumulation induced by RPC-specific expression of human FATP1 was non-toxic and promoted mitochondrial energy metabolism in RPCs and non-autonomously in photoreceptor cells. In contrast, the inhibition of LD accumulation by dFatp knockdown suppressed neurodegeneration in Aats-met(FB) Drosophila mutants, which carry elevated levels of reactive oxygen species (ROS). This suggests that abnormal turnover of LD may be toxic for photoreceptors cells of the retina under oxidative stress. Collectively, these findings indicate that FATP-mediated LD formation in RPCs promotes RPC and neuronal homeostasis under physiological conditions but could be deleterious for the photoreceptors under pathological conditions.       

SEP 2018     

DOI : 10.1371/journal.pgen.1007627 

Pubmed : 30199545

Abstract :

Biofilm has become a major topic of interest in medical, food, industrial, and environmental bacteriology. To be relevant, investigation of biofilm behavior requires effective and reliable techniques. We present herein a simple and robust method, adapted from the microplate technique, in which steam is used as a soft washing method to preserve biofilm integrity and to improve reproducibility of biofilm quantification. The kinetics of steam washing indicated that the method is adapted to remove both planktonic bacteria and excess crystal violet (CV) staining for S. aureus, S. epidermidis, S. carnosus, P. aeruginosa, and E. coli biofilm. Confocal laser scanning microscopy confirmed that steam washing preserved the integrity of the biofilm better than pipette-based washing. We also investigated the measurement of the turbidity of biofilm resuspended in phosphate-buffered saline (PBS) as an alternative to staining with CV. This approach allows the discrimination of biofilm producer strains from non-biofilm producer strains in a way similar to CV staining, and subsequently permits quantification of viable bacteria present in biofilm by culture enumeration from the same well. Biofilm quantification using steam washing and PBS turbidity reduced the technical time needed, and data were highly reproducible.

AUG 2018     

DOI : 10.1038/s41598-018-31437-y    

Pubmed : 30158585

Abstract :

The RNA exosome fulfills important functions in the processing and degradation of numerous RNAs species. However, the mechanisms of recruitment to its various nuclear substrates are poorly understood. Using Epstein-Barr virus mRNAs as a model, we have discovered a novel function for the splicing factor SRSF3 in the quality control of nuclear mRNAs. We have found that viral mRNAs generated from intronless genes are particularly unstable due to their degradation by the nuclear RNA exosome. This effect is counteracted by the viral RNA-binding protein EB2 which stabilizes these mRNAs in the nucleus and stimulates both their export to the cytoplasm and their translation. In the absence of EB2, SRSF3 participates in the destabilization of these viral RNAs by interacting with both the RNA exosome and its adaptor complex NEXT. Taken together, our results provide direct evidence for a connection between the splicing machinery and mRNA decay mediated by the RNA exosome. Our results suggest that SRSF3 aids the nuclear RNA exosome and the NEXT complex in the recognition and degradation of certain mRNAs.

AUG 2018     

DOI : 10.1038/s41598-018-31078-1    

Pubmed : 30150655

Abstract :

Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoproliferative disorder involving chronic NF-kB activation. Several mutations in the BCR and MyD88 signaling pathway components, such as MyD88 L265P, are implicated in this aberrant activation. Among heat shock proteins, HSP110 has recently been identified as a prosurvival and/or proliferation factor in many cancers, but its role in ABC-DLBCL survival mechanisms remained to be established. We observed that short hairpin RNA-mediated HSP110 silencing decreased the survival of several ABC-DLBCL cell lines and decreased immunoglobulin M-MyD88 co-localization and subsequent NF-kB signaling. Conversely, overexpression of HSP110 in ABC-DLBCL or non-DLBCL cell lines increased NF-kB signaling, indicating a tight interplay between HSP110 and the NF-kB pathway. By using immunoprecipitation and proximity ligation assays, we identified an interaction between HSP110 and both wild-type MyD88 and MyD88 L265P. HSP110 stabilized both MyD88 forms with a stronger effect on MyD88 L265P, thus facilitating chronic NF-kB activation. Finally, HSP110 expression was higher in lymph node biopsies from patients with ABC-DLBCL than in normal reactive lymph nodes, and a strong correlation was found between the level of HSP110 and MyD88. In conclusion, we identified HSP110 as a regulator of NF-kB signaling through MyD88 stabilization in ABC-DLBCL. This finding reveals HSP110 as a new potential therapeutic target in ABC-DLBCL.     

