Effects of doxorubicin cancer therapy on autophagy and the ubiquitin-proteasome system in long-term cultured adult rat cardiomyocytes

The clinical use of anthracyclines in cancer therapy is limited by dose-dependent cardiotoxicity that involves cardiomyocyte injury and death. We have tested the hypothesis that anthracyclines affect protein degradation pathways in adult cardiomyocytes. To this aim, we assessed the effects of doxorubicin (Doxo) on apoptosis, autophagy and the proteasome/ubiquitin system in long-term cultured adult rat cardiomyocytes. Accumulation of poly-ubiquitinated proteins, increase of cathepsin-D-positive lysosomes and myofibrillar degradation were observed in Doxo-treated cardiomyocytes. Chymotrypsin-like activity of the proteasome was initially increased and then inhibited by Doxo over a time-course of 48 h. Proteasome 20S proteins were down-regulated by higher doses of Doxo. The expression of MURF-1, an ubiquitin-ligase specifically targeting myofibrillar proteins, was suppressed by Doxo at all concentrations measured. Microtubule-associated protein 1 light chain 3B (LC3)-positive punctae and both LC3-I and -II proteins were induced by Doxo in a dose-dependent manner, as confirmed by using lentiviral expression of green fluorescence protein bound to LC3 and live imaging. The lysosomotropic drug chloroquine led to autophagosome accumulation, which increased with concomitant Doxo treatment indicating enhanced autophagic flux. We conclude that Doxo causes a downregulation of the protein degradation machinery of cardiomyocytes with a resulting accumulation of poly-ubiquitinated proteins and autophagosomes. Although autophagy is initially stimulated as a compensatory response to cytotoxic stress, it is followed by apoptosis and necrosis at higher doses and longer exposure times. This mechanism might contribute to the late cardiotoxicity of anthracyclines by accelerated aging of the postmitotic adult cardiomyocytes and to the susceptibility of the aging heart to anthracycline cancer therapy.

Involvement of the Ubiquitin/Proteasome System in Sorting of the Interleukin 2 Receptor β Chain to Late Endocytic Compartments

Down-regulation of cell surface growth factor receptors plays a key role in the tight control of cellular responses. Recent reports suggest that the ubiquitin system, in addition to participating in degradation by the proteasome of cytosolic and nuclear proteins, might also be involved in the down-regulation of various membrane receptors. We have previously characterized a signal in the cytosolic part of the interleukin 2 receptor β chain (IL2Rβ) responsible for its targeting to late endosomes/lysosomes. In this report, the role of the ubiquitin/proteasome system on the intracellular fate of IL2Rβ was investigated. Inactivation of the cellular ubiquitination machinery in ts20 cells, which express a thermolabile ubiquitin-activating enzyme E1, leads to a significant decrease in the degradation rate of IL2Rβ, with little effect on its internalization. In addition, we show that a fraction of IL2Rβ can be monoubiquitinated. Furthermore, mutation of the lysine residues of the cytosolic region of a chimeric receptor carrying the IL2Rβ targeting signal resulted in a decreased degradation rate. When cells expressing IL2Rβ were treated either by proteasome or lysosome inhibitors, a significant decrease in receptor degradation was observed. Our data show that ubiquitination is required for the sorting of IL2Rβ toward degradation. They also indicate that impairment of proteasome function might more generally affect intracellular routing.

Mice carrying ubiquitin-specific protease 2 (Usp2) gene inactivation maintain normal sodium balance and blood pressure

Ubiquitylation plays an important role in the control of Na⁺ homeostasis by the kidney. It is well established that the epithelial Na⁺ channel ENaC is regulated by the ubiquitin-protein ligase NEDD4-2, limiting ENaC cell surface expression and activity. Ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs). One such DUB, USP2-45, was identified previously as an aldosterone-induced protein in the kidney and is also a circadian output gene. In heterologous expression systems, USP2-45 binds to ENaC, deubiquitylates it, and enhances channel density and activity at the cell surface. Because the role of USP2-45 in renal Na⁺ transport had not been studied in vivo, we investigated here the effect of Usp2 gene inactivation in this process. We demonstrate first that USP2-45 protein has a rhythmic expression with a peak at ZT12. Usp2-KO mice did not show any differences from wild-type littermates with respect to the diurnal control of Na⁺ or K⁺ urinary excretion and plasma levels either on a standard diet or after acute and chronic changes to low- and high-Na⁺ diets, respectively. Moreover, they had similar aldosterone levels on either a low- or high-Na⁺ diet. Blood pressure measurements using telemetry did not reveal variations compared with control mice. Usp2-KO mice did not display alterations in expression of genes involved in sodium homeostasis or the ubiquitin system, as evidenced by transcriptome analysis in the kidney. Our data suggest that USP2 does not play a primary role in the control of Na⁺ balance or blood pressure.

