Identifying USPs regulating immune signals in Drosophila: USP2 deubiquitinates Imd and promotes its degradation by interacting with the proteasome.

International audienceBACKGROUND: Rapid activation of innate immune defences upon microbial infection depends on the evolutionary conserved NF-κB dependent signals which deregulation is frequently associated with chronic inflammation and oncogenesis. These signals are tightly regulated by the linkage of different kinds of ubiquitin moieties on proteins that modify either their activity or their stability. To investigate how ubiquitin specific proteases (USPs) orchestrate immune signal regulation, we created and screened a focused RNA interference library on Drosophila NF-κB-like pathways Toll and Imd in cultured S2 cells, and further analysed the function of selected genes in vivo. RESULTS: We report here that USP2 and USP34/Puf, in addition to the previously described USP36/Scny, prevent inappropriate activation of Imd-dependent immune signal in unchallenged conditions. Moreover, USP34 is also necessary to prevent constitutive activation of the Toll pathway. However, while USP2 also prevents excessive Imd-dependent signalling in vivo, USP34 shows differential requirement depending on NF-κB target genes, in response to fly infection by either Gram-positive or Gram-negative bacteria. We further show that USP2 prevents the constitutive activation of signalling by promoting Imd proteasomal degradation. Indeed, the homeostasis of the Imd scaffolding molecule is tightly regulated by the linkage of lysine 48-linked ubiquitin chains (K48) acting as a tag for its proteasomal degradation. This process is necessary to prevent constitutive activation of Imd pathway in vivo and is inhibited in response to infection. The control of Imd homeostasis by USP2 is associated with the hydrolysis of Imd linked K48-ubiquitin chains and the synergistic binding of USP2 and Imd to the proteasome, as evidenced by both mass-spectrometry analysis of USP2 partners and by co-immunoprecipitation experiments. CONCLUSION: Our work identified one known (USP36) and two new (USP2, USP34) ubiquitin specific proteases regulating Imd or Toll dependent immune signalling in Drosophila. It further highlights the ubiquitin dependent control of Imd homeostasis and shows a new activity for USP2 at the proteasome allowing for Imd degradation. This study provides original information for the better understanding of the strong implication of USP2 in pathological processes in humans, including cancerogenesis

Small flies to tackle big questions: assaying complex bacterial virulence mechanisms using D rosophila melanogaster

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A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila

BACKGROUND: Lysosomes are the major catabolic compartment within eukaryotic cells, and their biogenesis requires the integration of the biosynthetic and endosomal pathways. Endocytosis and autophagy are the primary inputs of the lysosomal degradation pathway. Endocytosis is specifically needed for the degradation of membrane proteins whereas autophagy is responsible for the degradation of cytoplasmic components. We previously identified the deubiquitinating enzyme UBPY/USP8 as being necessary for lysosomal biogenesis and productive autophagy in Drosophila. Because UBPY/USP8 has been widely described for its function in the endosomal system, we hypothesized that disrupting the endosomal pathway itself may affect the biogenesis of the lysosomes. RESULTS: In the present study, we blocked the progression of the endosomal pathway at different levels of maturation of the endosomes by expressing in fat body cells either dsRNAs or dominant negative mutants targeting components of the endosomal machinery: Shibire, Rab4, Rab5, Chmp1 and Rab7. We observed that inhibition of endosomal trafficking at different steps in vivo is systematically associated with defects in lysosome biogenesis, resulting in autophagy flux blockade. CONCLUSION: Our results show that the integrity of the endosomal system is required for lysosome biogenesis and productive autophagy in vivo

The Many Roles of Ubiquitin in NF-κB Signaling

The nuclear factor κB (NF-κB) signaling pathway ubiquitously controls cell growth and survival in basic conditions as well as rapid resetting of cellular functions following environment changes or pathogenic insults. Moreover, its deregulation is frequently observed during cell transformation, chronic inflammation or autoimmunity. Understanding how it is properly regulated therefore is a prerequisite to managing these adverse situations. Over the last years evidence has accumulated showing that ubiquitination is a key process in NF-κB activation and its resolution. Here, we examine the various functions of ubiquitin in NF-κB signaling and more specifically, how it controls signal transduction at the molecular level and impacts in vivo on NF-κB regulated cellular processes

A small switch has a large effect on autophagy.

