Structure-fonction de MARCH1, une E3 ubiquitine ligase régulant la présentation antigénique par le CMH II

Les molécules classiques du CMH de classe II sont responsables de la présentation de peptides exogènes par les cellules présentatrices d’antigène aux lymphocytes T CD4+. Cette présentation antigénique est essentielle à l’établissement d’une réponse immunitaire adaptative. Cependant, la reconnaissance d’auto-antigènes ainsi que l’élimination des cellules du Soi sont des problèmes à l’origine de nombreuses maladies auto-immunes. Notamment, le diabète et la sclérose en plaque. D’éventuels traitements de ces maladies pourraient impliquer la manipulation de la présentation antigénique chez les cellules dont la reconnaissance et l’élimination engendrent ces maladies. Il est donc primordial d’approfondir nos connaissances en ce qui concerne les mécanismes de régulation de la présentation antigénique. La présentation antigénique est régulée tant au niveau transcriptionnel que post-traductionnel. Au niveau post-traductionnel, diverses cytokines affectent le processus. Parmi celles-ci, l’IL-10, une cytokine anti-inflammatoire, cause une rétention intracellulaire des molécules du CMH II. Son mécanisme d’action consiste en l’ubiquitination de la queue cytoplasmique de la chaîne bêta des molécules de CMH II. Cette modification protéique est effectuée par MARCH1, une E3 ubiquitine ligase dont l’expression est restreinte aux organes lymphoïdes secondaires. Jusqu’à tout récemment, il y avait très peu de connaissance concernant la structure et les cibles de MARCH1. Considérant son impact majeur sur la présentation antigénique, nous nous sommes intéressé à la structure-fonction de cette molécule afin de mieux caractériser sa régulation ainsi que les diverses conditions nécessaires à son fonctionnement. Dans un premier article, nous avons étudié la régulation de l’expression de MARCH1 au niveau protéique. Nos résultats ont révélé l’autorégulation de la molécule par formation de dimères et son autoubiquitination. Nous avons également démontré l’importance des domaines transmembranaires de MARCH1 dans la formation de dimères et l’interaction avec le CMH II. Dans un second article, nous avons investigué l’importance de la localisation de MARCH1 pour sa fonction. Les résultats obtenus montrent la fonctionnalité des motifs de localisation de la portion C-terminale de MARCH1 ainsi que la présence d’autres éléments de localisation dans la portion N-terminale de la protéine. Les nombreux mutants utilisés pour ce projet nous ont permis d’identifier un motif ‘‘VQNC’’, situé dans la portion cytoplasmique C-terminale de MARCH1, dont la valine est requise au fonctionnement optimal de la molécule. En effet, la mutation de la valine engendre une diminution de la fonction de la molécule et des expériences de BRET ont démontré une modification de l’orientation spatiale des queues cytoplasmiques. De plus, une recherche d’homologie de séquence a révélé la présence de ce même motif dans d’autres ubiquitines ligases, dont Parkin. Parkin est fortement exprimée dans le cerveau et agirait, entre autre, sur la dégradation des agrégats protéiques. La dysfonction de Parkin cause l’accumulation de ces agrégats, nommés corps de Lewy, qui entraînent des déficiences au niveau du fonctionnement neural observé chez les patients atteints de la maladie de Parkinson. La valine comprise dans le motif ‘’VQNC’’ a d’ailleurs été identifiée comme étant mutée au sein d’une famille où cette maladie est génétiquement transmise. Nous croyons que l’importance de ce motif ne se restreint pas à MARCH1, mais serait généralisée à d’autres E3 ligases. Ce projet de recherche a permis de caractériser des mécanismes de régulation de MARCH1 ainsi que de découvrir divers éléments structuraux requis à sa fonction. Nos travaux ont permis de mieux comprendre les mécanismes de contrôle de la présentation antigénique par les molécules de CMH II.

