An amphipathic alpha-helix at the C terminus of hepatitis C virus nonstructural protein 4B mediates membrane association.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is an integral membrane protein that has been only poorly characterized to date. It is believed to comprise a cytosolic N-terminal part, a central part harboring four transmembrane passages, and a cytosolic C-terminal part. Here, we describe an amphipathic alpha-helix at the C terminus of NS4B (amino acid residues 229 to 253) that mediates membrane association and is involved in the formation of a functional HCV replication complex.

Molecular characterization of Unc5CL/ZUD

Résumé : Au cours de l'évolution, les organismes multicellulaires ont développé le système immunitaire afin de pouvoir se défendre contre les pathogènes tel que les bactéries, les virus, et les parasites. La réponse immunitaire doit être finement régulée par différentes voies de signalisation moléculaire, afin d'assurer une efficacité optimale, et d'éviter des dommages tissulaires indésirables. Les résultats expérimentaux décrits dans ce manuscrit, mettent en évidence que la protéine Unc5CL, qui contient un death domain (DD), est impliquée dans la régulation de la réponse immunitaire des muqueuses. Il a été démontré que cette protéine contient aussi un domaine transmembranaire de type III dans sa partie N-terminale, permettant ainsi de l'ancrer et d'exposer sa partie C-terminale dans le cytosol, un prérequis pour la signalisation dans ce compartiment cellulaire. De plus, cette protéine a la capacité d'activer le facteur de transcription NFxB, qui joue un rôle important dans le système immunitaire, ainsi que dans d'autres processus cellulaires essentiels. Le profil transcriptionnel révèle que l'activation de NF-κB induite par Unc5CL conduit principalement à une réponse inflammatoire, qui se caractérise par la production de diverses chimiokines (e.g. CXCL-1, IL-8 et CCL20). Il a également été démontré que Unc5CL requiert les mêmes molécules qui sont utilisées dans la voie de signalisation des récepteurs de la famille toll et de l'interleukine-1. De manière similaire à leur protéine adaptatrice MyD88, Unc5CL a la capacité de recruter, via une interaction homotypique DD-DD, les kinases IRAK1 et IRAK4 qui contiennent elles aussi un DD, permettant ainsi au signal d'être transmis. La production d'un anticorps polyclonal contre le DD de Unc5CL a permis d'identifier des lignées cellulaires et des tissus exprimant cette protéine, ainsi que de déterminer sa localisation sub-cellulaire. Unc5CL a été détecté dans les cellules de la muqueuse utérine et intestinale, ainsi que dans une lignée cellulaire issue d'un adénocarcinome colorectal humain, les CaCo-2. Dans chacun de ces cas, Unc5CL a été principalement détectée au niveau apical des cellules épithéliales polarisées. De manière similaire à PIDD, une protéine impliquée dans la réponse aux dommages à l'ADN, et au constituant des pores nucléaires Nup98, Unc5CL est constitutivement clivé de manière autoprotéolytique, au niveau d'un site HFS. Il est intéressant d'observer que les deux fragments ainsi générés restent fortement associés l'un à l'autre après clivage. Finalement, un criblage protéomique pour identifier un partenaire d'interaction, a mis en évidence l'ubiquitin ligase E3 ITCH, qui régule de manière négative Unc5CL en augmentant sa dégradation. Summary : Multicellular organisms have evolved the immune system in order to defend themselves against pathogens such as bacteria, viruses and eukaryotic parasites. Immune responses have to be tightly orchestrated by signaling mechanisms to achieve optimal effectiveness and minimal tissue damage. The experimental results in this thesis manuscript provide evidence that the death domain (DD)-containing protein Unc5CL might be involved in the regulation of mucosal immune responses. It could be shown that the protein contains an N-terminal type-III transmembrane domain that anchors the protein with its C-terminus exposed to the cytosol, a prerequisite for signaling events in this compartment. Furthermore, the protein has the capacity to activate the transcription factor NF-κB, which plays an important role in the immune system as well as in other essential cellular processes. Transcriptional profiling revealed that Unc5CL-mediated activation of NF-κB mainly leads to an inflammatory response, characterized by the production of chemokines (e.g. CXCL-l, IL-8 and CCL20). Furthermore, it could be shown that Unc5CL requires the same downstream signaling molecules as the evolutionarily ancient tolUinterleukin-1 receptor family. Similar to their adapter protein MyD88, Unc5CL has the capacity to recruit the DD-containing kinases IRAKI and IRAK4 for signaling and can interact with these proteins via homotypic DD-DD interactions. Generation of polyclonal antibodies raised against the DD of Unc5CL allowed the identification of cell lines and tissues that express the endogenous protein as well as to confine its subcellular localization. Unc5CL was detected in primary mucosal uterine and intestinal epithelial cells as well as in the human colorectal adenocarcinoma cell line CaCo-2. In all cases, the protein was mainly localized to the apical face of these polarized epithelial cells. Similar to PIDD, a protein critically involved in responses to DNA damage, and the nuclear pore component Nup98, Unc5CL is constitutively autoproteolytically processed at an HFS site. Interestingly, the two generated cleavage fragments remain tightly associated after processing. Finally, a proteomics screen for interaction partners identified the E3 ubiquitin ligase ITCH as a negative regulator of Unc5CL by targeting the protein for degradation.