International audienceAtg8 family proteins and Atg13 are important regulators of autophagy. In this issue of Structure, Suzuki and colleagues describe crystal structures of light chain 3 (LC3; Atg8) in complex with the Atg13 LC3 interacting region (LIR) motif and identify a subtle switch of side chain conformation regulating the LC3-LIR interaction and autophagosome formation in vivo

Identification des "Ubiquitin Specific proteases" impliquées dans la régulation des voies de l'immunité chez la drosophile

La dérégulation des facteurs NF-KB, impliqués dans la survie cellulaire et l'inflammation, peut entraîne des pathologies inflammatoires chroniques et des cancers. Dans ce contexte, l'objectif de ma thèse était d'identifier de régulateurs négatifs des voies NF-KB conservées au cours de l'évolution, Tollet Imd, chez la drosophile. De nombre investigation pour rechercher des régulateurs de ces voies. J'ai réalisé le crible d'une collectio d'ARN interférents permettant l'inactivation des 21 USPs de drosophile en cellules S2. Ce crible a mis en évidence trois regulateurs négatifs de la voie Imd, dont un montre également une activité sur la voie Toll. Parmi ces candidats, dUSP36 un homologue de la protéine humaine USP36, avait été préalablement sélectionné par un crible génétique au laboratoire. Des études de l'équipe auxquelles j'ai contribué, montrent son rôle in vivo dans la régulation négative de la protéin( adaptatrice Imd via son activité catalytique. Afin de caractériser la fonction des deux autres USPs, j'ai mené de! expériences de transgénèse chez la drosophile qui prouvent que ces deux USPs répriment la voie Imd en cas d'infectioI et qu'elles sont requises pour maintenir l'état inactif de la voie Imd en l'absence d'infection. J'ai également entrepris d( caractériser l'activité catalytique des deux USPs in vitro. L'originalité de mon travail a consisté à limiter le crible à un( famille de gènes, ce qui a permis de détecter de nouveaux régulateurs qui n'avaient pas été mis en évidence dans de: cribles antérieurs.The deregulation of NF-KB signalling pathways, involved in cell survival and inflammation, leads t< chronic inflammation and cancers. The aim of this thesis was to identify negative regulators of the conserved NF-KE pathways, Toll and Imd, in Drosophila melanogaster. The stability or activity of several compounds ofNF-KB pathway! is regulated by ubiquitination. Consequently, the ubiquitine specific proteases (USPs) constitute a new area to look fo regulators ofthese pathways. To do this, 1 constructed a collection of interfering RNA able to inactivate the 21 USPs 0 drosophila in S2 cells. The screening of this collection identified three negative regulators of the Imd pathway, one 0 them having also an effect on the Toll pathway. The target specificity of USPs could explain the small number 0 candidates. Among these candidates, dUSP36, a homologue of human USP36, was previously detected in the lab in : genetic screening. Team studies, in which 1 participated, show its in vivo effect on the adaptative protein Imd through it: catalytic activity. ln order to characterise the two other candidates 1 performed trangenesis experiments in drosophila These studies show that the two USPs are able to prevent activation of the Imd pathway in case of infection and that the: are required to maintain the inactivated state of the Imd pathway in absencê of infection. 1 also started to characterise th4catalytic activity of both candidates in vitro. My work's novelty was to limit the screening to only one gene family which allowed the detection of new regulating genes which had not been revealed in previous screenings performed on ; large part or the entire genome.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Genome-wide analysis of genes encoding core components of the ubiquitin system during cerebral cortex development

