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Chronic hepatitis B virus infection is a leading cause of cirrhosis and liver cancer. Hepatitis B virus encodes the regulatory HBx protein whose primary role is to promote transcription of the viral genome, which persists as an extrachromosomal DNA circle in infected cells. HBx accomplishes this task by an unusual mechanism, enhancing transcription only from extrachromosomal DNA templates. Here we show that HBx achieves this by hijacking the cellular DDB1-containing E3 ubiquitin ligase to target the 'structural maintenance of chromosomes' (Smc) complex Smc5/6 for degradation. Blocking this event inhibits the stimulatory effect of HBx both on extrachromosomal reporter genes and on hepatitis B virus transcription. Conversely, silencing the Smc5/6 complex enhances extrachromosomal reporter gene transcription in the absence of HBx, restores replication of an HBx-deficient hepatitis B virus, and rescues wild-type hepatitis B virus in a DDB1-knockdown background. The Smc5/6 complex associates with extrachromosomal reporters and the hepatitis B virus genome, suggesting a direct mechanism of transcriptional inhibition. These results uncover a novel role for the Smc5/6 complex as a restriction factor selectively blocking extrachromosomal DNA transcription. By destroying this complex, HBx relieves the inhibition to allow productive hepatitis B virus gene expression.

Differential DNA methylation exists in the epigenome of end-stage failing human hearts but whether it contributes to disease progression is presently unknown. Here, we report that cardiac specific deletion of Dnmt3b, the predominant DNA methyltransferase in adult mouse hearts, leads to an accelerated progression to severe systolic insufficiency and myocardial thinning without a preceding hypertrophic response. This was accompanied by widespread myocardial interstitial fibrosis and myo-sarcomeric disarray. By targeted candidate gene quantitative RT-PCR, we discovered an over-activity of cryptic splice sites in the sarcomeric gene Myh7, resulting in a transcript with 8 exons missing. Moreover, a region of differential methylation overlies the splice site locus in the hearts of the cardiac-specific conditional knockout (CKO) mice. Although abundant and complex forms of alternative splice variants have been reported in diseased hearts and the contribution of each remains to be understood in further detail, our results demonstrate for the first time that a link may exist between alternative splicing and the cardiac epigenome. In particular, this gives the novel evidence whereby the loss of an epigenome modifier promotes the development and progression of heart disease.

PEX5 is the shuttling receptor for newly synthesized peroxisomal matrix proteins. Alone, or with the help of an adaptor protein, this receptor binds peroxisomal matrix proteins in the cytosol and transports them to the peroxisomal membrane docking/translocation module (DTM). The interaction between cargo-loaded PEX5 and the DTM ultimately results in its insertion into the DTM with the concomitant translocation of the cargo protein across the organelle membrane. PEX5 is not consumed in this event; rather it is dislocated back into the cytosol so that it can promote additional rounds of protein transportation. Remarkably, the data collected in recent years indicate that dislocation is preceded by monoubiquitination of PEX5 at a conserved cysteine residue. This mandatory modification is not the only type of ubiquitination occurring at the DTM. Indeed, several findings suggest that defective receptors jamming the DTM are polyubiquitinated and targeted to the proteasome for degradation. This work was funded by FEDER funds through the Operational Competitiveness Programme e COMPETE and by National Funds through FCT e Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-019731 (PTDC/BIA-BCM/118577/2010). T.F., T.A.R., M.P.P. and C.P.G. are supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN, and Fundo Social Europeu. A.F.C. is supported by Programa Ciência, funded by Programa Operacional Potencial Humano do QREN, Tipologia 4.2, Promoção do Emprego Científico, by Fundo Social Europeu and by National Funds from Ministério da Ciência, Tecnologia e Ensino Superior.

