Intracellular Bacteria Encode Inhibitory SNARE-Like Proteins

Pathogens use diverse molecular machines to penetrate host cells and manipulate intracellular vesicular trafficking. Viruses employ glycoproteins, functionally and structurally similar to the SNARE proteins, to induce eukaryotic membrane fusion. Intracellular pathogens, on the other hand, need to block fusion of their infectious phagosomes with various endocytic compartments to escape from the degradative pathway. The molecular details concerning the mechanisms underlying this process are lacking. Using both an in vitro liposome fusion assay and a cellular assay, we showed that SNARE-like bacterial proteins block membrane fusion in eukaryotic cells by directly inhibiting SNARE-mediated membrane fusion. More specifically, we showed that IncA and IcmG/DotF, two SNARE-like proteins respectively expressed by Chlamydia and Legionella, inhibit the endocytic SNARE machinery. Furthermore, we identified that the SNARE-like motif present in these bacterial proteins encodes the inhibitory function. This finding suggests that SNARE-like motifs are capable of specifically manipulating membrane fusion in a wide variety of biological environments. Ultimately, this motif may have been selected during evolution because it is an efficient structural motif for modifying eukaryotic membrane fusion and thus contribute to pathogen survival

Screening out irrelevant cell-based models of disease

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell-and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates

CARACTERISATION FONCTIONNELLE DE LA PROTEINE A MOTIF AT-HOOK D1 DE DROSOPHILE

LE AT HOOK EST UN MOTIF DE RECONNAISSANCE STRUCTURALE DE L'ADN RICHE EN PAIRES DE BASES DA_DT. DANS LES GENOMES EUCARYOTES, CES ADNS SONT PARTICULIEREMENT ENRICHIS DANS DEUX GRANDES FAMILLES DE SEQUENCES : CERTAINS SATELLITES DE L'HETEROCHROMATINE ET PLUSIEURS DOMAINES FONCTIONNELS DE L'EUCHROMATINE TELS QUE LES SEQUENCES D'ATTACHEMENT A L'ECHAFAUDAGE NUCLEAIRE (SEQUENCES SAR) ET LES ELEMENTS FRONTIERES (BE). LES PROTEINES DE LA FAMILLE HMG-I CONTIENNENT PLUSIEURS MOTIFS AT HOOK, ASSOCIES A UN DOMAINE C-TERMINAL ACIDE. DANS LE BUT D'ABORDER LE ROLE DE CES PROTEINES IN VIVO, NOUS AVONS CARACTERISE EN DETAIL LA PROTEINE HMG-I LIKE D1 DE DROSOPHILA MELANOGASTER. D1 EST EXPRIMEE TOUT AU LONG DU DEVELOPPEMENT DE LA DROSOPHILE, MAIS SA LOCALISATION PRESENTE UN ASPECT PARTICULIEREMENT DYNAMIQUE : DISTRIBUEE DE MANIERE HOMOGENE DANS LES NOYAUX DES CELLULES TRANSCRIPTIONNELLEMENT INACTIVES, ELLE EST RETROUVEE ASSOCIEE A UN NOMBRE RESTREINT DE SITES SUR LES CHROMOSOMES DE CELLULES ACTIVES. CETTE CORRELATION ENTRE ACTIVITE TRANSCRIPTIONNELLE ET LOCALISATION DE D1 SUGGERE UN ROLE DANS LA FORMATION DE STRUCTURE CHROMATINIENNES ASSOCIEES A L'EXTINCTION DE L'EXPRESSION GENIQUE. D1 EST PAR AILLEURS PREFERENTIELLEMENT ASSOCIEE AUX SEQUENCES SATELLITE DE DENSITE 1.688 G/CM 2 RICHES EN DA_DT (SATIII) QUI CONSTITUENT L'HETEROCHROMATINE CENTROMERIQUE DU CHROMOSOME X DE LA DROSOPHILE. L'ETUDE DU PHENOMENE DE VARIEGATION DE L'EXPRESSION DU GENE WHITE PLACE A PROXIMITE DU SATIII NOUS A PERMIS DE DEMONTRER QUE LA SUREXPRESSION DE D1 CONDUISAIT A UNE DIMINUTION DE L'EXPRESSION DE WHITE, ALORS QU'UNE DIMINUTION DE SON EXPRESSION (PAR INSERTION DE L'ELEMENT EP 4 7 3 DANS LA REGION PROMOTRICE DU GENE) INDUIT UN ACCROISSEMENT DE L'EXPRESSION DE WHITE (SUPPRESSEUR DE VARIEGATION) SUGGERANT UNE IMPLICATION DIRECTE DE D1 DANS LA FORMATION D'UNE STRUCTURE CHROMATINIENNE INACTIVE. DE PLUS, L'EXPRESSION D'UNE FORME DE D1 PRIVEE DE SA PARTIE C-TERMINALE ACIDE OU D'UN POLYPEPTIDE COMPOSE DE VINGT REITERATIONS DU MOTIF AT HOOK A UN EFFET OPPOSE, SE TRADUISANT PAR UNE FORTE SUPPRESSION DE LA VARIEGATION DU GENE WHITE. NOS RESULTATS SUGGERENT QUE CET EFFET RESULTE D'UNE COMPETITION POUR LES CIBLES D'ADN QUE CONSTITUENT LE SATIII, SANS LA COMPLEMENTATION FONCTIONNELLE QUI SERAIT APPORTEE PAR LE DOMAINE C-TERMINAL ACIDE DE D1. NOUS AVONS DONC IDENTIFIE D1 COMME UN COMPOSANT A LA FOIS STRUCTURAL ET FONCTIONNEL DE L'HETEROCHROMATINE, ET DONT L'ASSOCIATION AVEC LES SEQUENCES RICHES EN DA_DT DU SATELLITE III PERMETTRAIT DE CIBLER LE RECRUTEMENT DE PARTENAIRES PROTEIQUES PAR INTERACTION AVEC SON DOMAINE C-TERMINAL.TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Next-Generation Phenotypic Screening in Early Drug Discovery for Infectious Diseases

