The Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 is the only viral protein expressed in all EBV-carrying malignancies, but its contribution to oncogenesis has remained enigmatic. We show that EBNA-1 induces chromosomal aberrations, DNA double-strand breaks, and engagement of the DNA damage response (DDR). These signs of genomic instability are associated with the production of reactive oxygen species (ROS) and are reversed by antioxidants. The catalytic subunit of the leukocyte NADPH oxidase, NOX2/gp91 phox , is transcriptionally activated in EBNA-1-expressing cells, whereas inactivation of the enzyme by chemical inhibitors or RNAi halts ROS production and DDR. These findings highlight a novel function of EBNA-1 and a possible mechanism by which expression of this viral protein could contribute to malignant transformation and tumor progression.is a human gamma-herpesvirus that establishes latent infections in B lymphocytes, where only a subset of viral genes is expressed and virus replication is suppressed (1). The proteins encoded by the latency genes, including 6 EBV-encoded nuclear antigens (EBNA-1, -2, -3A, -3B, -3C, and -5) and 3 latent membrane proteins (LMP1, -2A, and -2B), induce growth transformation by capturing multiple signaling pathways that control B cell proliferation and apoptosis. It is generally assumed that the continuous expression of viral genes underlies the association of EBV with a variety of human malignancies, including Burkitt's lymphoma (BL), Hodgkin's disease (HD), nasopharyngeal carcinoma (NPC), and posttransplant lymphoproliferative disease (PTLD) (2). Some EBVpositive tumors do not express all of the latency proteins, leading to restricted forms of latency in which EBNA-1 is detected either alone (latency I, found in BL) or together with the LMPs (latency II, found in HD and NPC). Thus, EBNA-1 is the only viral protein regularly expressed in all EBV-carrying malignancies.EBNA-1 binds to the viral origin of replication (oriP) and is required for the correct partitioning of the viral episomes in proliferating cells (3). It may confer a growth advantage to BL cells (4) and protect them from apoptosis (5) but does not act as an autonomous oncogene (6) and seems to be dispensable for B cell immortalization in vitro (7). Hence, the mechanism by which EBNA-1 may contribute to malignant transformation is not understood.Genomic instability is common in malignant cells and was observed in EBV-carrying tumors (8-10). EBNA-3C (11) and LMP-1 (12) may promote this phenotype through inhibition of DNA repair or inactivation of cell cycle checkpoints, which allow the propagation of DNA damage. However, these viral proteins are not expressed in EBV-carrying BLs, and only half of HDs and NPCs express detectable levels of LMP1, suggesting a limited role in EBV oncogenesis. A possible involvement of EBNA-1 in the induction of genomic instability is suggested by a significant increase of transient chromosomal aberrations, such as dicentric chromosomes, chromosome fragments, and gaps, in EBVpositiv...
Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic-reticulum-associated degradation (ERAD). Although the involvement of membraneassociated ubiquitin-conjugating enzymes and ligases in these processes is well documented, their regulation by ubiquitin deconjugases is less well understood. By screening a database of human deubiquitinating enzymes (DUBs), we have identified a putative transmembrane domain in ubiquitin-specific protease (USP)19. We show that USP19 is a tail-anchored ubiquitin-specific protease localized to the ER and is a target of the unfolded protein response. USP19 rescues the ERAD substrates cystic fibrosis transmembrane conductance regulator (CFTR)DF508 and T-cell receptor-a (TCRa) from proteasomal degradation. A catalytically inactive USP19 was still able to partly rescue TCRa but not CFTRDF508, suggesting that USP19 might also exert a non-catalytic function on specific ERAD substrates. Thus, USP19 is the first example of a membrane-anchored DUB involved in the turnover of ERAD substrates.
The large tegument proteins of herpesviruses encode conserved cysteine proteases of unknown function. Here we show that BPLF1, the Epstein-Barr-virus-encoded member of this protease family, is a deneddylase that regulates virus production by modulating the activity of cullin-RING ligases (CRLs). BPLF1 hydrolyses NEDD8 conjugates in vitro, acts as a deneddylase in vivo, binds to cullins and stabilizes CRL substrates. Expression of BPLF1 alone or in the context of the productive virus cycle induces accumulation of the licensing factor CDT1 and deregulates S-phase DNA synthesis. Inhibition of BPLF1 during the productive virus cycle prevents cellular DNA re-replication and inhibits virus replication. Viral DNA synthesis is restored by overexpression of CDT1. Homologues encoded by other herpesviruses share the deneddylase activity. Thus, these enzymes are likely to have a key function in the virus life cycle by inducing a replication-permissive S-phase-like cellular environment.
Ventilation-induced diaphragm dysfunction (VIDD) is a marked decline in diaphragm function in response to mechanical ventilation, which has negative consequences for individual patients' quality of life and for the health care system, but specific treatment strategies are still lacking. We used an experimental intensive care unit (ICU) model, allowing time-resolved studies of diaphragm structure and function in response to long-term mechanical ventilation and the effects of a pharmacological intervention (the chaperone co-inducer BGP-15). The marked loss of diaphragm muscle fiber function in response to mechanical ventilation was caused by posttranslational modifications (PTMs) of myosin. In a rat model, 10 days of BGP-15 treatment greatly improved diaphragm muscle fiber function (by about 100%), although it did not reverse diaphragm atrophy. The treatment also provided protection from myosin PTMs associated with HSP72 induction and PARP-1 inhibition, resulting in improvement of mitochondrial function and content. Thus, BGP-15 may offer an intervention strategy for reducing VIDD in mechanically ventilated ICU patients.
The translation initiation factor complex eIF3f has an intrinsic deubiquitinase activity and regulates the Notch signaling pathway.
Sarcoglycanopathies are progressive muscle-wasting disorders caused by genetic defects of four proteins, ␣-, -, ␥-, and ␦-sarcoglycan, which are elements of a key transmembrane complex of striated muscle. The proper assembly of the sarcoglycan complex represents a critical issue of sarcoglycanopathies, as several mutations severely perturb tetramer formation. Misfolded proteins are generally degraded through the cell's quality-control system; however, this can also lead to the removal of some functional polypeptides. To explore whether it is possible to rescue sarcoglycan mutants by preventing their degradation, we generated a heterologous cell system, based on human embryonic kidney (HEK) 293 cells, constitutively expressing three (, ␥, and ␦) of the four sarcoglycans. In these ␥␦-HEK cells, the lack of ␣-sarcoglycan prevented complex formation and cell surface localization, wheras the presence of ␣-sarcoglycan allowed maturation and targeting of the tetramer. As in muscles of sarcoglycanopathy patients, transfection of ␥␦-HEK cells with disease-causing ␣-sarcoglycan mutants led to dramatic reduction of the mutated proteins and the absence of the complex from the cell surface. Proteasomal inhibition reduced the degradation of mutants and facilitated the assembly and targeting of the sarcoglycan complex to the plasma membrane. These data provide important insights for the potential development of pharmacological therapies for sarcoglycanopathies.
The large tegument proteins of herpesviruses contain N-terminal cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8. Expression of BPLF1 promotes cullin degradation and the stabilization of cullin-RING ligases (CRLs) substrates in the nucleus, while cytoplasmic CRLs and their substrates are not affected. The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication. Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by EBV reactivation in promoting virus replication.
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