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International audience; Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disease caused by an expansion of 55-200 CGG repeats located in the FMR1 gene. The main clinical and neuropathological features of FXTAS are progressive intention tremor and gait ataxia associated with brain atrophy, neuronal cell loss and presence of ubiquitin-positive intranuclear inclusions in both neurons and astrocytes. At the molecular level, FXTAS is characterized by increased expression of FMR1 sense and antisense RNA containing expanded CGG or GGC repeats, respectively. Here, we discuss the putative molecular mechanisms underlying FXTAS and notably recent reports that expanded CGG and GGC repeats may be pathogenic through RAN translation into toxic proteins.

The mechanism of NEMO recruitment into supramolecular complexes and its dependence on ubiquitination differs in response to the proinflammatory cytokines TNF and IL-1., Nuclear factor κB (NF-κB) essential modulator (NEMO), a regulatory component of the IκB kinase (IKK) complex, controls NF-κB activation through its interaction with ubiquitin chains. We show here that stimulation with interleukin-1 (IL-1) and TNF induces a rapid and transient recruitment of NEMO into punctate structures that are anchored at the cell periphery. These structures are enriched in activated IKK kinases and ubiquitinated NEMO molecules, which suggests that they serve as organizing centers for the activation of NF-κB. These NEMO-containing structures colocalize with activated TNF receptors but not with activated IL-1 receptors. We investigated the involvement of nondegradative ubiquitination in the formation of these structures, using cells deficient in K63 ubiquitin chains or linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination. Our results indicate that, unlike TNF, IL-1 requires K63-linked and linear ubiquitin chains to recruit NEMO into higher-order complexes. Thus, different mechanisms are involved in the recruitment of NEMO into supramolecular complexes, which appear to be essential for NF-κB activation.

International audience; Analyzing protein ubiquitylation changes during physiological or pathological processes is challenging due to its high reversibility and dynamic turnover of modified targets. We have developed a protein microarray to assess endogenous ubiquitylation levels from cell cultures, employing tandem ubiquitin-binding entities (TUBEs) with three or four ubiquitin-associated (UBA) domains as capture probes. Adriamycin (ADR)-stimulated MCF7 cells were used to differentiate protein ubiquitylation levels between cells that are sensitive or resistant to ADR treatment. We show that TUBEs-based microarrays can be used for the analysis of cellular processes regulated by ubiquitylation and for the detection of pathologies with aberrant ubiquitylation levels.

International audience; The yeast Saccharomyces cerevisiae is able to form complex multicellular structures called mats on low-density agar Petri plates. Mat formation strictly depends on Flo11p, a cell surface mannoprotein that mediates the adhesion of yeast cells to the agar surface. Here, we show that Swa2p, an auxilin ortholog required for clathrin-coated vesicle uncoating, is strictly required for biofilm formation. We show that the maturation and cellular levels of Flo11p are affected in Deltaswa2 cells, yet without compromising invasive growth. Both the TPR and J-domains of Swa2p, but not its clathrin-binding and ubiquitin-association motifs, are required for its function in Flo11p processing.

International audience; Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded by pericentriolar material. The pericentriolar material contains factors that are involved in microtubule nucleation and organization, and its recruitment varies during the cell cycle. We report here that proteasome inhibition in HeLa cells induces the accumulation of several proteins at the pericentriolar material, including gamma-tubulin, GCP4, NEDD1, ninein, pericentrin, dynactin, and PCM-1. The effect of proteasome inhibition on centrosome proteins does not require intact microtubules and is reversed after removal of proteasome inhibitors. This accrual of centrosome proteins is paralleled by accumulation of ubiquitin in the same area and increased polyubiquitylation of nonsoluble gamma-tubulin. Cells that have accumulated centrosome proteins in response to proteasome inhibition are impaired in microtubule aster formation. Our data point toward a role of the proteasome in the turnover of centrosome proteins, to maintain proper centrosome function.

International audience; Ubiquitin inclusions represent a cytopathological hallmark of ALS. The ubiquitin-dependent protein degradation pathway may also be involved in the pathophysiology of SOD1 mutated ALS cases as demonstrated in transgenic animals. UBE2H is an ubiquitin conjugating enzyme known to act on histones and cytoskeletal proteins, both involved in the degenerative pathway of the motor neuron. We screened the whole coding sequence of the UBE2H gene in 24 sporadic ALS (SALS) patients using single strand conformation polymorphism (SSCP). All variants detected by SSCP were analysed by genomic DNA sequencing. We found one known polymorphism (rs12539800) and two new synonymous single nucleotide polymorphisms (SNP) (nG78A and nG501A). The allele distribution of the rs12539800 (A336G) SNP were tested for association in 252 SALS patients and 357 controls. The allele and genotype distributions were identical in the two groups. The UBE2H gene is not implicated in SALS; however, the ubiquitin pathway is worthy of further investigation in ALS.

Deltex (DTX) and AIP4 are the human orthologues of the Drosophila deltex and Suppressor of deltex, which have been genetically described as being antagonistically involved in the Notch signalling pathway. Both genes encode E3 ubiquitin ligases of the RING (Really interesting new gene)-H2 and HECT (Homologous to E6AP carboxyl terminus) families, respectively. In an attempt to understand the molecular basis of their genetic interactions, we studied the relationship between DTX and AIP4 in the absence of activation of the Notch pathway. We show here that both molecules interact and partially colocalize to endocytic vesicles, and that AIP4 targets DTX for lysosomal degradation. Furthermore, AIP4-generated polyubiquitin chains are mainly conjugated through lysine 29 of ubiquitin in vivo, indicating a link between this type of chain and lysosomal degradation.

International audience; Genes coding for cell cycle components predicted to be essential for its regulation have been shown to be dispensable in mice, at the whole organism level. Such studies have highlighted the extraordinary plasticity of the embryonic cell cycle and suggest that many aspects of in vivo cell cycle regulation remain to be discovered. Here, we discuss the particularities of the mouse early embryonic cell cycle and review the mutations that result in cell cycle defects during mouse early embryogenesis, including deficiencies for genes of the cyclin family (cyclin A2 and B1), genes involved in cell cycle checkpoints (Mad2, Bub3, Chk1, Atr), genes involved in ubiquitin and ubiquitin-like pathways (Uba3, Ubc9, Cul1, Cul3, Apc2, Apc10, Csn2) as well as genes the function of which had not been previously ascribed to cell cycle regulation (Cdc2P1, E4F and Omcg1).