International audienceCell-based phenotypic screening has proven to be valuable, notably in recapitulating relevant biological conditions, for example, the host cell/pathogen niche. However, the corresponding methodological complexity is not readily compatible with high-throughput pipelines, and fails to inform either molecular target or mechanism of action, which frustrates conventional drug-discovery roadmaps. We review the state-of-the-art and emerging technologies that suggest new strategies for harnessing value from the complexity of phenotypic screening and augmenting powerful utility for translational drug discovery. Advances in cellular, molecular, and bioinformatics technologies are converging at a cutting edge where the complexity of phenotypic screening may no longer be considered a hinderance but rather a catalyst to chemotherapeutic discovery for infectious diseases. Phenotypic Screening for Infectious Disease Drug Discovery The term 'phenotype' (Figure 1) was coined originally in 1903 by the Danish botanist Wilhelm Johannsen [1-3] and emerged as foundational in experimental, theoretical, and fundamental biology juxtaposed with 'genotype'. However, unlike genotype, for which a definition arises from tangible minimal information and molecular typing, the term phenotype lacks clear definition [4], relying on semantic description, for example, 'morphology', 'behavior', 'appearance', 'structure' etc. Indeed, there is an entire literature on the meaning of 'phenotype' but, more than 100 years after its conceptualization, it was only in recent years that the discipline and tools enabling ontology (see Glossary) for high-throughput and high-dimensional phenotyping began to emerge [4-7]. Specifically, for microbiology (MicroO [6]), there are efforts to align with other communitybased efforts establishing standards for ontology in genetics (GO i), phenotype (PATO ii), smallmolecule chemical entities of biological interest (ChEBI iii), and PubChem iv. Indeed, such efforts are invigorated, in part, by the realization that the performance of powerful natural language processing with neural-networks, machine-learning, and deep-learning methods is wholly dependent on the underlying means for data extraction that are linked to such ontologies, and ultimately the ability to relate them [7]. For the purposes of the current article we use the term 'phenotype' in the restrictive context of phenotypic screening [8,9]

The 4 Notch receptors play distinct and antagonistic roles in the proliferation and hepatocytic differentiation of liver progenitors

International audienceThe Notch signaling pathway is involved in liver development and regeneration. Here, we investigate the role of the 4 mammalian Notch paralogs in the regulation of hepatoblast proliferation and hepatocytic differentiation. Our model is based on bipotential mouse embryonic liver (BMEL) progenitors that can differentiate into hepatocytes or cholangiocytes in vitro and in vivo. BMEL cells were subjected to Notch antagonists or agonists. Blocking Notch activation with a γ-secretase inhibitor, at 50 μM for 48 h, reduced cell growth by 50%. S-phase entry was impaired, but no apoptosis was induced. A systematic paralog-specific strategy was set using lentiviral transduction with constitutively active forms of each Notch receptor along with inhibition of endogenous Notch signaling. This assay demonstrates that proliferation of BMEL cells requires Notch2 and Notch4 activity, resulting in significant down-regulation of p27(Kip1) and p57(Kip2) cyclin-dependent kinase inhibitors. Conversely, Notch3-expressing cells proliferate less and express 3-fold higher levels of p57(Kip2). The Notch3 cells present a hepatocyte-like morphology, enhanced multinucleation, and a ploidy shift. Moreover, Notch3 activity is conducive to hepatocytic differentiation in vitro, while its paralogs impede this fate. Our study provides the first evidence of a functional diversity among the mammalian Notch homologues in the proliferation and hepatocytic-lineage commitment of liver progenitors

Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene

The Drosophila melanogaster Nipped-B protein facilitates transcriptional activation of the cut and Ultrabithorax genes by remote enhancers. Sequence homologues of Nipped-B, Scc2 of Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces pombe are required for sister chromatid cohesion during mitosis. The evolutionarily conserved Cohesin protein complex mediates sister chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to associate with chromosomes. Here, we show that Nipped-B is also required for sister chromatid cohesion but that, opposite to the effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits long-range activation of cut. To explain these findings, we propose a model based on the chromatin domain boundary activities of Cohesin in which Nipped-B facilitates cut activation by alleviating Cohesin-mediated blocking of enhancer-promoter communication