Provirus integration into a gene encoding a ubiquitin-conjugating enzyme results in a placental defect and embryonic lethality.

Ubiquitin-conjugating enzymes (E2 or Ubc) constitute a family of conserved proteins that play a key role in ubiquitin-dependent degradation of proteins in eukaryotes. We describe here a transgenic mouse strain where retrovirus integration into an Ubc gene, designated UbcM4, results in a recessive-lethal mutation. UbcM4 is the mouse homologue of the previously described human UbcH7 that is involved in the in vitro ubiquitination of several proteins including the tumor suppressor protein p53. The provirus is located in the first intron of the gene. When both alleles are mutated the level of steady-state mRNA is reduced by about 70%. About a third of homozygous mutant embryos die around day 11.5 of gestation. Embryos that survive that stage are growth retarded and die perinatally. The lethal phenotype is most likely caused by impairment of placenta development as this is the only organ that consistently showed pathological defects. The placental labyrinth is drastically reduced in size and vascularization is disturbed. The UbcM4 mouse mutant represents the first example in mammals of a mutation in a gene involved in ubiquitin conjugation. Its recessive-lethal phenotype demonstrates that the ubiquitin system plays an essential role during mouse development.

Two Nuclear Localization Signals in USP1 Mediate Nuclear Import of the USP1/UAF1 Complex

9 p. : il.

Rôle de la déubiquitinase BAP1 dans la réponse cellulaire aux dommages à l'ADN

L'ubiquitination est une modification post-traductionnelle qui joue un rôle central dans divers processus biologiques. Elle peut être contrecarrée par les déubiquitinases (DUBs). "BRCA1-Associated Protein 1" (BAP1) est une déubiquitinase, qui fait partie de complexes multiprotéiques, possèdant une fonction de suppression tumorale ainsi qu'un potentiel anti-métastatique. De plus, BAP1 est phosphorylée suite aux dommages à l’ADN par les kinases ATM/ATR. En nous basant sur ces données, nous avons purifié les protéines associées à BAP1 dans des conditions de stress génotoxique. Bien que la composition du complexe et l’activité DUB semblent inchangées, nous avons pu identifier des changements critiques dans les niveaux et les sites de phosphorylation, confirmant la régulation de BAP1 suite aux dommages à l’ADN. En déplétant BAP1 par ARNi et en utilisant des mutants dominants négatifs, nous avons obtenu des résultats suggèrant que suite au stress génotoxique, cette DUB est requise pour prolonger le point de contrôle en G2/M et ce, en retardant la reprise du cycle cellulaire. D'un autre côté, l'expression de BAP1 dans des cellules cancéreuses qui en sont déficientes restore une ploïdie normale et diminue la fréquence d'aberrations nucléaires, suggérant que cette protéine joue un rôle dans la stabilité génomique. Nos résultats suggèrent fortement que BAP1 joue un rôle dans la réponse des cellules au stress génotoxique et la stabilité génomique. Nos travaux permettront ainsi d’identifier et de caractériser les voies de signalisation cellulaire régulant l’activité et la fonction de BAP1 durant les périodes d’exposition à des agents qui endommagent l’ADN. Les connaissances acquises seront donc d’une valeur tangible pour nôtre compréhension de la mutagenèse induite par des agents carcinogènes, un déterminant clé de la formation des tumeurs.

Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6gag, a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6gag. Consistent with this, viruses with mutations in PR or p6gag were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

The N-end rule: functions, mysteries, uses.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Similar but distinct versions of the N-end rule operate in all organisms examined, from mammals to fungi and bacteria. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system. I discuss the mechanisms and functions of this pathway, and consider its applications.

Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells

Background Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways in vivo. Methodology/Principal Findings Using an in vivo biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination. Conclusions/Significance These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.

Investigation of the function of the LNX family of E3 ubiquitin ligases in the nervous system

The Lnx1 (Ligand of Numb protein X 1) and Lnx2 genes belong to a family of PDZ domain-containing RING finger domain E3 ubiquitin ligases. mRNA expression for both genes have been reported in early murine central nervous system. However, there have been limited reports with regards to the expression patterns for both the proteins in vivo. Hence, we have attempted to characterize the significance of these proteins in the context of morphology and physiology of the central nervous system. Through our studies, we have attempted to examine closely the expression of these two genes in the murine central nervous system. We have also looked at novel interacting ligands for both proteins. Interacting partners have been examined with particular relevance to possible roles of their interactions with LNX1 and LNX2 in the functioning of the nervous system. Moreover, we have performed loss-of-function studies by way of creation and characterization of knockout mice.