Genetics of essential tremor.

Essential tremor (ET) is a prevalent condition manifesting with progressive action tremor. Although ET was traditionally viewed as a sporadic disease, a significant proportion of cases report a positive family history of tremor. Autosomal dominant inheritance can be demonstrated in many families. Previously, genome-wide linkage studies in families mapped three loci for ET, hereditary essential tremor-1 (ETM1), ETM2 and ETM3. However, no causal mutation has been replicated in candidate genes within these loci, including dopamine D3 receptor (DRD3) and HS1-binding protein 3 (HS1BP3). Recently, the first genome-wide association study in ET followed by replication studies conducted in diverse populations identified a significant association between the leucine-rich repeat and Ig domain containing 1 gene (LINGO1) SNP rs9652490 and risk for ET Although further novel variants were indentified in LINGO1 and its paralog LINGO2 that may be associated with risk for ET, the pathogenic mechanisms involved remain elusive. Given the possibility that ET as a complex trait may be influenced by the combined effects of rare variants, novel high-throughput technologies sequencing all exons across the genome (exome sequencing) or the whole genome (genome sequencing) may become crucial in understanding/deciphering the genetic background of ET.

Expression of foraging and Gp-9 are associated with social organization in the fire ant Solenopsis invicta.

The aim of this study was to investigate levels of expression of two major genes, the odorant binding protein Gp-9 (general protein-9) and foraging, that have been shown to be associated with behavioural polymorphisms in ants. We analysed workers and young nonreproductive queens collected from nests of the monogyne (single reproductive queen per nest) and polygyne (multiple reproductive queens) social forms of Solenopsis invicta. In workers but not young queens, the level of foraging expression was significantly associated with social form and the task performed (ie localization in the nest or foraging area). The level of expression of Gp-9 was also associated with social form and worker localization. In addition there was a higher level of expression of the Gp-9(b) allele compared with the Gp-9(B) allele in the heterozygote workers and the young nonreproductive queens. Finally, in the polygyne colonies the level of expression of foraging was not significantly associated with the Gp-9 genotype for either workers or young nonreproductive queens, suggesting that both genes have independent non-epistatic effects on behaviour in S. invicta.

Podocalyxin is a novel polysialylated neural adhesion protein with multiple roles in neural development and synapse formation

Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments in vitro and phenotypic analyses of podxl-deficient mice indicate that PC is involved in neurite growth, branching and axonal fasciculation, and that PC loss-of-function reduces the number of synapses in the CNS and in the neuromuscular system. We also show that whereas some of the brain PC functions require PSA, others depend on PC per se. Our results show that PC, the second highly sialylated neural adhesion protein, plays multiple roles in neural development.

Expression and function of the NALP3 inflammasome in rheumatoid synovium.

Summary The NACHT, LRR and PYD domains containing protein (NALP3) inflammasome is a key regulator of interleukin-1beta (IL-1beta) secretion. As there is strong evidence for a pro-inflammatory role of IL-1beta in rheumatoid arthritis (RA) and in murine models of arthritis, we explored the expression of the different components of the NALP3 inflammasome as well as other nucleotide oligomerization domain (NOD)-like receptors (NLRs) in synovium obtained from patients with RA. The expression of NLRs was also studied in fibroblast lines derived from joint tissue. By immunohistology, NALP3 and apoptosis-associated speck-like protein containing a CARD domain (ASC) were expressed in myeloid and endothelial cells and B cells. T cells expressed ASC but lacked NALP3. In synovial fibroblast lines, NALP3 expression was not detected at the RNA and protein levels and stimulation with known NALP3 agonists failed to induce IL-1beta secretion. Interestingly, we were unable to distinguish RA from osteoarthritis synovial samples on the basis of their basal level of RNA expression of known NLR proteins, though RA samples contained higher levels of caspase-1 assayed by enzyme-linked immunsorbent assay. These results indicate that myeloid and endothelial cells are the principal sources of inflammasome-mediated IL-1beta production in the synovium, and that synovial fibroblasts are unable to activate caspase-1 because they lack NALP3. The NALP3 inflammasome activity does not account for the difference in level of inflammation between RA and osteoarthritis.