Induction of Early Transcription in One-Cell Mouse Embryos by Microinjection of the Nonhistone Chromosomal Protein HMG-I

AbstractIn the mouse embryo, the onset of zygotic transcription occurs at the end of the first cell cycle, upon completion of DNA replication. We show that the nonhistone chromosomal protein HMG-I, whose translocation into the pronuclei of one-cell embryos is linked to this first round of DNA synthesis, plays a critical role in the activation of zygotic transcription. Indeed, microinjection of purified HMG-I results in a higher nuclear accumulation of the protein and triggers an earlier activation of zygotic transcription, an effect which is abolished by the preincubation of the protein with a specific antibody directed against its AT-hook DNA-binding motifs. Significantly, microinjection of this antibody also prevents the normal onset of transcription in the embryo, suggesting that endogenous HMG-I is similarly involved in this process. Finally, microinjection of the exogenous protein modifies chromatin structure as measured by in situ accessibility to DNase I. We propose that general chromosomal architectural factors such as HMG-I can modulate the accessibility of chromatin to specialized regulatory factors, thereby promoting a transcriptionally competent state

Shigella subverts the host recycling compartment to rupture its vacuole

International audienceShigella enters epithlial cells via internalization into a vacuole. Subsequent vacuolar membrane rupture allows bacterial escape into the cytosol for replication and cell-to-cell spread. Bacterial effectors such as IpgD, a PI(4,5)P2 phosphatase that generates PI(5)P and alters host actin, facilitate this internalization. Here, we identify host proteins involved in Shigella uptake and vacuolar membrane rupture by high-content siRNA screening and subsequently focus on Rab11, a constituent of the recycling compartment. Rab11-positive vesicles are recruited to the invasion site before vacuolar rupture, and Rab11 knockdown dramatically decreases vacuolar membrane rupture. Additionally, Rab11 recruitment is absent and vacuolar rupture is delayed in the ipgD mutant that does not dephosphorylate PI(4,5)P₂ into PI(5)P. Ultrastructural analyses of Rab11-positive vesicles further reveal that ipgD mutant-containing vacuoles become confined in actin structures that likely contribute to delayed vacular rupture. These findings provide insight into the underlying molecular mechanism of vacuole progression and rupture during Shigella invasion

Modification of position-effect variegation by competition for binding to Drosophila satellites

white-mottled (w(m4)) position-effect variegation (PEV) arises by translocation of the white gene near the pericentric AT-rich 1.688 g/cm(3) satellite III (SATIII) repeats of the X chromosome of Drosophila. The natural and artificial A•T-hook proteins D1 and MATH20 modify w(m4) PEV in opposite ways. D1 binds SATIII repeats and enhances PEV, presumably via a recruitment of protein partners, whereas MATH20 suppresses it. We show that D1 and MATH20 compete for binding to identical sites of SATIII repeats in vitro and that conditional MATH20 expression results in a displacement of D1 from pericentric heterochromatin in vivo. In the presence of intermediate levels of MATH20, we show that this displacement becomes selective for SATIII repeats. These results strongly suggest that the suppression of w(m4) PEV by MATH20 is due to a displacement of D1 from its preferred binding sites and provide additional support for a direct role of D1 in the assembly of AT-rich heterochromatin

The AT-Hook Protein D1 Is Essential for Drosophila melanogaster Development and Is Implicated in Position-Effect Variegation

We have analyzed the expression pattern of the D1 gene and the localization of its product, the AT hook-bearing nonhistone chromosomal protein D1, during Drosophila melanogaster development. D1 mRNAs and protein are maternally contributed, and the protein localizes to discrete foci on the chromosomes of early embryos. These foci correspond to 1.672- and 1.688-g/cm(3) AT-rich satellite repeats found in the centromeric heterochromatin of the X and Y chromosomes and on chromosomes 3 and 4. D1 mRNA levels subsequently decrease throughout later development, followed by the accumulation of the D1 protein in adult gonads, where two distributions of D1 can be correlated to different states of gene activity. We show that the EP473 mutation, a P-element insertion upstream of D1 coding sequences, affects the expression of the D1 gene and results in an embryonic homozygous lethal phenotype correlated with the depletion of D1 protein during embryogenesis. Remarkably, decreased levels of D1 mRNA and protein in heterozygous flies lead to the suppression of position-effect variegation (PEV) of the white gene in the white-mottled (w(m4h)) X-chromosome inversion. Our results identify D1 as a DNA-binding protein of known sequence specificity implicated in PEV. D1 is the primary factor that binds the centromeric 1.688-g/cm(3) satellite repeats which are likely involved in white-mottled variegation. We propose that the AT-hook D1 protein nucleates heterochromatin assembly by recruiting specialized transcriptional repressors and/or proteins involved in chromosome condensation