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Apoptosis; Autophagy; Proteasome inhibitor Apoptosis; Autofagia; Inhibidor del proteasoma Apoptosi; Autofàgia; Inhibidor del proteasoma Protein ubiquitylation coordinates crucial cellular events in physiological and pathological conditions. A comparative analysis of the ubiquitin proteome from bortezomib (BTZ)-sensitive and BTZ-resistant mantle cell lymphoma (MCL) revealed an enrichment of the autophagy–lysosome system (ALS) in BTZ-resistant cells. Pharmacological inhibition of autophagy at the level of lysosome-fusion revealed a constitutive activation of proteaphagy and accumulation of proteasome subunits within autophagosomes in different MCL cell lines with acquired or natural resistance to BTZ. Inhibition of the autophagy receptor p62/SQSTM1 upon verteporfin (VTP) treatment disrupted proteaphagosome assembly, reduced co-localization of proteasome subunits with autophagy markers and negatively impacted proteasome activity. Finally, the silencing or pharmacological inhibition of p62 restored the apoptosis threshold at physiological levels in BTZ-resistant cells both in vitro and in vivo. In total, these results demonstrate for the first time a proteolytic switch from the ubiquitin–proteasome system (UPS) to ALS in B-cell lymphoma refractory to proteasome inhibition, pointing out a crucial role for proteaphagy in this phenomenon and paving the way for the design of alternative therapeutic venues in treatment-resistant tumors. This work was supported at early stages by Spanish MINECO, CTQ2011–27874 grant. M.G.-S. is a fellow of the UbiCODE project funded by the EU’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 765445. M.S.R. and L.C. were also funded by the Institut National du Cancer, France (PLBIO16-251), CONACyT-SRE (Mexico) grant 0280365 and the REPERE program of Occitanie. O.C. is funded by “La Ligue contre le cancer du Gard”. ICFO authors were supported by funding from the Spanish MINECO “Severo Ochoa” program for Centres of Excellence in R&D (CEX2019-000910-S [MCIN/ AEI/10.13039/501100011033]), from Fundació Privada Cellex, Fundación Mig-Puig, from Generalitat de Catalunya CERCA program and from LASERLAB Europe (grant agreement No 871124;). G.R. was financially supported by Fondo de Investigación Sanitaria PI15/00102 and PI18/01383, European Regional Development Fund (ERDF) ‘Una manera de hacer Europa’. G.R. and D.R.G. are members of the Spanish Network of Excellence UBIRed funded by the Spanish Ministry Science, Innovation and Universities (SAF2017-90900-REDT).

DNA damage activated by Adriamycin (ADR) promotes ubiquitin–proteasome system-mediated proteolysis by stimulating both the activity of ubiquitylating enzymes and the proteasome. In ADR-resistant breast cancer MCF7 (MCF7ADR) cells, protein ubiquitylation is significantly reduced compared to the parental MCF7 cells. Here, we used tandem ubiquitin-binding entities (TUBEs) to analyze the ubiquitylation pattern observed in MCF7 or MCF7ADR cells. While in MCF7, the level of total ubiquitylation increased up to six-fold in response to ADR, in MCF7ADR cells only a two-fold response was found. To further explore these differences, we looked for cellular factors presenting ubiquitylation defects in MCF7ADR cells. Among them, we found the tumor suppressor p53 and its ubiquitin ligase, Mdm2. We also observed a drastic decrease of proteins known to integrate the TUBE-associated ubiquitin proteome after ADR treatment of MCF7 cells, like histone H2AX, HMGB1 or β-tubulin. Only the proteasome inhibitor MG132, but not the autophagy inhibitor chloroquine partially recovers the levels of total protein ubiquitylation in MCF7ADR cells. p53 ubiquitylation is markedly increased in MCF7ADR cells after proteasome inhibition or a short treatment with the isopeptidase inhibitor PR619, suggesting an active role of these enzymes in the regulation of this tumor suppressor. Notably, MG132 alone increases apoptosis of MCF7ADR and multidrug resistant ovarian cancer A2780DR1 and A2780DR2 cells. Altogether, our results highlight the use of ubiquitylation defects to predict resistance to ADR and underline the potential of proteasome inhibitors to treat these chemoresistant cells.