International audience; The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor alpha (ERalpha) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERalpha proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERalpha and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERalpha. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERalpha are mechanistically separable functions of ERalpha. We find that proteolysis of ERalpha correlates with the ability of ERalpha mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERalpha proteolysis and transcription.

International audience; Protein transduction domains (PTDs) are promising tools for transducing presynthesized polypeptides across the plasma membrane. However, the development and optimization of PTDs are hampered by many technical problems and artifacts resulting notably from the tight binding of PTDs to the cell surface and the difficulty in discriminating, through imagery analyses, truly cytosolic from cytoplasmic vesicular compartments. To circumvent these problems, we have developed an unambiguous enzymatic assay of the cytosolic uptake of PTD-driven proteins, based on the processing by ubiquitin-specific C-terminal proteases (DUBs). This method, coupled with fluorometry and fluorescence microscopy, shows that the TAT PTD derived from human immunodeficiency virus type 1 is rapidly taken up by cells but fails to reach their cytosol, except when dendritic cells, which are known to take up circulating antigens for cross-presentation, are used. In addition to its usefulness in assessing cytosolic uptake, DUB processing of PTD-linked proteins can ensure the intracellular release of cargo proteins, which might prove helpful for MHC-I-based vaccination or intracellular delivery of biologically active polypeptides.

International audience; Exposure of cells to stressful conditions results in the rapid synthesis of a subset of specialized proteins termed heat shock proteins (HSPs) which function in protecting the cell against damage. The stress-induced activation of hsp genes is controlled by the heat shock transcription factor 1 (HSF1). At the cellular level, one of the most striking effects of stress is the rapid and reversible redistribution of HSF1 into a few nuclear structures termed nuclear stress granules which form primarily on the 9q12 locus in humans. Within these structures, HSF1 binds to satellite III repeated elements and drives the RNA polymerase II-dependent transcription of these sequences into stable RNAs which remain associated with the 9q12 locus for a certain time after synthesis. Other proteins, in particular splicing factors, were also shown to relocalize to the granules upon stress. Here, we investigated the role of stress-induced satellite III transcripts in the relocalization of splicing factors to the granules. We show that the recruitment of the two serine/arginine-rich (SR) proteins SF2/ASF and SRp30c requires the presence of stress-induced satellite III transcripts. In agreement with these findings, we identified the second RNA-recognition motif (RRM2) of hSF2/ASF as the motif required for the targeting to the granules, and we showed by immunoprecipitation that the endogenous hSF2/ASF protein is present in a complex with satellite III transcripts in stressed cells in vivo. Interestingly, satellite III transcripts also immunoprecipitate together with small nuclear ribonucleoproteins (snRNPs) in vivo whereas the intronless hsp70 transcripts do not, supporting the proposal that these transcripts are subject to splicing. Altogether, these data highlight the central role for satellite III transcripts in the targeting and/or retention of splicing factors into the granules upon stress.

International audience; Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other gamma-secretase substrates.

International audience; The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y13 receptor (P2Y13-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y13-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y13-R. When transiently expressed, P2Y13-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y12 receptor (P2Y12-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y13-R in the ER, suggesting a close link between ubiquitination of P2Y13-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y13-R and P2Y12-R strongly restored plasma membrane expression of P2Y13-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y13-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y13-R in hepatocytes, and consequently stimulated P2Y13-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y13-R-deficient mice, thus reinforcing the role of P2Y13-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin-proteasome system might be a novel powerful HDL therapy to enhance P2Y13-R expression and consequently promote the overall RCT.

International audience; Hypomorphic mutations in the X-linked human NEMO gene result in various forms of anhidrotic ectodermal dysplasia with immunodeficiency. NEMO function is mediated by two distal ubiquitin binding domains located in the regulatory C-terminal domain of the protein: the coiled-coil 2-leucine zipper (CC2-LZ) domain and the zinc finger (ZF) domain. Here, we investigated the effect of the D406V mutation found in the NEMO ZF of an ectodermal dysplasia with immunodeficiency patients. This point mutation does not impair the folding of NEMO ZF or mono-ubiquitin binding but is sufficient to alter NEMO function, as NEMO-deficient fibroblasts and Jurkat T lymphocytes reconstituted with full-length D406V NEMO lead to partial and strong defects in NF-κB activation, respectively. To further characterize the ubiquitin binding properties of NEMO ZF, we employed di-ubiquitin (di-Ub) chains composed of several different linkages (Lys-48, Lys-63, and linear (Met-1-linked)). We showed that the pathogenic mutation preferentially impairs the interaction with Lys-63 and Met-1-linked di-Ub, which correlates with its ubiquitin binding defect in vivo. Furthermore, sedimentation velocity and gel filtration showed that NEMO ZF, like other NEMO related-ZFs, binds mono-Ub and di-Ub with distinct stoichiometries, indicating the presence of a new Ub site within the NEMO ZF. Extensive mutagenesis was then performed on NEMO ZF and characterization of mutants allowed the proposal of a structural model of NEMO ZF in interaction with a Lys-63 di-Ub chain.

Turnip yellow mosaic virus (TYMV) - a member of the alphavirus-like supergroup of viruses - serves as a model system for positive-stranded RNA virus membrane-bound replication. TYMV encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (PRO). We recently reported that PRO is actually a multifunctional enzyme with a specific ubiquitin hydrolase (DUB) activity that contributes to viral infectivity. Here, we report the crystal structure of the 150-residue PRO. Strikingly, PRO displays no homology to other processing proteinases from positive-stranded RNA viruses, including that of alphaviruses. Instead, the closest structural homologs of PRO are DUBs from the Ovarian tumor (OTU) family. In the crystal, one molecule's C-terminus inserts into the catalytic cleft of the next, providing a view of the N-terminal product complex in replication polyprotein processing. This allows us to locate the specificity determinants of PRO for its proteinase substrates. In addition to the catalytic cleft, at the exit of which the active site is unusually pared down and solvent-exposed, a key element in molecular recognition by PRO is a lobe N-terminal to the catalytic domain. Docking models and the activities of PRO and PRO mutants in a deubiquitylating assay suggest that this N-terminal lobe is also likely involved in PRO's DUB function. Our data thus establish that DUBs can evolve to specifically hydrolyze both iso- and endopeptide bonds with different sequences. This is achieved by the use of multiple specificity determinants, as recognition of substrate patches distant from the cleavage sites allows a relaxed specificity of PRO at the sites themselves. Our results thus shed light on how such a compact protein achieves a diversity of key functions in viral genome replication and host-pathogen interaction., Author Summary Positive-stranded RNA viruses are ultimate parasites. In order to replicate their genome, they first need to invade a host cell and, with usually very limited viral genetic material, subvert the host's molecular machinery. Turnip yellow mosaic virus (TYMV) is an excellent model system for studying positive-stranded RNA virus replication. As for many such viruses, TYMV genome replication is dependent on the activity of a viral proteinase (PRO) to properly process the virus' replication molecules. We have recently established that PRO is a multifunctional enzyme and is also used by TYMV to subvert a key host defense against pathogens. We report here the atomic structure of PRO as well as new functional data on PRO's interaction with the host. Our data shed light on how PRO can perform such multiple activities despite its small size, providing TYMV with a Swiss army knife in its ongoing fight with a vastly more complex host.