Many yeast and some mammalian multivesicular body (MVB) cargoes display modification by K63-linked ubiquitin chains (K63Ub), which are required for their efficient sorting. Yeast UBD-containing ESCRT proteins are modified by the ubiquitin ligase Rsp5—some likely by K63Ub. A failure to generate K63Ub in yeast leads to MVB ultrastructure alteration., In yeast, the sorting of transmembrane proteins into the multivesicular body (MVB) internal vesicles requires their ubiquitylation by the ubiquitin ligase Rsp5. This allows their recognition by the ubiquitin-binding domains (UBDs) of several endosomal sorting complex required for transport (ESCRT) subunits. K63-linked ubiquitin (K63Ub) chains decorate several MVB cargoes, and accordingly we show that they localize prominently to the class E compartment, which accumulates ubiquitylated cargoes in cells lacking ESCRT components. Conversely, yeast cells unable to generate K63Ub chains displayed MVB sorting defects. These properties are conserved among eukaryotes, as the mammalian melanosomal MVB cargo MART-1 is modified by K63Ub chains and partly missorted when the genesis of these chains is inhibited. We show that all yeast UBD-containing ESCRT proteins undergo ubiquitylation and deubiquitylation, some being modified through the opposing activities of Rsp5 and the ubiquitin isopeptidase Ubp2, which are known to assemble and disassemble preferentially K63Ub chains, respectively. A failure to generate K63Ub chains in yeast leads to an MVB ultrastructure alteration. Our work thus unravels a double function of K63Ub chains in cargo sorting and MVB biogenesis.

In this study we determine that the Not4 E3 ligase is important for proteasome integrity. Consequently, deletion of Not4 leads to an accumulation of polyubiquitinated proteins and reduced levels of free ubiquitin. In the absence of Not4, the proteasome regulatory particle (RP) and core particle (CP) form salt-resistant complexes, and all other forms of RPs are unstable. Not4 can associate with RP species present in purified proteasome holoenzyme but not with purified RP. Additionally, Not4 interacts with Ecm29, a protein that stabilizes the proteasome. Interestingly, Ecm29 is identified in RP species that are inactive and not detectable in cells lacking Not4. In the absence of Not4, Ecm29 interacts less well with the proteasome and becomes ubiquitinated and degraded. Our results characterize Ecm29 as a proteasome chaperone whose appropriate interaction with the proteasome requires Not4.

During division of metazoan cells, the nucleus disassembles to allow chromosome segregation, and then reforms in each daughter cell. Reformation of the nucleus involves chromatin decondensation and assembly of the double- membrane nuclear envelope around the chromatin; however, regulation of the process is still poorly understood(1,2). In vitro, nucleus formation requires p97 ( ref. 3), a hexameric ATPase implicated in membrane fusion and ubiquitin- dependent processes(4,5). However, the role and relevance of p97 in nucleus formation have remained controversial. Here we show that p97 stimulates nucleus reformation by inactivating the chromatin- associated kinase Aurora B. During mitosis, Aurora B inhibits nucleus reformation by preventing chromosome decondensation and formation of the nuclear envelope membrane. During exit from mitosis, p97 binds to Aurora B after its ubiquitylation and extracts it from chromatin. This leads to inactivation of Aurora B on chromatin, thus allowing chromatin decondensation and nuclear envelope formation. These data reveal an essential pathway that regulates reformation of the nucleus after mitosis and defines ubiquitin- dependent protein extraction as a common mechanism of Cdc48/p97 activity also during nucleus formation.

Trehalose serves as a storage source of carbon and plays important roles under various stress conditions. For example, in many organisms trehalose has a critical function in preserving membrane structure and fluidity during dehydration/rehydration. In the yeast Saccharomyces cerevisiae, trehalose accumulates in the cell when the nutrient supply is limited but is rapidly degraded when the supply of nutrients is renewed. Hydrolysis of trehalose in yeast depends on neutral trehalase and acid trehalase (Ath1). Ath1 resides and functions in the vacuole; however, it appears to catalyze the hydrolysis of extracellular trehalose. Little is known about the transport route of Ath1 to the vacuole or how it encounters its substrate. Here, through the use of various trafficking mutants we showed that this hydrolase reaches its final destination through the multivesicular body (MVB) pathway. In contrast to the vast majority of proteins sorted into this pathway, Ath1 does not require ubiquitination for proper localization. Mutagenesis analyses aimed at identifying the unknown targeting signal revealed that the transmembrane domain of Ath1 contains the information sufficient for its selective sequestration into MVB internal vesicles.