The function of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is tightly controlled by post-translational modifications (PTMs) including ubiquitin or Small Ubiquitin-related MOdifiers (SUMO). It is known that SUMOylation by SUMO-1, SUMO-2/-3, mono- or polyubiquitylation have a distinct impact on PTEN activity, localisation and/or stability, however the molecular mechanisms governing these processes are still unclear. Studying PTM regulated events has always been a difficult task due to their labile nature. Here, we propose an update on the role of these PTMs on PTEN function, as well as a methodological overview on the use of molecular traps named SUMO Binding Entities (SUBEs) or Tandem Ubiquitin Binding Entities (TUBEs) to capture SUMOylated or Ubiquitylated forms of PTEN respectively. When combined with in vitro SUMOylation or Ubiquitylation assays, the use of molecular traps facilitate the detection of modified forms of PTEN. SUMO and ubiquitin-traps are also suitable to capture endogenously modified forms of PTEN after expression of E3 ligases or treatment with chemical inhibitors. This versatile approach represents an interesting alternative to explore PTEN regulation by SUMO and ubiquitin under physiological or pathological conditions.

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.

Tandem ubiquitin-binding entities (TUBEs) act as molecular traps to isolate polyubiquitylated proteins facilitating the study of this highly reversible posttranslational modification. We provide here sample preparation and adaptations required for TUBE-based enrichment of the ubiquitin proteome from tumor cell lines or primary cells. Our protocol is suitable to identify ubiquitin substrates, enzymes involved in the ubiquitin proteasome pathway, as well as proteasome subunits by mass spectrometry. This protocol was adapted to prepare affinity columns, reduce background, and improve the protein recovery depending on the sample source and necessities.

The successful use of proteasome inhibitors in clinical trials revealed the potential of the Ubiquitin Proteasome System for drug development. Protein remodeling through ubiquitylation is known to regulate the stability and activity of essential cellular factors through largely uncharacterized mechanisms. Here, we used Tandem repeated Ubiquitin Binding Entities (TUBEs) under non-denaturing conditions followed by mass spectrometry analysis to study global ubiquitylation events that may lead to the identification of potential drug targets. Using this approach we identified 643 proteins including known and unknown ubiquitin targets from human breast adenocarcinoma MCF7 cells treated with Adriamycin. Coherent with a global cellular response to this genotoxic insult, cellular factors identified are involved in protein synthesis, cellular transport, RNA post-transcriptional modification and signaling pathways regulating early stress responses. This includes components of large macromolecular complexes such as subunits and regulators of the proteasome, supporting the use of this method to characterize networks of molecular interactions coordinated by ubiquitylation. Further in vitro and in silico analysis confirmed that 84% of the total proteins identified here, are ubiquitylated. More importantly the enrichment of known biomarkers and targets for drug development, underlined the potential of this approach for the identification of this clinically relevant information. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Ubiquitylation provides a rapid alternative to control the activity of crucial cellular factors through the remodelling of a target protein. Diverse ubiquitin chains are recognized by domains with affinity for UBDs (ubiquitin-binding domains) present in receptor/effector proteins. Interestingly, some proteins contain more than one UBD and the preservation of this structure in many species suggests an evolutionary advantage for this topology. Here, we review some typical proteins that naturally contain more than one UBD and emphasize how such structures contribute to the mechanism they mediate. Characteristics such as higher affinities for polyubiquitin chains and chain-linkage preferences can be replicated by the TUBEs (tandem ubiquitin-binding entities). Furthermore, TUBEs show two additional properties: protection of ubiquitylated substrates from deubiquitylating enzymes and interference with the action of the proteasome. Consequently, TUBEs behave as ‘ubiquitin traps’ that efficiently capture endogenous ubiquitylated proteins. Interpretations and hypothetical models proposed by different groups to understand the synchronous action of multiple UBDs are discussed herein.