14 páginas, 6 figuras. En material suplementario: 3 figuras, 2 tablas, SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination., This research was funded by grant BIO2005-04650 from the Spanish Ministry of Education and Science (MEC) and FQM-02838 from the Andalucia Regional Government. Work at J.B. laboratory was funded by the Spanish Ministry of Education and Innovation (SAF2009-10667). J.L.O.R. is supported by a Federation of European Biochemical Societies (FEBS) post-doctoral grant. S.C.A. is supported by a resettlement contract from the University of Granada. N.A.J.v.N is a group leader of the Vlaams Instituut voor Biotechnologie (VIB). The 600 MHz spectra were recorded in the Centre for Scientific Instrumentation (CIC) of the University of Granada and at the RALF Large Scale Facility in Grenoble, which is funded by the 'Access to Research Infrastructures' program of the European Union.

International audience; In plants, the tight regulation of plasma membrane transporters is essential to maintain nutrient homeostasis. The mechanisms controlling the abundance of transporters, and other integral plasma membrane proteins, now come to light. Ubiquitination appears as a major signal initiating cargo endocytosis and sorting into multivesicular bodies prior to degradation in the vacuole. We have indeed demonstrated that the root iron transporter IRT1 is subjected to ubiquitin-dependent trafficking in root epidermal cells. This control is crucial to keep IRT1 levels at the cell surface low and to cope with the toxicity associated with other readily available metal substrates of IRT1. Our work combined with recent report on the BOR1 boron transporter establishes ubiquitination as a conserved mechanism of plasma membrane protein trafficking in plants, and highlights its importance for plant nutrition.

Nuclear factor-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase complex, is a critical component of the NF-κB pathway. Hypomorphic mutations in the X-linked human NEMO gene cause various forms of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID). All known X-linked EDA-ID–causing mutations impair NEMO protein expression, folding, or both. We describe here 2 EDA-ID–causing missense mutations that affect the same residue in the CC2-LZ domain (D311N and D311G) that do not impair NEMO production or folding. Structural studies based on pull-down experiments showed a defect in noncovalent interaction with K63-linked and linear polyubiquitin chains for these mutant proteins. Functional studies on the patients' cells showed an impairment of the classic NF-κB signaling pathways after activation of 2 NEMO ubiquitin-binding–dependent receptors, the TNF and IL-1β receptors, and in the CD40-dependent NF-κB pathway. We report the first human NEMO mutations responsible for X-linked EDA-ID found to affect the polyubiquitin binding of NEMO rather than its expression and folding. These experiments demonstrate that the binding of human NEMO to polyubiquitin is essential for NF-κB activation. They also demonstrate that the normal expression and folding of NEMO do not exclude a pathogenic role for NEMO mutations in patients with EDA-ID.

International audience; The Notch signaling pathway regulates numerous aspects of metazoan development and tissue renewal. Deregulation or loss of Notch signaling is associated with a wide range of human disorders from developmental syndromes to cancer. Notch receptors and their ligands are widely expressed throughout development, yet Notch activation is robustly controlled in a spatio-temporal manner. Within the past decades, genetic screens and biochemical approaches led to the identification of more than 10 E3 ubiquitin ligases and deubiquitinating enzymes implicated in the regulation of the Notch pathway. In this review, we highlight the recent studies in Notch signaling that reveal how ubiquitination of components of the Notch pathway, ranging from degradation to regulation of membrane trafficking, impacts on the developmental control of the signaling activities of both Notch receptors and their ligands.

International audience; Hedgehog pathway regulates tissue patterning and cell proliferation. Gli1 transcription factor is the major effector of Hedgehog signaling and its deregulation is often associated to medulloblastoma formation. Proteolytic processes represent a critical mechanism by which this pathway is turned off. Here, we characterize the regulation of an ubiquitin-mediated mechanism of Gli1 degradation, promoted by the coordinated action of the E3 ligase Itch and the adaptor protein Numb. We show that Numb activates the catalytic activity of Itch, releasing it from an inhibitory intramolecular interaction between its homologous to E6-AP C-terminus and WW domains. The consequent activation of Itch, together with the recruitment of Gli1 through direct binding with Numb, allows Gli1 to enter into the complex, resulting in Gli1 ubiquitination and degradation. This process is mediated by a novel Itch-dependent degron, composed of a combination of two PPXYs and a phospho-serine/proline motifs, localized in Gli1 C-terminal region, indicating the role of two different WW docking sites in Gli1 ubiquitination. Remarkably, Gli1 protein mutated in these modules is no longer regulated by Itch and Numb, and determines enhanced Gli1-dependent medulloblastoma growth, migration and invasion abilities, as well as in vitro transforming activity. Our data reveal a novel mechanism of regulation of Gli1 stability and function, which influences Hedgehog/Gli1 oncogenic potential.