The Ccr4-Not complex consists of nine subunits and acts as a regulator of mRNA biogenesis in Saccharomyces cerevisiae. The human ortholog of yeast NOT4, CNOT4, displays UbcH5B-dependent ubiquitin-protein ligase (E3 ligase) activity in a reconstituted in vitro system. However, an in vivo role for this enzymatic activity has not been identified. Site-directed mutagenesis of the RING finger of yeast Not4p identified residues required for interaction with Ubc4p and Ubc5p, the yeast orthologs of UbcH5B. Subsequent in vitro assays with purified Ccr4-Not complexes showed Not4p-mediated E3 ligase activity, which was dependent on the interaction with Ubc4p. To investigate the in vivo relevance of this activity, we performed synthetic genetic array (SGA) analyses using not4Δ and not4L35A alleles. This indicates involvement of the RING finger of Not4p in transcription, ubiquitylation, and DNA damage responses. In addition, we found a phenotypic overlap between deletions of UBC4 and mutants encoding single-amino-acid substitutions of the RING finger of Not4p. Together, our results show that Not4p functions as an E3 ligase by modulating Ubc4p/Ubc5p-mediated stress responses in vivo.

Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S -transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

Modification by ubiquitin plays a major role in a broad array of cellular functions. Although reversal of this process, deubiquitination, likely represents an important regulatory step contributing to cellular homeostasis, functions of deubiquitination enzymes still remain poorly characterized. We have previously shown that the ubiquitin protease Ubp3p requires a co-factor, Bre5p, to specifically deubiquitinate the coat protein complex II (COPII) subunit Sec23p, which is involved in anterograde transport between endoplasmic reticulum and Golgi compartments. In the present report, we show that disruption of BRE5 gene also led to a defect in the retrograde transport from the Golgi to the endoplasmic reticulum. Further analysis indicate that the COPI subunit beta'-COP represents another substrate of the Ubp3p.Bre5p complex. All together, our results indicate that the Ubp3p.Bre5p deubiquitination complex co-regulates anterograde and retrograde transports between endoplasmic reticulum and Golgi compartments.

Protein ubiquitination, a major post-translational modification in eukaryotes, requires an adequate pool of free ubiquitin. Cells maintain this pool by two pathways, both involving deubiquitinases (DUBs): recycling of ubiquitin from ubiquitin conjugates and processing of ubiquitin precursors synthesized de novo. Although many advances have been made in recent years regarding ubiquitin recycling, our knowledge on ubiquitin precursor processing is still limited, and questions such as when are these precursors processed and which DUBs are involved remain largely unanswered. Here we provide data suggesting that two of the four mammalian ubiquitin precursors, UBA < inf > 52 < /inf > and UBA < inf > 80 < /inf > , are processed mostly post-translationally whereas the other two, UBB and UBC, probably undergo a combination of co-and post-translational processing. Using an unbiased biochemical approach we found that UCHL < inf > 3 < /inf > , USP < inf > 9 < /inf > X, USP < inf > 7 < /inf > , USP < inf > 5 < /inf > and Otulin/Gumby/FAM < inf > 105 < /inf > b are by far the most active DUBs acting on these precursors. The identification of these DUBs together with their properties suggests that each ubiquitin precursor can be processed in at least two different manners, explaining the robustness of the ubiquitin de novo synthesis pathway. We are grateful to Dr. Cheryl Arrowsmith (University of Toronto, Canada) for providing the plasmids pET28a-LIC-USP5 (Addgene plasmid 25299) and pET28a-LIC-USP5(C335A). We thank Dr. João M. Cabral (IBMC, University of Porto, Portugal) for critically reading the manuscript. This work was supported by national funds through FCT - Fundação para a Ciência e a Tecnologia/MEC – Ministério da Educação e Ciência and when applicable co-funded by Fundo de Desenvolvimento Regional (FEDER) funds within the partnership agreement PT2020 related with the research unit number 4293; by Project “NORTE-07-0124-FEDER-000003 -Cell homeotasis tissue organization and organism biology”, co-funded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), under the Quadro de Referência Estratégico Nacional (QREN), through FEDER and by FCT; by Portuguese National Mass Spectrometry Network (RNEM) through the project REDE/1504/REM/2005; and by Química Orgânica, Produtos Naturais e Agroalimentares (QOPNA) research unit funds provided by FCT, European Union, QREN, FEDER and Operational Competitiveness Programme (COMPETE) under the projects PEst-C/QUI/UI0062/2013 and FCOMP-01-0124-FEDER-037296. C.P.G. and M.P.P. were supported by FCT, COMPETE and Fundo Social Europeu. A.V.M. was supported by the project FCOMP-01-0124-FEDER-027627-EXPL/BEX-BCM/0320/2012 financed by national funds from FCT/Ministério da Educação e Ciência (PIDDAC) and co-funded by FEDER through COMPETE—Programa Operacional Factores de Competitividade (POFC).