Malaria, caused by Plasmodium falciparum (P. falciparum), ranks as one of the most baleful infectious diseases worldwide. New antimalarial treatments are needed to face existing or emerging drug resistant strains. Protein degradation appears to play a significant role during the asexual intraerythrocytic developmental cycle (IDC) of P. falciparum. Inhibition of the ubiquitin proteasome system (UPS), a major intracellular proteolytic pathway, effectively reduces infection and parasite replication. P. falciparum and erythrocyte UPS coexist during IDC but the nature of their relationship is largely unknown. We used an approach based on Tandem Ubiquitin-Binding Entities (TUBEs) and 1D gel electrophoresis followed by mass spectrometry to identify major components of the TUBEs-associated ubiquitin proteome of both host and parasite during ring, trophozoite and schizont stages. Ring-exported protein (REX1), a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, was found to reach a maximum level of ubiquitylation in trophozoites stage. The Homo sapiens (H. sapiens) TUBEs associated ubiquitin proteome decreased during the infection, whereas the equivalent P. falciparum TUBEs-associated ubiquitin proteome counterpart increased. Major cellular processes such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection.In this work we analyze for the first time the interconnection between Plasmodium and human red blood cells ubiquitin-regulated proteins in the context of infection. We identified a number of human and Plasmodium proteins whose ubiquitylation pattern changes during the asexual infective stage. We demonstrate that ubiquitylation of REX1, a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, peaks in trophozoites stage. The ubiquitin-proteome from P. falciparum infected red blood cells (iRBCs) revealed a significant host-parasite crosstalk, underlining the importance of ubiquitin-regulated proteolytic activities during the intraerythrocytic developmental cycle (IDC) of P. falciparum. Major cellular processes defined from gene ontology such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection. Given the importance of ubiquitylation in the development of infectious diseases, this work provides a number of potential drug-target candidates that should be further explored.

Background The ubiquitin proteasome system (UPS) is one of the main proteolytical pathways in eukaryotic cells and plays an essential role in key cellular processes such as cell cycle, stress response, signal transduction, and transcriptional regulation. Many components of this pathway have been implicated in diverse pathologies including cancer, neurodegeneration and infectious diseases, such as malaria. The success of proteasome inhibitors in clinical trials underlines the potential of the UPS in drug discovery. Methods Plasmodium falciparum, the malaria causative pathogen, has been used to develop two assays that allow the quantification of the parasite protein ubiquitylation levels in a high-throughput format that can be used to find new UPS inhibitors. Results In both assays tandem ubiquitin binding entities (TUBEs), also known as ubiquitin traps, have been used to capture ubiquitylated proteins from cell lysates. The primary assay is based on AlphaLISA technology, and the orthogonal secondary assay relies on a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) system. A panel of well-known proteasome inhibitors has been used to validate both technologies. An excellent correlation was obtained between these biochemical assays and the standard whole cell assay that measures parasite growth inhibition. Conclusions The two assays presented can be used in a high-throughput format to find new UPS inhibitors for P. falciparum and could help to identify new targets within this system. This methodology is also applicable to other cellular contexts or pathologies.