International audience; The age-related altered expression of neuron-related proteins as seen in other regions of the central nervous system is expected in the aging retina. Using immunohistochemical techniques, we characterized the distribution and aggregation of tau, βA4-amyloid, α-synuclein, and ubiquitin in human retina obtained from 19 enucleated eyes of patients aged 49 to 87 years and correlated the findings with the ages. Using a phosphorylation-independent antibody, tau aggregates were observed within the cytoplasm of several photoreceptor cells, and there was a positive correlation between age and the number of tau-positive ganglionic cells. Tau deposits were immunonegative with a phosphorylation-dependent antibody. We did not observe βA4-amyloid in subretinal pigment epithelium deposits or in neuroepithelial layers. α-Synuclein and ubiquitin inclusions were found in the inner nuclear layer, and there was colocalization of these proteins. The proportion of patients displaying such α-synuclein and/or ubiquitin intracytoplasmic inclusions was significantly higher with aging. The presence of ubiquitin deposits within drusen was remarkable, but diffuse ubiquitin aggregates between the retinal pigment epithelium and Bruch membrane were also noticed. These results indicate that protein aggregation in the retina increases with aging and that tau, α-synuclein, and ubiquitin should be the subjects of future investigations.

International audience; Ubp3/Bre5 complex is a substrate-specific deubiquitylating enzyme which mediates deubiquitylation of Sec23, a component of the COPII complex involved in the transport between endoplasmic reticulum and Golgi apparatus. Here we show that ubiquitylation of Sec23 is controlled by the Rsp5 ubiquitin ligase both in vivo and in vitro. We have recently identified Cdc48, a chaperone-like that plays a key role in the proteasomal escort pathway, as a partner of the Ubp3/Bre5 complex. We now found that cdc48 thermosensitive mutant cells not only accumulate ubiquitylated form of Sec23 but also display a stabilization of this protein at the restrictive temperature. This indicates that Cdc48 controls the proteasome-mediated degradation of Sec23. Our data favor the idea that Cdc48 plays a key role in deciphering fates of ubiquitylated Sec23 to degradation or deubiquitylation/stabilization via its cofactors.

International audience; A single heat shock factor (HSF), mediating the heat shock response, exists from yeast to Drosophila, whereas several related HSFs have been found in mammals. This raises the question of the specific or redundant functions of the different members of the HSF family and in particular of HSF1 and HSF2, which are both ubiquitously expressed. Using immortalized mouse embryonic fibroblasts (iMEFs) derived from wild-type, Hsf1(-/-), Hsf2(-/-) or double-mutant mice, we observed the distinctive behaviors of these mutants with respect to proteasome inhibition. This proteotoxic stress reduces to the same extent the viability of Hsf1(-/-)- and Hsf2(-/-)-deficient cells, but through different underlying mechanisms. Contrary to Hsf2(-/-) cells, Hsf1(-/-) cells are unable to induce pro-survival heat shock protein expression. Conversely, proteasome activity is lower in Hsf2(-/-) cells and the expression of some proteasome subunits, such as Psmb5 and gankyrin, is decreased. As gankyrin is an oncoprotein involved in p53 degradation, we analyzed the status of p53 in HSF-deficient iMEFs and observed that it was strongly stabilized in Hsf2(-/-) cells. This study points a new role for HSF2 in the regulation of protein degradation and suggests that pan-HSF inhibitors could be valuable tools to reduce chemoresistance to proteasome inhibition observed in cancer therapy.

International audience; The COP9 signalosome (CSN) is an essential multisubunit complex that regulates the activity of cullin-RING ubiquitin ligases by removing the ubiquitin-like peptide NEDD8 from cullins. Here, we demonstrate that the CSN can affect other components of the ubiquitination cascade. Down-regulation of human CSN4 or CSN5 induced proteasome-mediated degradation of the ubiquitin-conjugating enzyme UBC3/Cdc34. UBC3 was targeted for ubiquitination by the cullin-RING ubiquitin ligase SCF(betaTrCP). This interaction required the acidic C-terminal extension of UBC3, which is absent in ubiquitin-conjugating enzymes of the UBCH5 family. Conversely, the UBC3 acidic domain was sufficient to impart sensitivity to SCF(betaTrCP)-mediated ubiquitination to UBCH5 enzymes. Our work indicates that the CSN is necessary to ensure the stability of selected ubiquitin-conjugating enzymes and uncovers a novel pathway of regulation of ubiquitination processes.

International audience; Post-translational modification of proteins via ubiquitination plays a crucial role in numerous vital functions of the cell. Polyubiquitination is one of the key regulatory processes involved in regulation of mitotic progression. Here we describe a differential proteomic screen dedicated to identification of novel proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryo. Mutated recombinant His6-tagged ubiquitin (Ubi (K48R)) was added to mitotic extract from which we purified conjugated proteins, as well as associated proteins in nondenaturing conditions by cobalt affinity chromatography. Proteins eluted from Ubi (K48R) supplemented and control extracts were compared by LC-MS/MS analysis after monodimensional SDS-PAGE. A total of 144 proteins potentially ubiquitinated or associated with them were identified. Forty-one percent of these proteins were shown to be involved in ubiquitination and/or proteasomal degradation pathway confirming the specificity of the screen. Twelve proteins, among them ubiquitin itself, were shown to carry a "GG" or "LRGG" remnant tag indicating their direct ubiquitination. Interestingly, sequence analysis of ubiquitinated substrates carrying these tags indicated that in Xenopus cell-free embryo extract supplemented with Ubi (K48R) the majority of polyubiquitination occurred through lysine-11 specific ubiquitin chain polymerization. The potential interest in this atypical form of ubiquitination as well as usefulness of our method in analyzing atypical polyubiquitin species is discussed.

International audience; Linear endocytic motifs of signaling receptors as well as their ubiquitination determine the rate of ligand-induced endocytosis that mediates down-regulation of these receptors and restricts the magnitude and duration of their respective signal transduction pathways. We previously hypothesized that, in the absence of its cognate ligand, type I interferon (IFN), the IFNalpha receptor chain 1 (IFNAR1) receptor chain is protected from basal endocytosis by a hypothetical masking complex that prevents the Tyr-based endocytic motif within IFNAR1 from interacting with components of the adaptin protein complex 2 (AP2). Here we identify a member of the Janus kinase (Jak) family, Tyk2, as a component of such a masking complex. In the absence of ligand or of receptor chain ubiquitination, binding of Janus kinase Tyk2 within the proximity of the Tyr-based linear motif of IFNAR1 is required to prevent IFNAR1 internalization and to maintain its cell surface expression. Furthermore, interaction experiments revealed that Tyk2 physically shields this Tyr-based motif from the recognition by the AP50 subunit of AP2. These data delineate a long-sought ligand- and ubiquitin-independent mechanism by which Tyk2 contributes to both the regulation of total IFNAR1 levels as well as the regulation of the cell surface density of this receptor chain.