International audience; TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

We have reported that a novel c-Myc-binding protein, MM-1, repressed the E-box-dependent transcription activity of c-Myc by recruiting the HDAC1 complex via TIF1s/ KAP1, a transcriptional corepressor. We have also reported that a mutation of A157R in MM-1, which is often observed in patients with leukemia or lymphoma, abrogated all of the repressive activities of MM-1 toward c-Myc, indicating that MM-1 is a novel tumor suppressor. In this study, we found that MM-1 was bound to a component of proteasome and stimulated degradation of c-Myc in human cells. Knockdown of endogenous MM-1 in human HeLa cells by introduction of siRNA against MM-1 stabilized the endogenous c-Myc. To identify proteins that participate in c-Myc degradation by MM-1, in vivo and in vitro binding assays were carried out. The results showed that MM-1 directly bound to Rpt3, a subunit of 26S proteasome, and that c-Myc directly bound to Skp2, which recruited ElonginC, ElonginB and Cullin2, thereby forming a novel ubiquitin E3 ligase. Knockdown of endogenous Cullin2 stabilized the endogenous c-Myc. Thus, MM-1 is a factor that connects c-Myc to the ubiquitin E3 ligase and the proteasome. Plasmids. pcDNA3-FLAG-Elongin B, pcDNA3-FLAG- Rpt3, pcDNA3-FLAG-Cullin2, pcDNA3-T7-Rpn12 and pcDNA3-T7-Skp2, corresponding cDNAs starting from the first ATG, were inserted in-frame into the EcoRI-XhoI sites of pcDNA3-FLAG and pcDNA3-T7, respectively. Plasmids of various kinds of c-Myc and MM-1 were described previously (11-13).

International audience; Monitoring Editor: Thomas Sommer The ubiquitin-associated (UBA) domain of the mRNA nuclear export receptor Mex67 helps coordinating transcription elongation and nuclear export by interacting both with ubiquitin conjugates and specific targets, such as Hpr1, a component of the THO complex. Here we analyzed substrate specificity and ubiquitin selectivity of the Mex67 UBA domain. UBA-Mex67 is formed by three helices arranged in a classical UBA fold plus a fourth helix, H4. Deletion or mutation of helix H4 strengthens the interaction between UBA-Mex67 and ubiquitin, but decreases its affinity for Hpr1. Interaction with Hpr1 is required for Mex67 UBA domain to bind poly-ubiquitin, possibly by inducing an H4-dependent conformational change. In vivo, deletion of helix H4 reduces cotranscriptional recruitment of Mex67 on activated genes and also shows a mRNA export defect. Based on these results, we propose that H4 functions as a molecular switch that coordinates the interaction of Mex67 with ubiquitin bound to specific substrates, defines the selectivity of the Mex67 UBA domain for poly-ubiquitin and prevents its binding to nonspecific substrates.

To regulate the spatiotemporal expression of their target genes, the transcription factors undergo post-translational modifications of which the most studied is phosphorylation. Acetylation and ubiquitinylation on lysine residues also exert a role in the transcription, as it is the case for the regulation of the activity of the huge family of Ets transcription factors. Recently, sumoylation, a post-translational modification similar to ubiquitinylation, was described as playing a crucial role in the inhibition of the activity of these factors.

The mRNA nuclear export receptor Mex67/Mtr2 is recruited to mRNAs through RNA-binding adaptors, including components of the THO/TREX complex that couple transcription to mRNA export. Here we show that the ubiquitin-associated (UBA) domain of Mex67 is not only required for proper nuclear export of mRNA but also contributes to recruitment of Mex67 to transcribing genes. Our results reveal that the UBA domain of Mex67 directly interacts with polyubiquitin chains and with Hpr1, a component of the THO/TREX complex, which is regulated by ubiquitylation in a transcription-dependent manner. This interaction transiently protects Hpr1 from ubiquitin/proteasome-mediated degradation and thereby coordinates recruitment of the mRNA export machinery with transcription and early messenger ribonucleoproteins assembly.