The Ubiquitin-Proteasome System (UPS) plays an important role in the setting of the cellular response to multiple stress signals. Although the primary function of ubiquitin was initially associated with proteolysis, it is now considered as a key regulator of protein function controlling, among other functions, signalling cascades, transcription, apoptosis or oncogenesis. Failure at any level of the UPS is associated with the development of multiple pathologies including metabolic problems, immune diseases, inflammation and cancer. The successful use of the proteasome inhibitor Bortezomib (Velcade) in the treatment of multiple myeloma (MM) and mantle cell lymphoma (MCL) revealed the potential of the UPS as pharmacological target. Ten years later, new inhibitors tackling not only the proteasome but also different subsets of enzymes which conjugate or de-conjugate ubiquitin or ubiquitin-like molecules, have been developed. Most of them are excellent tools to characterize better the emerging molecular mechanisms regulating distinct critical cellular processes. Some of them have been launched already while many others are still in pre-clinical development. This chapter updates some of the most successful efforts to develop and characterize inhibitors of the UPS which tackle mechanisms involved in cancer. Particular attention has been dedicated to updating the status of the clinical trials of these inhibitors.

The transcription factor NF-kappaB is a key modulator of apoptosis in a variety of cell types, but to date this specific function of NF-kappaB has not been demonstrated in epithelia. Here, we describe the activation of NF-kappaB during post-lactational involution of the mouse mammary gland, a period of extensive apoptosis of luminal epithelial cells. Significantly, active NF-kappaB localized exclusively to nonapoptotic epithelial cells both in vivo and in the mammary epithelial cell line, KIM-2, transduced with an NF-kappaB-dependent green fluorescent protein reporter. Activation of NF-kappaB in vitro coincided with a decrease in the cytosolic repressor, IkappaBalpha. Furthermore, induction of NF-kappaB either by extracellular ligands or, more specifically, by inhibition of the IkappaB repressor with adenoviral constructs expressing antisense mRNA, resulted in enhanced survival of KIM-2 cells. Therefore, although coincident with induction of apoptosis both in vivo and in vitro, NF-kappaB appeared to exert a selective survival function in epithelial cells. This study highlights for the first time a role for NF-kappaB in modulating apoptosis in epithelium.

This work aims at characterizing the interplay between human immunodeficiency virus type 1 (HIV-1) and the antiapoptotic cellular protein Bcl-2 responsible for a persistent infection in lymphoblastoid T (J.Jhan) or monocytic (U937) cells. We report that the kinetics of Bcl-2 protein level during the establishment of a chronic infection is biphasic, characterized by a transient decrease followed by restoration to the initial level. The extent and duration of this transient decrease were inversely correlated with the basal level of Bcl-2 as shown by kinetics of Bcl-2 levels in J.Jhan or U937 clones exhibiting different levels of Bcl-2. Using these clones, we also showed that Bcl-2 downregulates HIV-1 replication. Therefore, the cells overexpressing Bcl-2 are characterized by a low viral burden which, in turn, has little effect on the level of this protein. The observed bipasic kinetics is the result of a dual regulation of Bcl-2 induced by HIV-1 infection itself: an upregulation at the transcriptional level of the bcl-2 gene concomitant with a downregulation at the protein level. Convergent data suggest that this downregulation is caused by the oxidative stress induced by the infection itself as shown by the associated modulations of glutathione and thioredoxin levels and by the prevention of these dysregulations by N -acetylcysteine. Altogether, these data indicate that infection first results in a decrease of Bcl-2, permitting an initial boost of replication. Then, as the synthesis at the transcriptional level proceeds, the replication is negatively controlled by Bcl-2 to reach a balance characterized by low virus production and a level of Bcl-2 compatible with cell survival. We suggest that the basal level of Bcl-2, together with infection-inducible transcription factors able to activate bcl-2 gene transcription, is a critical cellular determinant in the tendency toward an acute or a persistent infection.