International audience; Fibroblast growth factor receptor (FGFR) signaling plays an important role in skeletogenesis. The molecular mechanisms triggered by activated FGFR in bone forming cells are however not fully understood. In this study, we identify a role for phosphatidylinositol 3-kinase (PI3K) signaling in cell apoptosis induced by FGFR2 activation in osteoblasts. We show that FGFR2 activation leads to decrease PI3K protein levels, resulting in attenuation of PI3K signaling in human osteoblasts. Biochemical and molecular analyses revealed that the attenuated PI3K signaling induced by FGFR2 activation is due to increased Cbl-PI3K molecular interaction mediated by the Cbl Y731 residue, which results in increased PI3K ubiquitination and proteasome degradation. Biochemical and immunocytochemical analyses showed that FGFR2 and Cbl interact in raft micro-domains at the plasma membrane. FGFR2 activation increases FGFR2 and Cbl recruitment in micro-domains, resulting in increased molecular interactions. Consistently, functional analyses showed that the attenuation of PI3K/Akt signaling triggered by FGFR2 activation results in increased osteoblast apoptosis. These results identify a functional molecular mechanism by which activated FGFR2 recruits Cbl in raft micro-domains to trigger PI3K ubiquitination and proteasome degradation, and reveal a novel role for PI3K/Akt attenuation in the control of osteoblast survival by FGFR2 signaling.

International audience; Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination, and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance.

International audience; The Fos family of transcription factors comprises c-Fos, Fra-1, Fra-2 and FosB, which are all intrinsically unstable proteins. Fos proteins heterodimerize with a variety of other transcription factors to control genes encoding key cell regulators. Their best known partners are the Jun family proteins (c-Jun, JunB, and JunD). At the cellular level, Fos-involving dimers control proliferation, differentiation, apoptosis and responses to environmental cues. At the organism level, they play paramount parts in organogenesis, immune responses and cognitive functions, among others. fos family genes are subjected to exquisite, complex and intermingled transcriptional and post-transcriptional regulations, which are necessary to avoid pathological effects. In particular, the Fos proteins undergo to numerous post-translational modifications, such as phosphorylations and sumoylation, regulating their transcriptional activity, their subcellular localization and their turnover. The mechanisms whereby c-Fos and Fra-1 are degraded have been studied in detail. Contrasting with the classical scenario, according to which most unstable key cell regulators are hydrolyzed by the proteasome after conjugation of polyubiquitin chains, the bulk of c-Fos and Fra-1 can be hydrolyzed independently of any prior ubiquitylation in different situations. c-Fos and Fra-1 share a common destabilizing domain whose primary sequence is conserved in Fra-2 and FosB, suggesting that similar breakdown mechanisms might be at play in the latter two proteins. However, a database search indicates that this domain is not found in any other protein, suggesting that the mechanisms underlying Fos protein destruction may be specific to this family. Interestingly, under particular conditions, a fraction of cytoplasmic c-Fos is ubiquitylated, leading to faster turnover. This poses the question of the multiplicity of degradation pathways that can target the same substrate depending on its activation state, its protein partnership and/or its intracellular localization. This issue is discussed here together with the, thus far, overlooked roles of the various proteasomal complexes found in all cells.

International audience; Listeria monocytogenes surface proteins internalin (Inl)A and InlB interact with the junctional protein E-cadherin and the hepatocyte growth factor (HGF) receptor Met, respectively, on the surface of epithelial cells to mediate bacterial entry. Here we show that InlA triggers two successive E-cadherin posttranslational modifications, i.e. the Src-mediated tyrosine phosphorylation of E-cadherin followed by its ubiquitination by the ubiquitin-ligase Hakai. E-cadherin ubiquitination induces the recruitment of clathrin that is required for optimal bacterial internalization. We also show that the initial clustering of E-cadherin at the bacterial entry site requires caveolin, a protein normally involved in clathrinindependent endocytosis. Strikingly clathrin and caveolin are also recruited at the site of entry of E-cadherin-coated sepharose beads and functional experiments demonstrate that these two proteins are required for bead entry. Together these results not only document how the endocytosis machinery is recruited and involved in the internalization of a zippering bacterium, but also strongly suggest a functional link between E-cadherin endocytosis and the formation of adherens junctions in epithelial cells.

International audience; BTBD1 is a recently cloned BTB-domain-containing protein particularly expressed in skeletal muscle and interacting with DNA topoisomerase 1 (Topo1), a key enzyme of cell survival. We have previously demonstrated that stable overexpression of a N-terminal truncated BTBD1 inhibited ex vivo myogenesis but not adipogenesis of pluripotent C2C12 cells. Here, BTBD1 expression was studied in three models of cellular differentiation: myogenesis (C2C12 cells), adipogenesis (3T3-L1 cells) and osteogenesis (hMADS cells). BTBD1 mRNA was found to be upregulated during myogenesis. At the opposite, we have not observed BTBD1 upregulation in an altered myogenesis cellular model and we observed a downregulation of BTBD1 mRNA expression in adipogenesis. Interestingly, amounts of Topo1 protein, but not Topo1 mRNA, were found to be modulated at the opposite of BTBD1 mRNA. No variation of BTBD1 expression was measured during osteogenesis. Taken together, these results indicate that BTBD1 mRNA is specifically regulated during myogenic and adipogenic differentiation, in relation with Topo1 expression. Moreover, they corroborate observations made previously with truncated BTBD1 and show that BTBD1 is a key protein of balance between adipogenesis and myogenesis. Finally, a transcriptome analysis gave molecular clues to decipher BTBD1 role, with an emphasis on the involvement in ubiquitin/proteasome degradation pathway.