Frontotemporal dementia (FTD) with ubiquitin-immunoreactive neuronal inclusions ( both cytoplasmic and nuclear) of unknown nature has been linked to a chromosome 17q21 region (FTDU-17) containing MAPT (microtubule-associated protein tau)(1-3). FTDU-17 patients have consistently been shown to lack a tau-immunoreactive pathology(1-3), a feature characteristic of FTD with parkinsonism linked to mutations in MAPT (FTDP-17)(4). Furthermore, in FTDU-17 patients, mutations in MAPT and genomic rearrangements in the MAPT region have been excluded by both genomic sequencing(5) and fluorescence in situ hybridization on mechanically stretched chromosomes(6). Here we demonstrate that FTDU-17 is caused by mutations in the gene coding for progranulin (PGRN), a growth factor involved in multiple physiological and pathological processes including tumorigenesis(7). Besides the production of truncated PGRN proteins due to premature stop codons(8), we identified a mutation within the splice donor site of intron 0 (IVS0+5G> C), indicating loss of the mutant transcript by nuclear degradation. The finding was made within an extensively documented Belgian FTDU-17 founder family(3). Transcript and protein analyses confirmed the absence of the mutant allele and a reduction in the expression of PGRN. We also identified a mutation ( c. 3G>A) in the Met1 translation initiation codon, indicating loss of PGRN due to lack of translation of the mutant allele. Our data provide evidence that PGRN haploinsufficiency leads to neurodegeneration because of reduced PGRN-mediated neuronal survival. Furthermore, in a Belgian series of familial FTD patients, PGRN mutations were 3.5 times more frequent than mutations in MAPT, underscoring a principal involvement of PGRN in FTD pathogenesis.

Ubiquitination is important for a broad array of cellular functions. Although reversal of this process, de-ubiquitination, most probably represents an important regulatory step contributing to cellular homeostasis, the specificity and properties of de-ubiquitination enzymes remain poorly understood. Here, we show that the Saccharomyces cerevisiae ubiquitin protease Ubp3 requires an additional protein, Bre5, to form an active de-ubiquitination complex that cleaves ubiquitin from specific substrates. In particular, this complex rescues Sec23p, a COPII subunit essential for the transport between the endoplasmic reticulum and the Golgi apparatus, from degradation by the proteasome. This probably contributes to maintaining and adapting a Sec23 expression level that is compatible with an efficient secretion pathway, and consequently with cell growth and viability.

Sendai virus infection strongly induces interferon (IFN) production and has recently been shown to interdict the subsequent IFN signaling through the Jak/Stat pathway. This anti-IFN activity of SeV is due to its “C” proteins, a nested set of four proteins (C′, C, Y1, Y2) that carry out a nested set of functions in countering the innate immune response. We previously reported that all four C proteins interact with Stat1 to prevent IFN signaling through the Jak/Stat pathway. Nevertheless, only the longer C proteins reduced Stat1 levels and prevented IFN from inducing an antiviral (VSV) state, or apoptosis, in IFN-competent murine cells. Here, we investigate the mechanism by which the various C proteins differentially affect the host antiviral defenses. All four C proteins were found to physically associate with Stat1 during cell culture infections, and in vitro in the absence of other viral gene products (as evidenced by co-immunoprecipitation). In addition, the inability of a null mutant (CF170S) to bind Stat1 suggests that this interaction is physiologically relevant. We have also shown that the proteasomal inhibitor MG132 can prevent the C protein-induced dismantling of the antiviral (VSV) state in murine cells; thus, the turnover of Stat1 correlates with the C protein-mediated counteraction of the antiviral (VSV) state. The C protein-induced instability of Stat1 was accompanied by a clear increase in the level of mono-ubiquinated Stat1, an unexpected hallmark of protein degradation. Finally, we show that a rSeV with mutant C proteins but wild-type Y proteins (CΔ10–15, that does not counteract the endogenous antiviral (VSV) state of MEFs even though their C proteins bind Stat1 and prevent its activity) is also unable to decrease bulk Stat1 levels or to increase the level of ubiquinated Stat1.