Effets cellulaires de l’activation de ligases de l’ubiquitine par la protéine lysosomale LITAF

LITAF (lipopolysaccharide-induced tumor necrosis factor-alpha factor), une protéine lysosomale, possède deux motifs PPXY capables d’interagir avec les domaines WW d’un sous-groupe spécifique de trois ligases de l’ubiquitine. Ces ligases sont impliquées dans l’ubiquitylation ainsi que la dégradation de diverses protéines cellulaires aux lysosomes et aux protéasomes. Les travaux menés dans le cadre de cette étude visaient à démontrer que LITAF active ces ligases et à déterminer les conséquences de cette activation sur les substrats de ces ligases. Pour y parvenir, des expériences d’ubiquitylation in vivo et in vitro ont été menées en présence de LITAF ainsi que des ligases et des substrats appropriés. L’activation des ligases a été mesurée par leur taux d’autoubiquitylation et celle de leurs substrats par leur taux d’ubiquitylation et de dégradation. Les résultats obtenus montrent que l’activité des ligases est augmentée en présence de LITAF et que l’ubiquitylation et la dégradation des substrats de ces ligases sont partiellement augmentées. LITAF semble donc jouer un rôle de régulateur des ligases de l’ubiquitine. L’importance de ces résultats réside dans le fait que l'expression et la localisation intracellulaire de LITAF sont affectées dans plusieurs pathologies. Nos résultats amènent un éclairage nouveau sur le rôle physiologique de cette protéine et pourraient expliquer en partie comment l'altération de l'expression de LITAF affecte l'équilibre cellulaire.

La modulation du métabolisme cellulaire par l'E3 Ubiquitine Ligase MARCH-1

La relocalisation et la dégradation médiée par ubiquitination sont utilisées par la cellule pour contrôler la localisation et l’expression de ses protéines. L’E3 ubiquitine ligase MARCH1 est impliqué dans la régulation post-traductionnelle de CMH-II et de CD86. Dans ce mémoire, on propose un rôle additionnel à MARCH1. Nos résultats expérimentaux nous portent à croire que MARCH1 pourrait moduler le métabolisme cellulaire en favorisant la relocalisation et la dégradation d’enzymes impliquées dans la glycolyse. La grande majorité des cellules utilise la phosphorylation oxydative pour générer de l’ATP en présence d’oxygène. Dans un environnement hypoxique, cette dernière est non fonctionnelle et la cellule doit utiliser la glycolyse anaérobique pour produire son ATP. Une cellule cancéreuse à des besoins énergétiques supérieurs en raison de l’augmentation de sa biomasse et de sa prolifération incontrôlée. Pour subvenir à ces besoins, elle maximise sa production d’énergie en modifiant son métabolisme; c’est l’effet Warburg. On retrouve dans les cellules immunitaires des modifications similaires au métabolisme cellulaire suite à un signal d’activation. Ici, nous montrons que la respiration mitochondriale maximale, la réserve respiratoire et la glycolyse maximale sont diminuées dans les cellules présentatrice d’antigènes qui expriment MARCH1. Nous avons montré que MARCH1 était localisable au niveau de la mitochondrie, ce qui lui permet d’interagir avec les enzymes de la glycolyse. Finalement, nous avons quantifié l’expression de Eno1 et de LDHB par Western Blot, pour montrer une augmentation de l’expression de ces enzymes en absence de MARCH1. À la lumière de ces résultats, nous discutons des avantages que procure la diminution de l’expression de MARCH1 dans un contexte inflammatoire, suite à l’activation des cellules présentatrices d’antigènes. Ce phénomène permettrait une présentation antigénique plus efficace, une augmentation de la production d’énergie et une meilleure résistance aux ROS produits lors de la réponse inflammatoire.

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.

KEAP1 E3 ligase-mediated downregulation of NF-kappaB signaling by targeting IKKbeta.