Hypoxia and the Net ternary complex factor (TCF) regulate similar processes (angiogenesis, wound healing, and cellular migration) and genes (PAI-1, c-fos, erg-1, NOS-2, HO-1, and vascular endothelial growth factor genes), suggesting that they are involved in related pathways. We show here that hypoxia regulates Net differently from the other TCFs and that Net plays a role in the hypoxic response in vivo in mice and in cells. Hypoxia induces Net depletion from target promoters, nuclear export, ubiquitylation, and proteasomal degradation. Key mediators of the hypoxic response, the prolyl-4-hydroxylases containing domain proteins (PHDs), regulate Net. PHD downregulation in normoxia leads to Net degradation, and PHD overexpression delays Net downregulation by hypoxia. Net inhibition by RNA interference or mutation leads to altered regulation by hypoxia of the Net targets PAI-1, c-fos, and egr-1. We propose that hypoxia stimulates transcription of target promoters through removal of the repressor function of Net. Interestingly, the hematocrit response to a chemical inducer of hypoxia-like responses (cobalt chloride) is strongly altered in Net mutant mice. Our results show that the Net TCF is part of the biological response to hypoxia, adding a new component to an important pathological and physiological process.

Activation of the transcription factor NF-kappaB after stimulation through antigen receptors is important for lymphocyte differentiation, activation, proliferation, and protection against apoptosis. Much progress has been made in understanding the molecular events leading to NF-kappaB activation, but how this activation is eventually down-regulated is less well understood. Recent studies have indicated that Bcl10 functions downstream of lymphocyte antigen receptors to promote the activation of the IkappaB kinase complex leading to the phosphorylation and degradation of the IkappaB inhibitors of NF-kappaB. Bcl10 has also been implicated in the pathogenesis of mucosa-associated lymphoid tissue lymphoma, possibly in association with its nuclear localization. Here, we provide evidence that the IkappaB kinase complex phosphorylates Bcl10 after T cell antigen receptor stimulation and causes its proteolysis via the beta-TrCP ubiquitin ligase/proteasome pathway. These findings document a negative regulatory activity of the IKK complex and suggest that Bcl10 degradation is part of the regulatory mechanisms that precisely control the response to antigens. Mutants of Bcl10 in the IKK phosphorylation site are resistant to degradation, accumulate in the nucleus, and lead to an increase in IL-2 production after T cell antigen receptor stimulation.

International audience; p53 functions to prevent malignant progression, in part by inhibiting proliferation or inducing the death of potential tumour cells. One of the most important regulators of p53 is MDM2, a RING domain E3 ligase that ubiquitinates p53, leading to both proteasomal degradation and relocation of p53 from the nucleus to the cytoplasm. Previous studies have suggested that although polyubiquitination is required for degradation, monoubiquitination of p53 is sufficient for nuclear export. Using a p53-ubiquitin fusion protein we show that ubiquitination contributes to two steps before export: exposure of a carboxy-terminal nuclear export sequence (NES), and dissociation of MDM2. Monoubiquitination can directly promote further modifications of p53 with ubiquitin-like proteins and MDM2 promotes the interaction of the SUMO E3 ligase PIASy with p53, enhancing both sumoylation and nuclear export. Our results suggest that modifications such as sumoylation can regulate the strength of the p53-MDM2 interaction and participate in driving the export of p53.

International audience; A cellular defense mechanism counteracts the deleterious effects of misfolded protein accumulation by eliciting a stress response. The cytoplasmic deacetylase HDAC6 (histone deacetylase 6) was previously shown to be a key element in this response by coordinating the clearance of protein aggregates through aggresome formation and their autophagic degradation. Here, for the first time, we demonstrate that HDAC6 is involved in another crucial cell response to the accumulation of ubiquitinated protein aggregates, and unravel its molecular basis. Indeed, our data show that HDAC6 senses ubiquitinated cellular aggregates and consequently induces the expression of major cellular chaperones by triggering the dissociation of a repressive HDAC6/HSF1 (heat-shock factor 1)/HSP90 (heat-shock protein 90) complex and a subsequent HSF1 activation. HDAC6 therefore appears as a master regulator of the cell protective response to cytotoxic protein aggregate formation.

International audience; Many bacteria pathogenic for plants or animals, including Shigella spp., which is responsible for shigellosis in humans, use a type III secretion apparatus to inject effector proteins into host cells. Effectors alter cell signaling and host responses induced upon infection; however, their precise biochemical activities have been elucidated in very few cases. Utilizing Saccharomyces cerevisiae as a surrogate host, we show that the Shigella effector IpaH9.8 interrupts pheromone response signaling by promoting the proteasome-dependent destruction of the MAPKK Ste7. In vitro, IpaH9.8 displayed ubiquitin ligase activity toward ubiquitin and Ste7. Replacement of a Cys residue that is invariant among IpaH homologs of plant and animal pathogens abolished the ubiquitin ligase activity of IpaH9.8. We also present evidence that the IpaH homolog SspH1 from Salmonella enterica can ubiquitinate ubiquitin and PKN1, a previously identified SspH1 interaction partner. This study assigns a function for IpaH family members as E3 ubiquitin ligases.

International audience; The interaction of the P-beta-Cat(19-44) peptide, a 26 amino acid peptide (K(19)AAVSHWQQQSYLDpSGIHpSGATTTAP(44)) that mimics the phosphorylated beta-Catenin antigen, has been studied with its monoclonal antibody BC-22, by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. This antibody is specific to diphosphorylated beta-Catenin and does not react with the non-phosphorylated protein. Phosphorylation of beta-Catenin at sites Ser33 and Ser37 on the DSGXXS motif is required for the interaction of beta-Catenin with the ubiquitin ligase SCF(beta-TrCP). beta-TrCP is involved in the ubiquitination and proteasome targeting of the oncogenic protein beta-Catenin, the accumulation of which has been implicated in various human cancers. The three-dimensional structure of the P-beta-Cat(19-44) in the bound conformation was determined by TRNOESY NMR experiments; the peptide adopts a compact structure in the presence of mAb with formation of turns around Trp25 and Gln26, with a tight bend created by the DpS(33)GIHpS(37) motif; the peptide residues (D32-pS37) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentate association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of beta-Catenin with the SCF(beta-TrCP) protein.

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.