AUG 2018     

DOI : 10.1182/blood-2017-12-819706              

Pubmed : 29871863

Abstract :

Roses have high cultural and economic importance as ornamental plants and in the perfume industry. We report the rose whole-genome sequencing and assembly and resequencing of major genotypes that contributed to rose domestication. We generated a homozygous genotype from a heterozygous diploid modern rose progenitor, Rosa chinensis 'Old Blush'. Using single-molecule real-time sequencing and a metaassembly approach, we obtained one of the most comprehensive plant genomes to date. Diversity analyses highlighted the mosaic origin of 'La France', one of the first hybrids combining the growth vigor of European species and the recurrent blooming of Chinese species. Genomic segments of Chinese ancestry identified new candidate genes for recurrent blooming. Reconstructing regulatory and secondary metabolism pathways allowed us to propose a model of interconnected regulation of scent and flower color. This genome provides a foundation for understanding the mechanisms governing rose traits and should accelerate improvement in roses, Rosaceae and ornamentals.    

JUN 2018     

DOI : 10.1038/s41588-018-0110-3      

Pubmed : 29713014

Abstract :

The staphylococcal bi-component leukocidins Panton-Valentine leukocidin (PVL) and gamma-haemolysin CB (HlgCB) target human phagocytes. Binding of the toxins' S-components to human complement C5a receptor 1 (C5aR1) contributes to cellular tropism and human specificity of PVL and HlgCB. To investigate the role of both leukocidins during infection, we developed a human C5aR1 knock-in (hC5aR1(KI)) mouse model. HlgCB, but unexpectedly not PVL, contributed to increased bacterial loads in tissues of hC5aR1(KI) mice. Compared to humans, murine hC5aR1(KI) neutrophils showed a reduced sensitivity to PVL, which was mediated by the toxin's F-component LukF-PV. By performing a genome-wide CRISPR-Cas9 screen, we identified CD45 as a receptor for LukF-PV. The human-specific interaction between LukF-PV and CD45 provides a molecular explanation for resistance of hC5aR1(KI) mouse neutrophils to PVL and probably contributes to the lack of a PVL-mediated phenotype during infection in these mice. This study demonstrates an unsuspected role of the F-component in driving the sensitivity of human phagocytes to PVL.         

JUN 2018     

DOI : 10.1038/s41564-018-0159-x      

Pubmed : 29736038

Abstract :

Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking, and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidylinositol-4-phosphate (PI4P), phosphatidic acidic (PA), and phosphatidylserine (PS) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of organelles not only defines the electrostatic territory but also distinguishes different functional compartments within this territory by specifying their varying surface charges.

MAY 2018     

DOI : 10.1016/j.devcel.2018.04.011    

Pubmed : 29754803

Abstract :

Since the identification of the antisense protein of HTLV-2 (APH-2) and the demonstration that APH-2 mRNA is expressed in vivo in most HTLV-2 carriers, much effort has been dedicated to the elucidation of similarities and/or differences between APH-2 and HBZ, the antisense protein of HTLV-1. Similar to HBZ, APH-2 negatively regulates HTLV-2 transcription. However, it does not promote cell proliferation. In contrast to HBZ, APH-2 half-life is very short. Here, we show that APH-2 is addressed to PML nuclear bodies in T-cells, as well as in different cell types. Covalent SUMOylation of APH-2 is readily detected, indicating that APH-2 might be addressed to the PML nuclear bodies in a SUMO-dependent manner. We further show that silencing of PML increases expression of APH-2, while expression of HBZ is unaffected. On the other hand, SUMO-1 overexpression leads to a specific loss of APH-2 expression that is restored upon proteasome inhibition. Furthermore, the carboxy-terminal LAGLL motif of APH-2 is responsible for both the targeting of the protein to PML nuclear bodies and its short half-life. Taken together, these observations indicate that natural APH-2 targeting to PML nuclear bodies induces proteasomal degradation of the viral protein in a SUMO-dependent manner. Hence, this study deciphers the molecular and cellular bases of APH-2 short half-life in comparison to HBZ and highlights key differences in the post-translational mechanisms that control the expression of both proteins.      