IkappaB kinase beta (IKKbeta) is involved in tumor development and progression through activation of the nuclear factor (NF)-kappaB pathway. However, the molecular mechanism that regulates IKKbeta degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)-based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKbeta ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKbeta and to upregulation of NF-kappaB-derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKbeta degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKbeta ubiquitination may contribute to tumorigenesis.

Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin–proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3α, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3α-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

Functions of the DNA damage response pathway target Ho endonuclease of yeast for degradation via the ubiquitin-26S proteasome system

Ho endonuclease of Saccharomyces cerevisiae is a homing endonuclease that makes a site-specific double-strand break in the MAT gene in late G1. Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2Rad6 and UBC3Cdc34. UBC2Rad6 is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in rad18 mutants. We show that the Ho degradation pathway involving UBC3Cdc34 goes through the Skp1/Cdc53/F-box (SCF) ubiquitin ligase complex and identify a F-box protein, Yml088w, that is required for Ho degradation. Components of a defined pathway of the DNA damage response, MEC1, RAD9, and CHK1, are also necessary for Ho degradation, whereas functions of the RAD24 epistasis group and the downstream effector RAD53 have no role in degradation of Ho. Our results indicate a link between the endonuclease function of Ho and its destruction.

E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins.

Ubiquitin-dependent proteolysis of the mitotic cyclins A and B is required for the completion of mitosis and entry into the next cell cycle. This process is catalyzed by the cyclosome, an approximately 22S particle that contains a cyclin-selective ubiquitin ligase activity, E3-C, that requires a cyclin-selective ubiquitin carrier protein (UBC) E2-C. Here we report the purification and cloning of E2-C from clam oocytes. The deduced amino acid sequence of E2-C indicates that it is a new UBC family member. Bacterially expressed recombinant E2-C is active in in vitro cyclin ubiquitination assays, where it exhibits the same substrate specificities seen with native E2-C. These results demonstrate that E2-C is not a homolog of UBC4 or UBC9, proteins previously suggested to be involved in cyclin ubiquitination, but is a new UBC family member with unique properties.

Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP).

The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.

Phosphomimetic Mutation of the N-Terminal Lid of MDM2 Enhances the Polyubiquitination of p53 through Stimulation of E2-Ubiquitin Thioester Hydrolysis.

Mouse double minute 2 (MDM2) has a phosphorylation site within a lid motif at Ser17 whose phosphomimetic mutation to Asp17 stimulates MDM2-mediated polyubiquitination of p53. MDM2 lid deletion, but not Asp17 mutation, induced a blue shift in the λmax of intrinsic fluorescence derived from residues in the central domain including Trp235, Trp303, Trp323, and Trp329. This indicates that the Asp17 mutation does not alter the conformation of MDM2 surrounding the tryptophan residues. In addition, Phe235 mutation enhanced MDM2 binding to p53 but did not stimulate its ubiquitination function, thus uncoupling increases in p53 binding from its E3 ubiquitin ligase function. However, the Asp17mutation inMDM2 stimulated its discharge of the UBCH5a-ubiquitin thioester adduct (UBCH5a is a ubiquitin-conjugating enzyme E2D 1 UBC4/5 homolog yeast). This stimulation of ubiquitin discharge fromE2 was independent of the p53 substrate. There are now four known effects of the Asp17 mutation on MDM2: (i) it alters the conformation of the isolated N-terminus as defined by NMR; (ii) it induces increased thermostability of the isolated N-terminal domain; (iii) it stimulates the allosteric interaction ofMDM2 with the DNA-binding domain of p53; and (iv) it stimulates a novel protein–protein interaction with the E2-ubiquitin complex in the absence of substrate p53 that, in turn, increases hydrolysis of theE2-ubiquitin thioester bond. These data also suggest a new strategy to disrupt MDM2 function by targeting the E2-ubiquitin discharge reaction.