Next Section Abstract In yeast, the Met4 transcription factor and its cofactors Cbf1, Met28, Met31, and Met32 control the expression of sulfur metabolism and oxidative stress response genes. Met4 activity is tuned to nutrient and oxidative stress conditions by the SCFMet30 ubiquitin ligase. The mechanism whereby SCFMet30-dependent ubiquitylation of Met4 controls Met4 activity remains contentious. Here, we have demonstrated that intracellular cysteine levels dictate the degradation of Met4 in vivo, as shown by the ability of cysteine, but not methionine or S-adenosylmethionine (AdoMet), to trigger Met4 degradation in an str4Δ strain, which lacks the ability to produce cysteine from methionine or AdoMet. Met4 degradation requires its nuclear localization and activity of the 26 S proteasome. Analysis of the regulated degradation of a fully functional Met4-Cbf1 chimera, in which Met4 is fused to the DNA binding domain of Cbf1, demonstrates that elimination of Met4 in vivo can be triggered independently of both its normal protein interactions. Strains that harbor the Met4-Cbf1 fusion as the only source of Cbf1 activity needed for proper kinetochore function exhibit high rates of methionine-dependent chromosomal instability. We suggest that SCFMet30 activity or Met4 utilization as a substrate may be directly regulated by intracellular cysteine concentrations.

Plasma membrane transport of single amino-acid methionine in yeast is shown to be mediated by at least seven different permeases whose activities are transcriptionaly and post-transcriptionaly regulated by different ubiquitin-dependent mechanisms. Upon high extracellular methionine exposure, three methionine-permease genes are repressed while four others are induced. SCF(Met30), SCF(Grr1) and Rsp5 ubiquitin ligases are the key actors of the ubiquitin-dependent remodeling of methionine transport. In addition to regulating the activity of Met4, the SCF(Met30) ubiquitin ligase is shown to convey an intracellular signal to a membrane initiated signaling pathway by controlling the nuclear concentration of the Stp1 transcription factor. By coupling intra- and extracellular metabolite sensing, SCF(Met30) thus allows yeast cells to accurately adjust the intermediary sulfur metabolism to the growth conditions. The multiple ubiquitin-dependent mechanisms that function in methionine transport regulation further exemplify the pervasive role of ubiquitin in the adaptation of single-cell organisms to environmental modifications.

Inherited prion diseases are neurodegenerative pathologies related to genetic mutations in the prion protein (PrP) gene, which favour the conversion of PrPC into a conformationally altered pathogenic form, PrPSc. The molecular basis of PrPC/PrPSc conversion, the intracellular compartment where it occurs and how this process leads to neurological dysfunction are not yet known. We have studied the intracellular synthesis, degradation and localization of a PrP mutant associated with a genetic form of Creutzfeldt-Jakob disease (CJD), PrPT182A, in transfected FRT cells. PrPT182A is retained in the endoplasmic reticulum (ER), is mainly associated with detergent-resistant microdomains (DRMs) and is partially resistant to proteinase K digestion. Although an untranslocated form of this mutant is polyubiquitylated and undergoes ER-associated degradation, the proteasome is not responsible for the degradation of its misfolded form, suggesting that it does not have a role in the pathogenesis of inherited diseases. On the contrary, impairment of PrPT182A association with DRMs by cholesterol depletion leads to its accumulation in the ER and substantially increases its misfolding. These data support the previous hypothesis that DRMs are important for the correct folding of PrP and suggest that they might have a protective role in pathological scrapie-like conversion of PrP mutants.

The N-terminus of any protein may be used as a destabilization signal for targeted protein degradation. In the eukaryotic cytosol, the signal - the so-called N-degron--is recognized for degradation by (i) the N-end rule, a well-described degradation process involving epsilon-ubiquitination; or (ii) N-terminal ubiquitination, a more recently described pathway. Dedicated E3 ubiquitin ligases known as N-recognins then act on the protein. The proteolytic pathways involve ATP-dependent chambered proteases, such as the 26S proteasome in the cytosol, which generate short oligopeptides. The N-terminus of the polypeptide chain is also important for post-proteasome degradation by specific aminopeptidases, which complete peptide cleavage to generate free amino acids. Finally, in each compartment of the eukaryotic cell, N-terminal methionine excision creates a variety of N-termini for mature proteins. It has recently been shown that the N-terminal methionine excision pathway has a major impact early in targeted protein degradation.

Mutations in the CSA or CSB complementation genes cause the Cockayne syndrome, a severe genetic disorder that results in patients’ death in early adulthood. CSA and CSB act in a transcription-coupled repair (TCR) pathway, but their functional relationship is not understood. We have previously shown that CSA is a subunit of an E3 ubiquitin ligase complex. Here we demonstrate that CSB is a substrate of this ligase: Following UV irradiation, CSB is degraded at a late stage of the repair process in a proteasome- and CSA-dependent manner. Moreover, we demonstrate the importance of CSB degradation for post-TCR recovery of transcription and for the Cockayne syndrome. Our results unravel for the first time the functional relationship between CSA and CSB.

International audience; Viruses are obligate parasites of host cells. Virus-host coevolution has selected virus for growth despite antiviral defenses set up by hosting cells and organisms. Ubiquitin conjugation onto proteins, through a cascade of reactions mediated by E1 (ubiquitin-activating enzyme) and E2 and E3 (ubiquitin- conjugating ligases), is one of the major regulatory systems that, in particular, tightly controls the concentration of cellular proteins by sorting them for degradation. The combined diversity of E2 and E3 ligases ensures the selective/specific ubiquitination of a large number of protein substrates within the cell interior. Therefore it is not surprising that several viruses encode proteins with E3 ubiquitin ligase activities that target cellular proteins playing a key role in innate antiviral mechanisms.

The common cytokine receptor gamma(c) is shared by the interleukin-2, -4, -7, -9, -15, and -21 receptors, and is essential for lymphocyte proliferation and survival. The regulation of gamma(c) receptor expression level is therefore critical for the ability of cells to respond to these cytokines. We previously reported that gamma(c) is efficiently constitutively internalized and addressed towards a degradation endocytic compartment. We show that gamma(c) is ubiquitinated and also associated to ubiquitinated proteins. We report that the ubiquitin-ligase c-Cbl induces gamma(c) down-regulation. In addition, the ubiquitin-hydrolase, DUB-2, counteracts the effect of c-Cbl on gamma(c) expression. We show that an increase in DUB-2 expression correlates with an increased gamma(c) half-life, resulting in the up-regulation of the receptor. Altogether, we show that gamma(c) is the target of an ubiquitination mechanism and its expression level can be regulated through the activities of a couple of ubiquitin-ligase/ubiquitin-hydrolase enzymes, namely c-Cbl/DUB-2.