MAY 2018     

DOI : 10.1038/s41388-018-0163-x      

Pubmed : 29507418

Abstract :

Conjugation of fluorescent polymer chains synthesized by RAFT controlled radical polymerization with native proteins was achieved by introducing an activated ester function at their omega-chain-end via a very efficient thiol-ene click post-polymerization functionalization strategy. These fluorescent polymer probes covalently reacted with the lysine amino groups of native proteins to form bright protein-polymer fluorescent conjugates. In particular, streptavidin was labeled with various polymer probes and it was shown that it retained in each case its ability to selectively bind biotinylated compounds. Finally, streptavidin labeled with fluorescent polymer chains was used to reveal biotinylated antibodies, enabling the detection of CD40 receptors at the surface of live dendritic cells.

APR 2018     

DOI : 10.1039/c7py02064c     

Abstract :

We report on the modification of the electric properties of the imidazolium-based [BMIM] [NTf2] ionic liquid upon surface confinement in the sub-monolayer regime. Solid like insulating nanostructures of [BMIM][NTf2] spontaneously form on a variety of insulating substrates, at odds with the liquid and conductive nature of the same substances in the bulk phase. A systematic spatially resolved investigation by atomic force microscopy of the morphological, mechanical, and electrical properties of [BMIM] [NTf2] nanostructures showed that this liquid substance rearranges into lamellar nanostructures with a high degree of vertical order and enhanced resistance to mechanical compressive stresses and very intense electric fields, denoting a solid-like character. The morphological and structural reorganization has a profound impact on the electric properties of supported [BMIM] [NTf2] islands, which behave like insulator layers with a relative dielectric constant between 3 and 5, comparable to those of conventional ionic solids and significantly smaller than those measured in the bulk ionic liquid. These results suggest that, in the solid-like ordered domains confined either at surfaces or inside the pores of the nanoporous electrodes of photoelectrochemical devices, the ionic mobility and the overall electrical properties can be significantly perturbed with respect to the bulk liquid phase, which would likely influence the performance of the devices.     

APR 2018     

DOI : 10.1021/acs.jpcc.7b12600          

Abstract :

Lamin B Receptor (LBR) is an integral protein of the interphase inner nuclear membrane that is implicated in chromatin anchorage to the nuclear envelope. Phosphorylation of a stretch of arginine-serine (RS) dipeptides in the amino-terminal nucleoplasmic domain of LBR regulates the interactions of the receptor with other nuclear proteins, DNA and RNA and thus modulates tethering of heterochromatin to the nuclear envelope. While phosphorylation has been extensively studied, very little is known about other post-translational modifications of the protein. There is only one report on the O-beta-linked N-acetyl-glucosaminylation (O-GlcNAcylation) of a serine residue downstream of the RS domain of rat LBR. In the present study we identify additional O-GlcNAcylation sites by using as substrates of O-beta-N-acetylglucosaminyltransferase (OGT) a set of peptides containing the entire LBR RS domain or parts of it as well as flanking sequences. The in vitro activity of OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Furthermore, we provide evidence that O-GlcNAcylation hampers DNA binding while it marginally affects RS domain phosphorylation mediated by SRPK1, Akt2 and cdk1 kinases. General significance: Our methodology providing a quantitative description of O-GlcNAc patterns based on a combination of mass spectrometry and high resolution NMR spectroscopy on short peptide substrates allows subsequent functional analyses. Hence, our approach is of general interest to a wide audience of biologists aiming at deciphering the functional role of O-GlcNAc glycosylation and its crosstalk with phosphorylation.

APR 2018     

DOI : 10.1016/j.bbagen.2018.01.007  

Pubmed : 29337275

Abstract :

Living systems control cell growth dynamically by processing information from their environment. Although responses to a single environmental change have been intensively studied, little is known about how cells react to fluctuating conditions. Here, we address this question at the genomic scale by measuring the relative proliferation rate (fitness) of 3,568 yeast gene deletion mutants in out-of-equilibrium conditions: periodic oscillations between two environmental conditions. In periodic salt stress, fitness and its genetic variance largely depended on the oscillating period. Surprisingly, dozens of mutants displayed pronounced hyperproliferation under short stress periods, revealing unexpected controllers of growth under fast dynamics. We validated the implication of the high-affinity cAMP phosphodiesterase and of a regulator of protein translocation to mitochondria in this group. Periodic oscillations of extracellular methionine, a factor unrelated to salinity, also altered fitness but to a lesser extent and for different genes. The results illustrate how natural selection acts on mutations in a dynamic environment, highlighting unsuspected genetic vulnerabilities to periodic stress in molecular processes that are conserved across all eukaryotes.  