In the human lymphoblastoid T cell line JJhan-5.1, stably transfected with a human immunodeficiency virus-1 long terminal repeat luciferase vector, the level of luciferase activity is dependent on activation of the nuclear factor kappaB (NF-kappaB) transcription factor. Tumor necrosis factor-induced luciferase activity was not modified in JJhan-5.1 cells co-cultivated with murine adenocarcinoma EMT-6 cells but was strongly decreased when nitric oxide (NO) synthase 2 expression was induced in these cells. Two NO synthase inhibitors counteracted this inhibitory effect. Tumor necrosis factor-alpha binding to JJhan-5.1 cells was not modified after incubation with EMT-6 cells. Viability and protein synthesis in JJhan-5.1 cells were also unchanged. Induction of NF-kappaB DNA binding activity was inhibited when EMT-6 cells expressed NO synthase 2 activity. Aminoguanidine, which completely abolished nitrite production, prevented this inhibition. NF-kappaB activation was also strongly inhibited by S-nitrosoglutathione but was marginally affected by N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1, 2-ethylenediamine. Taken together, these results indicated that NO-related species, released by EMT-6 effector cells and probably different from NO itself, inhibited NF-kappaB activation in human lymphoblastoid target cells. Consequently, transcriptional activity of a long terminal repeat-driven luciferase gene construct was markedly diminished.

The tumor suppressor p53 regulates the expression of genes involved in cell cycle progression, senescence and apoptosis. Here, we investigated the effect of single point mutations in the oligomerization domain (OD) on tetramerization, transcription, ubiquitylation and stability of p53. As predicted by docking and molecular dynamics simulations, p53 OD mutants show functional defects on transcription, Mdm2-dependent ubiquitylation and 26S proteasome-mediated degradation. However, mutants unable to form tetramers are well degraded by the 20S proteasome. Unexpectedly, despite the lower structural stability compared to WT p53, p53 OD mutants form heterotetramers with WT p53 when expressed transiently or stably in cells wild type or null for p53. In consequence, p53 OD mutants interfere with the capacity of WT p53 tetramers to be properly ubiquitylated and result in changes of p53-dependent protein expression patterns, including the pro-apoptotic proteins Bax and PUMA under basal and adriamycin-induced conditions. Importantly, the patient derived p53 OD mutant L330R (OD1) showed the more severe changes in p53-dependent gene expression. Thus, in addition to the well-known effects on p53 stability, ubiquitylation defects promote changes in p53-dependent gene expression with implications on some of its functions.

6 p. [+ 7 p. Supplementary Information] SUMO-modified proteins are recognized by SUMO interacting motifs (SIMs), thus triggering diverse cellular responses. Here SIMs were used to develop SUMO-traps to capture endogenous SUMOylated proteins. Our results show that these small peptides are transferable motifs that maintain their SUMO binding capacity when fused to the heterologous carrier protein GST. The tandem disposition of SIMs increases the binding capacity of SUMO-traps to specifically interact with polySUMO but not poly-Ubiquitin chains. We demonstrate that this SUMO capturing system purifies SUMOylated proteins such as I kappa B alpha, PTEN, PML or p53 in vitro and in vivo. These properties can be used to explore the many critical functions regulated by protein SUMOylation We would like to thank C. Gwizdek, M. Matunis and R.T. Hay for reagents and tools provided. This work was funded by the MINECO (Spain) grant BFU2008-01108/BMC and BFU2011-28536 (MSR), Department of Industry of the Government of the Basque Country (Etortek Research Programmes 2011/2012). Our group at Inbiomed is supported by the "Obra Social KUTXA'' and the Diputacion Foral de Guipuzcoa. E.DS-F is fellow from Fundacao para a Ciencia e Tecnologia, Ministerio da Educacao e Ciencia-Portugal, Grant SFRH/BD/71514/2010.

Studying postubiquitylation events has always been a difficult task due to the labile nature of these posttranslational modifications. When utilized in tandem, ubiquitin-binding entities (TUBEs) not only increase up to thousand times the affinity for poly-ubiquitin chains but also protect ubiquitylated proteins from the action of the proteasome and de-ubiquitylating enzymes.

Studying postubiquitylation events has always been a difficult task due to the labile nature of these posttranslational modifications. When utilized in tandem, ubiquitin-binding entities (TUBEs) not only increase up to thousand times the affinity for poly-ubiquitin chains but also protect ubiquitylated proteins from the action of the proteasome and de-ubiquitylating enzymes.