International audience; Retroviruses use endosomal machinery to bud out of infected cells, and various Gag proteins recruit this machinery by interacting with either of three cellular factors as follows: ubiquitin ligases of the Nedd4 family, Tsg101, or Alix/Aip1. Here we show that the murine leukemia virus Gag has the unique ability to interact with all three factors. Small interfering RNAs against Tsg101 or Alix and dominant-negative forms of Nedd4 can all reduce production of virus-like particles. However, inactivating the Nedd4-binding site abolishes budding, whereas disrupting Tsg101 or Alix binding has milder effects. Nedd4 ubiquitin ligases are therefore essential, and Tsg101 and Alix play auxiliary roles. Most interestingly, overexpression of Alix can stimulate the release of Gag, and this occurs independently of most Alix partners Tsg101, Cin85, Alg-2, and endophilins. In addition, Gag mutants that do not bind Tsg101 or Alix concentrate on late endosomes and become very sensitive to dominant-negative forms of Nedd4 that do not conjugate ubiquitin. This suggests that the direct interaction of Gag with Tsg101 and Alix favors budding from the plasma membrane and relieves a requirement for ubiquitination by Nedd4.1. Other Nedd4-dependent Gag proteins also contain binding sites for Tsg101 or Alix, suggesting that this could be a common feature of retroviruses.

Several viral proteins expressed by DNA or RNA transforming viruses have the particular property of binding via their C-terminal end to various cellular proteins with PDZ domains. This study is focused on the PDZ protein TIP-2/GIPC, which was originally identified in two-hybrid screens performed with two different baits: the human T-cell leukemia virus type 1 Tax oncoprotein and the regulator of G signaling RGS-GAIP. Further studies have shown that TIP-2/GIPC is also able to associate with the cytoplasmic domains of various transmembrane proteins. In this report we show that TIP-2/GIPC interacts with the E6 protein of human papillomavirus type 18 (HPV-18). This event triggers polyubiquitination and proteasome-mediated degradation of the cellular protein. In agreement with this observation, silencing of E6 by RNA interference in HeLa cells causes an increase in the intracellular TIP-2/GIPC level. This PDZ protein has been previously found to be involved in transforming growth factor β (TGF-β) signaling by favoring expression of the TGF-β type III receptor at the cell membrane. In line with this activity of TIP-2/GIPC, we observed that depletion of this protein in HeLa cells hampers induction of the Id3 gene by TGF-β treatment and also diminishes the antiproliferative effect of this cytokine. Conversely, silencing of E6 increases the expression of Id3 and blocks proliferation of HeLa cells. These results support the notion that HPV-18 E6 renders cells less sensitive to the cytostatic effect of TGF-β by lowering the intracellular amount of TIP-2/GIPC.

International audience; The ubiquitin-proteasomal pathway of degradation of proteins is activated or repressed in response to a number of environmental stresses and thereby plays an essential role in cell function and survival. Hypoxic stress, resulting from a decrease in the concentration of oxygen in tissues, is encountered in both physiological and pathological situations, in particular in cancer. The transcriptional complex hypoxia-inducible factor (HIF) is the key player in the signalling pathway that controls the hypoxic response of mammalian cells. Under hypoxic conditions it transactivates an impressive number of genes involved in a multitude of cellular functions. Tight regulation of this response in part involves the ubiquitin-proteasomal system where oxygen-dependent prolyl-4-hydroxylation of the alpha subunit of HIF triggers a cascade of events that leads to its degradation by the 26S proteasome. Inhibition of the proteasome in conjunction with topoisomerase inhibition has shown some promise in the treatment of experimental cancer. Such treatment may impact on the hypoxic adaptation of tumour cells.

International audience; At the late endosomes, cargoes destined for the interior of the vacuole are sorted into invaginating vesicles of the multivesicular body. Both PtdIns(3,5)P(2) and ubiquitin are necessary for proper sorting of some of these cargoes. We show that Ent5p, a yeast protein of the epsin family homologous to Ent3p, localizes to endosomes and specifically binds to PtdIns(3,5)P(2) via its ENTH domain. In cells lacking Ent3p and Ent5p, ubiquitin-dependent sorting of biosynthetic and endocytic cargo into the multivesicular body is disrupted, whereas other trafficking routes to the vacuole are not affected. Ent3p and Ent5p are associated with Vps27p, a FYVE domain containing protein that interacts with ubiquitinated cargoes and is required for protein sorting into the multivesicular body. Therefore, Ent3p and Ent5p are the first proteins shown to be connectors between PtdIns(3,5)P(2)- and the Vps27p-ubiquitin-driven sorting machinery at the multivesicular body.At the late endosomes, cargoes destined for the interior of the vacuole are sorted into invaginating vesicles of the multivesicular body. Both PtdIns(3,5)P(2) and ubiquitin are necessary for proper sorting of some of these cargoes. We show that Ent5p, a yeast protein of the epsin family homologous to Ent3p, localizes to endosomes and specifically binds to PtdIns(3,5)P(2) via its ENTH domain. In cells lacking Ent3p and Ent5p, ubiquitin-dependent sorting of biosynthetic and endocytic cargo into the multivesicular body is disrupted, whereas other trafficking routes to the vacuole are not affected. Ent3p and Ent5p are associated with Vps27p, a FYVE domain containing protein that interacts with ubiquitinated cargoes and is required for protein sorting into the multivesicular body. Therefore, Ent3p and Ent5p are the first proteins shown to be connectors between PtdIns(3,5)P(2)- and the Vps27p-ubiquitin-driven sorting machinery at the multivesicular body.

International audience; Clusterin is a puzzling protein upregulated in many diseased tissues, presented as either a survival or a death protein. The role of clusterin might depend on the final maturation and localization of the protein, which can be secreted or reside inside cells, either after in situ synthesis or uptake of extracellular clusterin. We studied the biological effects of intracellular clusterin and observed that clusterin forms containing the alpha-chain region strongly accumulated in an ubiquitinated form in juxtanuclear aggregates meeting the main criterions of aggresomes and leading to profound alterations of the mitochondrial network. The viability of cells transfected by intracellular forms of clusterin was improved by overexpression of Bcl-2, and caspase inhibition was capable of rescuing cells expressing clusterin, which presented an altered mitochondrial permeability. We propose that, although it might be an inherently pro-survival and anti-apoptotic protein expressed by cells under stress in an attempt to protect themselves, clusterin can become highly cytotoxic when accumulated in the intracellular compartment. This activity might reconcile the opposite purported influences of clusterin on cell survival and explain how clusterin can be causally involved in neurodegeneration.