MAR 2018     

DOI : 10.15252/msb.20177823            

Pubmed : 29507053

Abstract :

Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses.

MAR 2018     

DOI : 10.1371/journal.ppat.1006908           

Pubmed : 29505618

Abstract :

Highly virulent Helicobacter pylori strains contain the cag pathogenicity island (cagPAI). It codes for about 30 proteins forming a type IV secretion system (T4SS) which translocates the pro-inflammatory protein CagA into epithelial host cells. While CagA and various other Cag proteins have been extensively studied, several cagPAI proteins are poorly characterized or of unknown function. CagN (HP0538) is of unknown function but highly conserved in the cagPAI suggesting an important role. cagM (HP0537) is the first gene of the cagMN operon and its product is part of the CagT4SS core complex. Both proteins do not have detectable homologs in other type IV secretion systems. We have characterized the biochemical and structural properties of CagN and CagM and their interaction. We demonstrate by circular dichroism, Multi-Angle Light Scattering (MALS) and small angle X-ray scattering (SAXS) that CagN is a folded, predominantly monomeric protein with an elongated shape in solution. CagM is folded and forms predominantly dimers that are also elongated in solution. We found by various in vivo and in vitro methods that CagN and CagM directly interact with each other. CagM self-interacts stably with a low nanomolar KD and can form stable multimers. Finally, in vivo experiments show that deletion of CagM reduces the amounts of CagN and other outer CagPAI proteins in H. pylori cells.

MAR 2018     

DOI : 10.1016/j.ijmm.2018.02.005         

Pubmed : 29572102

Abstract :

Facultative animal-bacteria symbioses, which are critical determinants of animal fitness, are largely assumed to be mutualistic. However, whether commensal bacteria benefit from the association has not been rigorously assessed. Using a simple and tractable gnotobiotic model-Drosophila mono-associated with one of its dominant commensals, Lactobacillus plantarum-we reveal that in addition to benefiting animal growth, this facultative symbiosis has a positive impact on commensal bacteria fitness. We find that bacteria encounter a strong cost during gut transit, yet larvae-derived maintenance factors override this cost and increase bacterial population fitness, thus perpetuating symbiosis. In addition, we demonstrate that the maintenance of the association is required for achieving maximum animal growth benefits upon chronic undernutrition. Taken together, our study establishes a prototypical case of facultative nutritional mutualism, whereby a farming mechanism perpetuates animal-bacteria symbiosis, which bolsters fitness gains for both partners upon poor nutritional conditions.

FEB 2018     

DOI : 10.1016/j.cmet.2017.11.011      

Pubmed : 29290388

Abstract :

Muscle stem cells or satellite cells are required for skeletal muscle regeneration. It has been shown that the satellite cell microenvironment, including neighboring cells such as endothelial cells, macrophages or fibroblasts are essential for complete and efficient regeneration. A deficient behavior of these cells compromises regeneration. Therefore, there is a strong interest in understanding the cellular and molecular interactions at work between these cell types during muscle regeneration. Fluorescence-activated cell sorting allows to isolate these four cell types at different time points of regeneration, for further high throughput or behavioral experiments. We present here a method for the concomitant isolation of 4 cell types present in the regenerating skeletal muscle: muscle stem cells, endothelial cells, fibro-adipogenic precursor cells and macrophages.