The NF-κB pathway is regulated by SUMOylation at least at three levels: the inhibitory molecule IκBα, the IKK subunit γ/NEMO and the p52 precursor p100. Here we investigate the role of SUMO-2/3 in the degradation of IκBα and activation of NF-κB mediated by TNFα. We found that under conditions of deficient SUMOylation, an important delay in both TNFα-mediated proteolysis of IκBα and NF-κB dependent transcription occurs. In vitro and ex vivo approaches, including the use of ubiquitin-traps (TUBEs), revealed the formation of chains on IκBα containing SUMO-2/3 and ubiquitin after TNFα stimulation. The integration of SUMO-2/3 appears to promote the formation of ubiquitin chains on IκBα after activation of the TNFα signalling pathway. Furthermore, heterologous chains of SUMO-2/3 and ubiquitin promote a more efficient degradation of IκBα by the 26S proteasome in vitro compared to chains of either SUMO-2/3 or ubiquitin alone. Consistently, Ubc9 silencing reduced the capture of IκBα modified with SUMO-ubiquitin hybrid chains that display a defective proteasome-mediated degradation. Thus, hybrid SUMO-2/3-ubiquitin chains increase the susceptibility of modified IκBα to the action of 26S proteasome, contributing to the optimal control of NF-κB activity after TNFα-stimulation.

Antioxidant molecules have been suggested to be of therapeutic value in the treatment of HIV-infected patients. To evaluate this possibility, we examined in vitro the effects of two types of antioxidant molecules in terms of inhibition of HIV replication in monocytes, one of the main reservoirs of HIV, and also in terms of modulation of the immune competence as measured by PBMC proliferation. We tested the effects of BHA, a phenolic, lipid-soluble, chain-breaking antioxidant, and NAC, a known glutathione precursor with some direct free-radical scavenging properties as well, on the regulation of HIV-1 expression in latently infected U1 cells and in productively and chronically infected U937 cells. Both antioxidants inhibited TNF- or PMA-induced NF-kappa B activity in U1 cells, as well as the sustained NF-kappa B activity permanently induced by the virus itself in chronically HIV-infected U937 cells. This resulted in only a partial inhibition of TNF- or PMA-induced HIV replication in U1 cells, and no detectable effect on HIV replication in chronically infected U937 cells. This may be the first limitation to potential antiviral effects of antioxidant therapies. Another limitation is that antioxidant concentrations high enough to block NK-kappa B activation were shown to have a suppressive effect on immune functions in vitro, because NAC and BHA blocked IL-2-induced PBMC proliferation. These data warrant prudence in the design of antioxidant-based therapies aimed at suppressing HIV replication.

In normal cells p53 is maintained at low level through the action of the ubiquitin-proteasome system. As a consequence of p53 transcriptional activity, various regulators of this tumor suppressor are produced, forming a negative feedback loop tightly controlling p53 stability. One of the most prominent is the ubiquitin-ligase Mdm2. Here, we have used a transfer of signals strategy to study the p53 degradation process promoted by Mdm2 in the absence of p53 transcriptional activity. Our results show that in a p53 null background, transcriptionally silent p53-fusions require multiple N- and C-terminal signals to be optimally targeted to proteasomal degradation. As for WT p53, p53-fusions able to form tetramers are polyubiquitylated and optimally degraded by the proteasome. However, p53 molecules unable to oligomerize, show Mdm2-mediated polyubiquitylation deficiency but are still targeted to proteasome degradation in vitro and ex vivo. In the presence of Mdm2, nuclear shuttling of p53 monomeric fusions favours proteasome-dependent degradability but not its polyubiquitylation. In vitro, 265 proteasome fails to drive degradation of OD mutants in the presence of Mdm2, suggesting the contribution of additional cellular factors in this process. All together, our results indicate that Mdm2-mediated proteasome-dependent degradation of polyubiquitylation deficient p53 monomers is mechanistically possible, taking alternative pathways to better achieve their proteolysis. These results support the existence of additional levels to regulate p53 stability and activity acting on individual subunits of the functional tetramer. (C) 2010 Elsevier Ltd. All rights reserved.