International audienceAbstract Ubiquitination involves three types of enzymes (E1, E2, and E3) that sequentially attach ubiquitin (Ub) to target proteins. This posttranslational modification controls key cellular processes, such as the degradation, endocytosis, subcellular localization and activity of proteins. Ubiquitination, which can be reversed by deubiquitinating enzymes (DUBs), plays important roles during brain development. Furthermore, deregulation of the Ub system is linked to the pathogenesis of various diseases, including neurodegenerative disorders. We used a publicly available RNA-seq database to perform an extensive genome-wide gene expression analysis of the core components of the ubiquitination machinery, covering Ub genes as well as E1, E2, E3 and DUB genes. The ubiquitination network was governed by only Uba1 and Ube2m , the predominant E1 and E2 genes, respectively; their expression was positively regulated during cortical formation. The principal genes encoding HECT (homologous to the E6-AP carboxyl terminus), RBR (RING-in-between-RING), and RING (really interesting new gene) E3 Ub ligases were also highly regulated. Pja1 , Dtx3 (RING ligases) and Stub1 (U-box RING) were the most highly expressed E3 Ub ligase genes and displayed distinct developmental expression patterns. Moreover, more than 80 DUB genes were expressed during corticogenesis, with two prominent genes, Uch-l1 and Usp22, showing highly upregulated expression. Several components of the Ub system overexpressed in cancers were also highly expressed in the cerebral cortex under conditions not related to tumour formation or progression. Altogether, this work provides an in-depth overview of transcriptomic changes during embryonic formation of the cerebral cortex. The data also offer new insight into the characterization of the Ub system and may contribute to a better understanding of its involvement in the pathogenesis of neurodevelopmental disorders

Rôle de l'exoenzyme S de Pseudomonas aeruginosa dans la virulence bactérienne (étude fonctionnelle du domaine GAP et de ses cibles sur la réponse immunitaire chez Drosophila melanogaster)

L'exoenzyme S, \Ille toxine de type III, de Pseudomonas aeruginosa possède W1 domaine GAP (GTPase Adivating Protein) (ExoSGAP) inhibant les Rho GTPases (Rho, Rac, Cdc42) et la phagocytose dans les cellules de Mammifères en culture. J'ai utilisé une approche de transgenèse chez Drosopbila melanogaster en utilisant un système d'expression tissu-spécifique inductible (U AS-GaJ4) afm d'exprimer ExoSGAP. Nous avons montré qu'ExoSGAP cible in vivo les Rho GTPases Rho, Rac1, Rac2 et Cdc42. ExoSGAP affecte la résistence des mouches aux infections en inhibant la phagocytose des bactéries par les plasmatocytes, des cellules de type macrophage, mais n'a pas d'effet sur les voies NF-kB. Une approche génétique a permis d'identifier de nouvelles cibles de la toxine, en recherchant des gènes dont la déréguIation modifie le phéndype d' œil ou d'aile induit par l'expression d'ExoSGAP. Nous avons identifié plusieurs gènes pouvant avoir un rôle dans les voies des JNK et NF-kB Ces résultats valident une stratégie d'étude des toxines de type III par transgenèse chez la drosophile.J'ai pa.raIIèlement momré la spécificité de la GTPase Ra.c2 dans la résistance des mouches aux infections bactériennes. Rac2 participe notamment à la phagocytose. Les travaux du Dr. H. Tricoire ont permis d'identifier 180 gènes dont la dérégulation modifie la réponse des mouches à un stress oxydant. J'ai testé 105 de ces lignées pour leur résistance aux infections, afin d'étudier une corrélation possible entre la réponse aux stress oxydant et infectieux. Ce crible a permis de montrer l'implication d'une protéine à domaine lectine PSLR (Pseudomonas Sensitive Lectin Receptor) dans la réponse imnmnitaire de la drosophile.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

The <b><i>Drosophila</i></b> Deubiquitinating Enzyme dUSP36 Acts in the Hemocytes for Tolerance to <b><i>Listeria monocytogenes</i></b> Infections

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Deubiquitinating Enzymes Related to Autophagy: New Therapeutic Opportunities?

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