FEB 2018     

DOI : 10.1016/j.ymeth.2017.12.013    

Pubmed : 29269151

Abstract :

Epstein-Barr virus (EBV) expresses several mRNAs produced from intronless genes that could potentially be unfavorably translated compared to cellular spliced mRNAs. To overcome this situation, the virus encodes an RNA-binding protein (RBP) called EB2, which was previously found to both facilitate the export of nuclear mRNAs and increase their translational yield. Here, we show that EB2 binds both nuclear and cytoplasmic cap-binding complexes (CBC and eukaryotic initiation factor 4F [eIF4F], respectively) as well as the poly(A)-binding protein (PABP) to enhance translation initiation of a given messenger ribonucleoparticle (mRNP). Interestingly, such an effect can be obtained only if EB2 is initially bound to the native mRNPs in the nucleus. We also demonstrate that the EB2-eIF4F-PABP association renders translation of these mRNPs less sensitive to translation initiation inhibitors. Taken together, our data suggest that EB2 binds and stabilizes cap-binding complexes in order to increase mRNP translation and furthermore demonstrate the importance of the mRNP assembly process in the nucleus to promote protein synthesis in the cytoplasm. IMPORTANCE Most herpesvirus early and late genes are devoid of introns. However, it is now well documented that mRNA splicing facilitates recruitment on the mRNAs of cellular factors involved in nuclear mRNA export and translation efficiency. To overcome the absence of splicing of herpesvirus mRNAs, a viral protein, EB2 in the case of Epstein-Barr virus, is produced to facilitate the cytoplasmic accumulation of viral mRNAs. Although we previously showed that EB2 also specifically enhances translation of its target mRNAs, the mechanism was unknown. Here, we show that EB2 first is recruited to the mRNA cap structure in the nucleus and then interacts with the proteins eIF4G and PABP to enhance the initiation step of translation.             

FEB  2018     

DOI : 10.1128/JVI.01917-17   

Pubmed : 29142127

Abstract :

Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome. 

JAN 2018     

DOI : 10.1038/s41467-017-02793-6    

Pubmed : 29382845

Abstract :

Caspase-4/5 in humans and caspase-11 in mice bind hexa-acylated lipid A, the lipid moeity of lipopolysaccharide (LPS), to induce the activation of non-canonical inflammasome. Pathogens such as Francisella novicida express an under-acylated lipid A and escape caspase-11 recognition in mice. Here, we show that caspase-4 drives inflammasome responses to F. novicida infection in human macrophages. Caspase-4 triggers F. novicida-mediated, gasdermin D-dependent pyroptosis and activates the NLRP3 inflammasome. Inflammasome activation could be recapitulated by transfection of under-acylated LPS from different bacterial species or synthetic tetra-acylated lipid A into cytosol of human macrophage. Our results indicate functional differences between human caspase-4 and murine caspase-11. We further establish that human Guanylate-binding proteins promote inflammasome responses to under-acylated LPS. Altogether, our data demonstrate a broader reactivity of caspase-4 to under-acylated LPS than caspase-11, which may have important clinical implications for management of sepsis.

JAN 2018     

DOI : 10.1038/s41467-017-02682-y    

Pubmed : 29339744

Abstract :

In this report, we present an improved protocol for CRISPR/Cas9 genome editing in mice. The procedure consists in the electroporation of intact mouse zygotes with ribonucleoprotein complexes prepared in vitro from recombinant Cas9 nuclease and synthetic dual guide RNA. This simple cloning-free method proves to be extremely efficient for the generation of indels and small deletions by non-homologous end joining, and for the generation of specific point mutations by homology-directed repair. The procedure, which avoids DNA construction, in vitro transcription and oocyte microinjection, greatly simplifies genome editing in mice.        

JAN 2018     

DOI : 10.1038/s41598-017-18826-5    

Pubmed : 29323173

Abstract :

Huntington's disease (HD), a devastating neurodegenerative disorder, strongly affects the corticostriatal network, but the contribution of pre- and postsynaptic neurons in the first phases of disease is unclear due to difficulties performing early subcellular investigations in vivo. Here, we have developed an on-a-chip approach to reconstitute an HD corticostriatal network in vitro, using microfluidic devices compatible with subcellular resolution. We observed major defects in the different compartments of the corticostriatal circuit, from presynaptic dynamics to synaptic structure and transmission and to postsynaptic traffic and signaling, that correlate with altered global synchrony of the network. Importantly, the genetic status of the presynaptic compartment was necessary and sufficient to alter or restore the circuit. This highlights an important weight for the presynaptic compartment in HD that has to be considered for future therapies. This disease-on-a-chip microfluidic platform is thus a physiologically relevant in vitro system for investigating pathogenic mechanisms and for identifying drugs.        