Background/Aims We have integrated gene expression profiling of liver biopsies of NASH patients with liver samples of a mouse model of steatohepatitis (MAT1A-KO) to identify a gene-pathway associated with NASH. Methods Affymetrix U133 Plus 2.0 microarrays were used to evaluate nine patients with NASH, six patients with steatosis, and six control subjects; Affymetrix MOE430A microarrays were used to evaluate wild-type and MAT1A-KO mice at 15 days, 1, 3, 5 and 8 months after birth. Transcriptional profiles of patients with NASH and MAT1A-KO mice were compared with those of their proficient controls. Results We identified a gene-pathway associated with NASH, that accurately distinguishes between patients with early-stage NASH and controls. Patients with steatosis have a gene expression pattern intermediate between that of NASH and controls. Promoter analysis revealed that 34 of the genes associated with NASH contained an Sp1 element. We found that Sp1 binding to these genes is increased in MAT1A-KO mice. Sp1 is also hyperphosphorylated in MAT1A-KO as well as in patients with NASH and steatosis. Conclusions A gene-pathway associated with NASH has been identified. We speculate that hyperphosphorylation of Sp1 may be involved in the genesis of steatosis and that other factors, such as oxidative stress, may trigger its progression to NASH.

Macrophages play a major role in HIV-1 persistence. In the present paper, we demonstrate that the absence of apoptosis in HIV-1-infected primary human monocyte-differentiated macrophages (MDM) correlates with an increase in anti-apoptotic (Bcl-2 and Bcl-x(L)) and a decrease in pro-apoptotic (Bax and Bad) proteins. This is associated with macrophage activation as shown by tumor necrosis factor (TNF) production and NF-kappaB activation upon infection. TNF production was shown to be involved in the upregulation of Bcl-2 and Bcl-x(L) because this increase was abolished by an anti-TNF anti-serum or an inhibitor of TNF synthesis. In parallel, inhibition of TNF production induced an increase in the number of apoptotic cells. Furthermore, using an inhibitor of NF-kappaB activation, we demonstrated that TNF-induced upregulation of Bcl-x(L) and Bcl-2 occurs, respectively, through a NF-kappaB-dependent and an NF-kappaB-independent pathway.

The NF-kappaB/Rel family of transcription factors regulates a wide variety of genes whose products play a fundamental role in inflammatory and immune responses. The implication of NF-kappaB/Rel proteins and their IkappaB regulatory subunits in the control of cellular growth and oncogenesis, was suggested by the induction of fatal lymphomas in birds by the v-rel oncoprotein, and the rearrangement and amplification of several genes encoding the NF-kappaB/Rel/IkappaB signal transduction factors in human malignancies, primarily of lymphoid origin. Hodgkin's disease (HD) is a lymphoma characterized by a low frequency of malignant Hodgkin and Reed-Sternberg (H/RS) cells in a reactive background of non-neoplastic cells. The peculiar activated phenotype of Hodgkin and Reed-Sternberg cells and their pattern of cytokine secretion are believed to be a consequence of constitutive activation of the NF-kappaB transcription factor. Here, we report the detection of mutations of the IkBa gene, in two HD-derived cell lines and in two out of eight biopsy samples from patients with relapsed Hodgkin's disease. The presence of defective IkappaBalpha is thus likely to explain the constitutive activation of NF-kappaB in these cells and suggests that IkappaBalpha is a tumour suppressor controlling the oncogenic activation of NF-kappaB in Hodgkin and Reed-Sternberg cells.