Molecular mechanisms implicated in NF-κB activation and antiviral defense

SUMMARY Nuclear factor kappa B (NF-κB) transcription factors control many aspects of cell fate through induction of inflammatory, immune or survival molecules. We have identified two novel proteins, named receptor interacting protein (RIP)-4 and caspase recruitment domain (CARD) adaptor inducing interferon-β (Cardif), which activate NF-κB. Further, we have found that Cardif plays a prominent antiviral function. Antiviral innate immunity is mounted upon recognition by the host of virally associated structures like double-stranded (ds) RNA, which constitutes a viral replication product of many viruses within infected cells. dsRNA, depending on its subcellular localization, can be sensed by two separate arms of host defense. Firstly, Toll-like receptor (TLR)-3, a member of the type I transmembrane TLR family, recognizes endosomally-located dsRNA. Secondly, cytoplasmic dsRNA is detected by the recently identified RNA helicase retinoic acid inducible gene I (RIG-I). Triggering of TLR3- and RIG-I-dependent pathways results in the activation of the transcription factors NF-κB and Interferon regulatory factor (IRF)-3, which cooperatively transduce antiviral immune responses. We have demonstrated that RIP1, a kinase previously shown to be required for TNF signaling, transmits TLR3-dependent NF-κB activation. Further we have identified and characterized Cardif as an essential adaptor transmitting RIG-I-mediated antiviral responses, including activation of NF-κB and IRF3. In addition, we showed that Cardif is cleaved and inactivated by a serine protease of hepatitis C virus, and therefore may represent an attractive target for this virus to escape innate immune responses. RESUME Les facteurs de transcription "nuclear factor kappa B" (NF-κB) contrôlent divers aspects du devenir cellulaire à travers l'induction de molécules inflammatoires, immunitaires ou de survie. Nous avons identifié deux nouvelles protéines, nommées "receptor interacting protein" (RIP)-4 et "caspase recruitment domain (CARD) adaptor inducing interferon-β" (Cardif), qui activent NF-κB. En outre, nous avons trouvé que Cardif joue un rôle antiviral crucial. L'immunité innée antivirale s'établit au moment de la reconnaissance par l'hôte de structures virales, comme l'ARN double brin, qui constitue un produit de réplication de beaucoup de virus à l'intérieur de cellules infectées. L'ARN double brin, dépendant de sa localisation subcellulaire, peut être détecté par deux branches de défense distinctes. Premièrement, le récepteur transmembranaire "Toll-like" (TLR), TLR3, reconnaît l'ARN double brin lorsque localisé dans les endosomes. Deuxièmement, l'ARN double brin cytoplasmique est reconnu par l'ARN hélicase récemment décrite "retinoic acid inducible gene I" (RIG-I). Le déclenchement de voies dépendantes de TLR3 et RIG-I active les facteurs de transcription NF-κB et IRF3, qui coopèrent afin de transduire des réponses immunitaires antivirales. Nous avons démontré que RIP1, une kinase décrite précédemment dans le signalement du TNF, transmet l'activation de NF-κB dépendante de TLR3. De plus, nous avons identifié et caractérisé Cardif comme un adapteur essentiel transmettant les réponses antivirales médiées par RIG-I, qui incluent l'activation de NF-κB et IRF3. De surcroît, Cardif est clivé et inactivé par une sérine protéase du virus de l'hépatite C, et ainsi pourrait représenter une cible attractive pour ce virus afin d'échapper aux réponses immunitaires innées.

NLRC5, at the Heart of Antigen Presentation.

Nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs) are intracellular proteins mainly involved in pathogen recognition, inflammatory responses, and cell death. Until recently, the function of the family member NLR caspase recruitment domain (CARD) containing 5 (NLRC5) has been a matter of debate. It is now clear that NLRC5 acts as a transcriptional regulator of the major-histocompatibility complex class I. In this review we detail the development of our understanding of NLRC5 function, discussing both the accepted and the controversial aspects of NLRC5 activity. We give insight into the molecular mechanisms, and the potential implications, of NLRC5 function in health and disease.

Podocalyxin is a novel polysialylated neural adhesion protein with multiple roles in neural development and synapse formation

Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments in vitro and phenotypic analyses of podxl-deficient mice indicate that PC is involved in neurite growth, branching and axonal fasciculation, and that PC loss-of-function reduces the number of synapses in the CNS and in the neuromuscular system. We also show that whereas some of the brain PC functions require PSA, others depend on PC per se. Our results show that PC, the second highly sialylated neural adhesion protein, plays multiple roles in neural development.

Differential expression of NLRP3 among hematopoietic cells.

Although the importance of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in health and disease is well appreciated, a precise characterization of NLRP3 expression is yet undetermined. To this purpose, we generated a knock-in mouse in which the Nlrp3 coding sequence was substituted for the GFP (enhanced GFP [egfp]) gene. In this way, the expression of eGFP is driven by the endogenous regulatory elements of the Nlrp3 gene. In this study, we show that eGFP expression indeed mirrors that of NLRP3. Interestingly, splenic neutrophils, macrophages, and, in particular, monocytes and conventional dendritic cells showed robust eGFP fluorescence, whereas lymphoid subsets, eosinophils, and plasmacytoid dendritic cells showed negligible eGFP levels. NLRP3 expression was highly inducible in macrophages, both by MyD88- and Trif-dependent pathways. In vivo, when mice were challenged with diverse inflammatory stimuli, differences in both the number of eGFP-expressing cells and fluorescence intensity were observed in the draining lymph node. Thus, NLRP3 levels at the site of adaptive response initiation are controlled by recruitment of NLRP3-expressing cells and by NLRP3 induction.

From protein sequences to 3D-structures and beyond: the example of the UniProt knowledgebase.

With the dramatic increase in the volume of experimental results in every domain of life sciences, assembling pertinent data and combining information from different fields has become a challenge. Information is dispersed over numerous specialized databases and is presented in many different formats. Rapid access to experiment-based information about well-characterized proteins helps predict the function of uncharacterized proteins identified by large-scale sequencing. In this context, universal knowledgebases play essential roles in providing access to data from complementary types of experiments and serving as hubs with cross-references to many specialized databases. This review outlines how the value of experimental data is optimized by combining high-quality protein sequences with complementary experimental results, including information derived from protein 3D-structures, using as an example the UniProt knowledgebase (UniProtKB) and the tools and links provided on its website ( http://www.uniprot.org/ ). It also evokes precautions that are necessary for successful predictions and extrapolations.