JAN 2018     

DOI : 10.1016/j.celrep.2017.12.013    

Pubmed : 29298414

Abstract :

Background: Resistance to thyroid hormone due to THRA mutations (RTH) is a recently discovered genetic disease, displaying important variability in its clinical presentation. The mutations alter the function of TR1, one of the two nuclear receptors for thyroid hormone. Methods: The aim of this study was to understand the relationship between specific THRA mutations and phenotype. CRISPR/Cas9 genome editing was used to generate five new mouse models of RTH, with frameshift or missense mutations. Results: Like human patients, mutant mice displayed a hypothyroid-like phenotype, with altered development. Phenotype severity varied between the different mouse models, mainly depending on the ability of the mutant receptor to interact with transcription corepressor in the presence of thyroid hormone. Conclusion: The present mutant mice represent highly relevant models for the human genetic disease which will be useful for future investigations.

JAN  2018     

DOI : 10.1089/thy.2017.0389 

Pubmed : 29205102

Abstract :

Monocytes from FMF patients secreted significantly more IL-1β and IL-18 and died significantly faster than HD monocytes in response to low concentrations of Clostridium difficile toxin B (TcdB), a Pyrin-activating stimulus. Monocytes from patients bearing two MEFV exon 10 pathogenic variants displayed an increased Pyrin inflammasome response compared with monocytes from patients with a single exon 10 pathogenic variant indicating a gene-dosage effect. Using a short priming step, the response of monocytes from FMF patients to NLRP3- and NLRC4-activating stimuli was normal indicating that MEFV mutations trigger a specific hypersensitivity of monocytes to low doses of a Pyrin-engaging stimulus.

JAN  2018     

DOI :  10.1093/rheumatology/kex373

Pubmed : 29040788

Abstract :

Extant cetaceans are fully aquatic mammals with deep modifications of their sensory organs, notably of the sound perception pathway. Early diverging cetaceans, known as archaeocetes, show a diversity of morphologies of the petrotympanic complex and middle ear ossicles, documenting a variety of sound transmission mechanisms from a mostly terrestrial configuration to a fully aquatic layout. Protocetids are a paraphyletic assemblage of semi-aquatic archaeocetes known from the Eocene. The auditory region of these transitional' forms is only partly known. The middle Eocene locality of Kpogame, Togo (46-43 Ma) has yielded abundant material of protocetid whales documenting the auditory region, including isolated bullae, one petrosal and a skull fragment preserving the petrotympanic complex. Detailed study of this material leads us to reassess the original taxonomic attribution of this material, first attributed to Togocetus traversei, and to recognize three different protocetid taxa on the basis of bullar and petrosal remains: ?Carolinacetus sp., Togocetus traversei and Protocetidae indeterminate (morphotype ). Associating isolated petrosals or bullae with dental remains solely based on size criteria can be misleading and we show here that there is no correlation between the size of tympanic bulla and size of dental remains in protocetids. CT-scan investigation of the in situ petrotympanic complex reveals that Protocetidae retained a complete tympanic ring similar to that of terrestrial artiodactyls and that the involucrum could probably articulate with the medial side of the ventral surface of the petrosal. This bulla/petrosal articulation is absent in fully aquatic cetaceans. Finally, the close phylogenetic relationships between protocetids from Kpogame and North American protocetids suggest successive dispersals of these non-fully aquatic archaeocetes from African coasts to North America.

JAN 2018     

DOI : 10.1080/14772019.2017.1328378      

Abstract :

More and more natural DNA variants are being linked to physiological traits. Yet, understanding what differences they make on molecular regulations remains challenging. Important properties of gene regulatory networks can be captured by computational models. If model parameters can be "personalized" according to the genotype, their variation may then reveal how DNA variants operate in the network. Here, we combined experiments and computations to visualize natural alleles of the yeast GAL3 gene in a space of model parameters describing the galactose response network. Alleles altering the activation of Gal3p by galactose were discriminated from those affecting its activity (production/degradation or efficiency of the activated protein). The approach allowed us to correctly predict that a non-synonymous SNP would change the binding affinity of Gal3p with the Gal80p transcriptional repressor. Our results illustrate how personalizing gene regulatory models can be used for the mechanistic interpretation of genetic variants.             

JAN 2018     

DOI : 10.15252/msb.20177803            

Pubmed : 29335276

Abstract :

A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.          

JAN 2018     

DOI : 10.1021/acssynbio.7b00280           

Pubmed : 28915016