We used BHA, a phenolic, lipid-soluble, chain-breaking antioxidant to show that peroxyl radical scavenging blocks NF-1(B activation and HIV1 enhancer activity in PMA- or TNF-stimulated lymphoblastoid T (J.Jhan) and monocytic (U937) cell lines. The anti-oxidative effect of BHA was accompanied by an increase in thiol, but not glutathione, content in stimulated and unstimulated T cells, whereas TNF stimulation itself barely modified the cellular thiol level. Oxidative stress obtained by the addition of H2O2 to the culture medium of JJhan or U937 cells could not by itself induce NF-κB activation. These observations suggest that TNF and PMA do not lead to NF-1(B activation through induction of changes in the cell redox status. Rather, TNF and PMA can exert a full activation effect only if cells are in a basal redox equilibrium tending towards oxidation since prior modification towards reduction by BHA treatment prevents their activation effects.

9 p. The NF-κB pathway is regulated by multiple post-translational modifications including phosphorylation, ubiquitylation and SUMOylation. Many of these modifications act on the natural inhibitor IκBα modulating its capacity to control signal-mediated NF-κB activity. While the canonical pathway involving the phosphorylation and polyubiquitylation of IκBα has been well characterized, the role of these post-translational modifications in the control of basal NF-κB activity has not been deeply explored. Using the recently developed Tandem-repeated Ubiquitin Binding Entities (also known as ubiquitin traps) to capture ubiquitylated proteins, we identified monoubiquitylated forms of IκBα from multiple rat organs and cell types. The identification of these forms was demonstrated through different procedures such as immunoprecipitations with specific ubiquitin antibodies or His6-Ubiquitin pull downs. Monoubiquitylated forms of IκBα are resistant to TNFα-mediated degradation and can be captured using TUBEs, even after proteasome inhibitors treatment. As it occurs for monoSUMOylation, monoubiquitylation is not dependent of the phosphorylation of IκBα on the serines 32/36 and is not optimally degraded after TNFα stimulation. A ubiquitin-IκBα fusion exhibits phosphorylation defects and resistance to TNFα mediated degradation similar to the ones observed for endogenous monoubiquitylated IκBα. The N-terminal attachment of a single ubiquitin moiety on the IκBα fusion results in a deficient binding to the IKKβ kinase and recruitment of the SCF ligase component βTrCP, promoting a negative impact on the NF-κB activity. Altogether, our results suggest the existence of a reservoir of monoubiquitylated IκBα resistant to TNFα-induced proteolysis, which is able to interact and repress DNA binding and NF-κB transcriptional activity. Such pool of IκBα may play an important role in the control of basal and signal-mediated NF-κB activity. This work was funded by the Ramón y Cajal Program, Ministerio de Educación y Ciencia grant BFU2006-12991 and BFU2008-01108/BMC, Fondo de Investigaciones Sanitarias (FIS) CIBERhed, Government of the Autonomous Community of the Basque Country grant PI09-05, Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Etortek Research Programs 2008/2009) and from the Innovation Technology Department of the Bizkaia Country. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Accurate regulation of nuclear factor-κB (NF-κB) activity is crucial to prevent a variety of disorders including immune and inflammatory diseases. Active NF-κB promotes IκBα and A20 expression, important negative regulatory molecules that control the NF-κB response. In this study, using two-hybrid screening we identify the RING-type zinc-finger protein 114 (RNF114) as an A20-interacting factor. RNF114 interacts with A20 in T cells and modulates A20 ubiquitylation. RNF114 acts as negative regulator of NF-κB-dependent transcription, not only by stabilizing the A20 protein but also IκBα. Importantly, we demonstrate that in T cells, the effect of RNF114 is linked to the modulation of T-cell activation and apoptosis but is independent of cell cycle regulation. Altogether, our data indicate that RNF114 is a new partner of A2O involved in the regulation of NF-κB activity that contributes to the control of signaling pathways modulating T cell-mediated immune response.