Targeting the ubiquitin proteasome system in haematological malignancies

The ubiquitin proteasome system (UPS) plays a central role in cellular protein homeostasis through the targeted destruction of damaged/misfolded proteins and regulatory proteins that control critical cellular functions. The UPS comprises a sequential series of enzymatic activities to covalently attach ubiquitin to proteins to target them for degradation through the proteasome. Aberrancies within this system have been associated with transformation and tumourigenesis and thus, the UPS represents an attractive target for the development of anti-cancer therapies. The use of the first-in-class proteasome inhibitor, bortezomib, in the treatment of Plasma Cell Myeloma and Mantle Cell Lymphoma has validated the UPS as a therapeutic target. Following on its success, efforts are focused on the development of second-generation proteasome inhibitors and small molecule inhibitors of other components of the UPS. This review will provide an overview of the UPS and discuss current and novel therapies targeting the UPS.

Manipulation of the ubiquitin-proteasome system by HIV-1 : role of the accessory protein Vpr

Le virus de l’immunodéficience humaine de type 1 (VIH-1), l’agent étiologique du SIDA, est un rétrovirus complexe arborant plusieurs protéines accessoires : Nef, Vif, Vpr, et Vpu. Celles-ci sont impliquées dans la modulation de la réplication virale, dans l’évasion immunitaire et dans la progression de la pathogenèse du SIDA. Dans ce contexte, il a été démontré que la protéine virale R (Vpr) induit un arrêt de cycle cellulaire en phase G2. Le mécanisme par lequel Vpr exerce cette fonction est l’activation, ATR (Ataxia telangiectasia and Rad3 related)-dépendante, du point de contrôle de dommage à l’ADN, mais les facteurs et mécanismes moléculaires directement impliqués dans cette activité demeurent inconnus. Afin d’identifier de nouveaux facteurs cellulaires interagissant avec Vpr, nous avons utilisé une purification d’affinité en tandem (TAP) pour isoler des complexes protéiques natifs contenant Vpr. Nous avons découvert que Vpr s’associait avec CRL4A(VprBP), un complexe cellulaire d’E3 ubiquitine ligase, comprenant les protéines Cullin 4A, DDB1 (DNA damage-binding protein 1) et VprBP (Vpr-binding protein). Nos études ont mis en évidence que le recrutement de la E3 ligase par Vpr était nécessaire mais non suffisant pour l’induction de l’arrêt de cycle cellulaire en G2, suggérant ainsi que des événements additionnels seraient impliqués dans ce processus. À cet égard, nous apportons des preuves directes que Vpr détourne les fonctions de CRL4A(VprBP) pour induire la polyubiquitination de type K48 et la dégradation protéosomale de protéines cellulaires encore inconnues. Ces événements d’ubiquitination induits par Vpr ont été démontrés comme étant nécessaire à l’activation d’ATR. Finalement, nous montrons que Vpr forme des foyers ancrés à la chromatine co-localisant avec VprBP ainsi qu’avec des facteurs impliqués dans la réparation de l’ADN. La formation de ces foyers représente un événement essentiel et précoce dans l’induction de l’arrêt de cycle cellulaire en G2. Enfin, nous démontrons que Vpr est capable de recruter CRL4A(VprBP) au niveau de la chromatine et nous apportons des preuves indiquant que le substrat inconnu ciblé par Vpr est une protéine associée à la chromatine. Globalement, nos résultats révèlent certains des ménanismes par lesquels Vpr induit des perturbations du cycle cellulaire. En outre, cette étude contribue à notre compréhension de la modulation du système ubiquitine-protéasome par le VIH-1 et son implication fonctionnelle dans la manipulation de l’environnement cellulaire de l’hôte.

A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system

The aim of this study was to evaluate the anti-tumor activity of Amblyomin-X, a serine protease Kunitz-type inhibitor. Amblyomin-X induced tumor mass regression and decreased number of metastatic events in a B16F10 murine melanoma model. Alterations on expression of several genes related to cell cycle were observed when two tumor cell lines were treated with Amblyomin-X. PSMB2, which encodes a proteasome subunit, was differentially expressed, in agreement to inhibition of proteasomal activity in both cell lines. In conclusion, our results indicate that Amblyomin-X selectively acts on tumor cells by inducing apoptotic cell death, possibly by targeting the ubiquitin-proteasome system. (C) 2010 Elsevier Ltd. All rights reserved.