Characterization of cytoplasmic antiviral signaling pathways

Summary Multicellular organisms have evolved the immune system to protect from pathogen such as viruses, bacteria, fungi or parasites. Detection of invading pathogens by the host innate immune system is crucial for mounting protective responses and depends on the recognition of microbial components by specific receptors. The results presented in this manuscript focus on the signaling pathways involved in the detection of viral infection by the sensing of viral nucleic acids. First, we describe a new regulatory mechanism controlling RNA-sensing antiviral pathways. Our results indicate that TRIF and Cardif, the crucial adaptor proteins for endosomal and cytoplasmic RNA detection signaling pathway, are processed and inactivated by caspases. The second aspect investigated here involves a signaling pathway triggered upon cytosolic DNA sensing. The interferon inducible protein DAI was recently described as a DNA sensor able to induce the activation of IRFs and NF-κΒ transcription factors leading to type I interferon production. Here we identify two RIP homotypic interaction motifs (RHIMs) in DAI and demonstrate that they mediate the recruitment of RIP1 and RIP3 and the subsequent NF-κΒ activation. Moreover, we observed that the mouse cytomegalovirus RHIM- containing protein M45 has the potential to block this signaling cascade by interfering with the formation of the DAI-RIP1/3 signaling complex. Finally, we report the generation and the initial characterization of NLRX1-deficient mice. NLRX1 is a member of the NOD-like receptor family localized to the mitochondria. The function of NLRX1 is still controversial: one study proposed that NLRX1 acts as an inhibitor of the RIG-like receptor (RLR) antiviral pathway by binding the adaptor protein Cardif, whereas another report implicated NLRX1 in the generation of reactive oxygen species (ROS) and the amplification of NF-κΒ and JNK triggered by TNF-α, poly(I:C) or Shigella infection. Collectively, our results indicate that NLRX1-deficiency does not affect RLR signaling nor TNF-α induced responses. Proteomics analysis identified UQCRC2, a subunit of the complex III of the mitochondrial respiratory chain, as a NLRX1 binding partner. This observation might reveal a possible functional link between NLRX1 and mitochondrial respiration and/or ROS generation. Résumé Au cours de l'évolution, les organismes multicellulaires ont développé le système immunitaire afin de se protéger contre les pathogènes. Une étape cruciale pour le déclenchement des réponses protectrices est la reconnaissance par les cellules du système immunitaire de molécules propres aux microbes grâce à des récepteurs spécifiques. Les résultats présentés dans cette thèse décrivent des nouveaux aspects concernant les voies de signalisation impliquées dans la détection des virus. Le premier projet décrit un mécanisme de régulation des voies activées par la détection d'ARN virale. Nos résultats montrent que TRIF et Cardif, des protéines adaptatrices des voies déclenchées par la reconnaissance de ces acides nucléiques au niveau des endosomes et du cytoplasme, sont clivés et inactivés par les caspases. Le projet suivant de notre recherche concerne une voie de signalisation activée par la détection d'ADN au niveau du cytoplasme. La protéine DAI a été récemment décrite comme un senseur pour cet ADN capable d'activer les facteurs de transcription IRF et NF-κΒ et d'induire ainsi la production des interférons de type I. Ici on démontre que DAI interagit avec RIP1 et RIP3 par le biais de domaines appelés RHIM et que ce complexe est responsable de l'activation de NF-κΒ. On a aussi identifié une protéine du cytomégalovirus de la souris, M45, qui contient ce même domaine et on a pu démontrer qu'elle a la capacité d'interférer avec la formation du complexe entre DAI et RIP1/RIP3 bloquant ainsi l'activation de NF-κΒ. Enfin on décrit ici la génération de souris déficientes pour le gène qui code pour la protéine NLRX1. Cette protéine fait partie de la famille des récepteurs NOD et est localisée dans la mitochondrie. Une étude a suggéré que NLRX1 agit comme un inhibiteur des voies antivirales activées par les récepteurs du type RIG-I (RLR) en interagissant avec la protéine adaptatrice Cardif. Une autre étude propose par contre que NLRX1 participe à la production des dérivés réactifs de l'oxygène et contribue ainsi à augmenter l'activation de NF- κΒ et JNK induite par le TNF-α ou le poly(I:C). Nos résultats montrent que l'absence de NLRX1 ne modifie ni la voie de signalisation RLR ni les réponses induites par le TNF-α. Des analyses ultérieures ont permis d'identifier comme partenaire d'interaction de NLRX1 la protéine UQCRC2, une des sous-unités qui composent le complexe III de la chaîne respiratoire mitochondriale. Cette observation pourrait indiquer un lien fonctionnel entre NLRX1 et la respiration mitochondriale ou la production des dérivés réactifs de l'oxygène au niveau de cette organelle.

Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors.

The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-gamma (IFN-gamma)-producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X(7) purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1beta (IL-1beta). The priming of IFN-gamma-producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3(-/-)) or caspase-1-deficient (Casp-1(-/-)) mice unless exogenous IL-1beta is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7(-/-) or Nlrp3(-/-) or Casp1(-/-) hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

Ash2 acts as an Ecdysone Receptor coactivator by stabilizing the histone methyltransferase Trr.

The molting hormone ecdysone triggers chromatin changes via histone modifica- tions that are important for gene regulation. On hormone activation, the ecdysone receptor (EcR) binds to the SET domain-containing histone H3 methyltransferase trithorax-related protein (Trr). Methylation of histone H3 at lysine 4 (H3K4me), which is associated with tran- scriptional activation, requires several cofactors, including Ash2. We find that ash2 mutants have severe defects in pupariation and metamorphosis due to a lack of activation of ecdy- sone-responsive genes. This transcriptional defect is caused by the absence of the H3K4me3 marks set by Trr in these genes. We present evidence that Ash2 interacts with Trr and is re- quired for its stabilization. Thus we propose that Ash2 functions together with Trr as an ecdysone receptor coactivator.

Spontaneous Cdc42 polarization independent of GDI-mediated extraction and actin-based trafficking.

The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.