Design and synthesis of E3 ligase RNF5 inhibitors as innovative strategy to trigger mutant CFTR rescue in Cystic Fibrosis

In Cystic Fibrosis (CF) the deletion of phenylalanine 508 (F508del) in the CFTR anion channel is associated to misfolding and defective gating of the mutant protein. Among the known proteins involved in CFTR processing, one of the most promising drug target is the ubiquitin ligase RNF5, which normally promotes F508del-CFTR degradation. In this context, a small molecule RNF5 inhibitor is expected to chemically mimic a condition of RNF5 silencing, thus preventing mutant CFTR degradation and causing its stabilization and plasma membrane trafficking. Hence, by exploiting a virtual screening (VS) campaign, the hit compound inh-2 was discovered as the first-in-class inhibitor of RNF5. Evaluation of inh-2 efficacy on CFTR rescue showed that it efficiently decreases ubiquitination of mutant CFTR and increases chloride current in human primary bronchial epithelia. Based on the promising biological results obtained with inh-2, this thesis reports the structure-based design of potential RNF5 inhibitors having improved potency and efficacy. The optimization of general synthetic strategies gave access to a library of analogues of the 1,2,4-thiadiazol-5-ylidene inh-2 for SAR investigation. The new analogues were tested for their corrector activity in CFBE41o- cells by using the microfluorimetric HS-YFP assay as a primary screen. Then, the effect of putative RNF5 inhibitors on proliferation, apoptosis and the formation of autophagic vacuoles was evaluated. Some of the new analogs significantly increased the basal level of autophagy, reproducing RNF5 silencing effect in cell. Among them, one compound also displayed a greater rescue of the F508del-CFTR trafficking defect than inh-2. Our preliminary results suggest that the 1,2,4-thiadiazolylidene could be a suitable scaffold for the discovery of potential RNF5 inhibitors able to rescue mutant CFTRs. Biological tests are still ongoing to acquire in-depth knowledge about the mechanism of action and therapeutic relevance of this unprecedented pharmacological strategy.

FBXO25-associated nuclear domains: A novel subnuclear structure

Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.

Functional characterization of the putative Aspergillus nidulans DNA damage binding protein homologue DdbA

Nucleotide excision repair (NER) eliminates helix-distorting DNA base lesions. Seven XP-deficient genetic complementation groups (XPA to XPG) have already been identified in mammals, and their corresponding genes have been cloned. Hereditary defects in NER are associated with several diseases, including xeroderma pigmentosum (XP). UV-DDB (XPE) is formed by two associated subunits, DDB1 and DDB2. UV-DDB was identified biochemically as a protein factor that exhibits very strong and specific binding to ultraviolet (UV)-treated DNA. As a preliminary step to characterize the components of the NER in the filamentous fungus Aspergillus nidulans, here we identified a putative DDB1 homologue, DdbA. Deletion and expression analysis indicated that A. nidulans ddbA gene is involved in the DNA damage response, more specifically in the UV light response and 4-nitroquinoline oxide (4-NQO) sensitivity. Furthermore, the Delta ddbA strain cannot self-cross and expression analysis showed that ddbA can be induced by oxidative stress and is developmentally regulated in both asexual and sexual processes. The Delta ddbA mutation can genetically interact with uvsB(ATR), atmA(ATM), nkuA(KU70), H2AX-S129A (a replacement of the conserved serine in the C-terminal of H2AX with alanine), and cshB (a mutation in CSB Cockayne`s syndrome protein involved in the transcription-coupled repair subpathway of NER) mutations. Finally, to determine the DdbA cellular localization, we constructed a GFP:DdbA strain. In the presence and absence of DNA damage, DdbA was mostly detected in the nuclei, indicating that DdbA localizes to nuclei and its cellular localization is not affected by the cellular response to DNA damage induced by 4-NQO and UV light.

PHOSPHODIESTERASE-4 INHIBITION REDUCES PROTEOLYSIS AND ATROGENES EXPRESSION IN RAT SKELETAL MUSCLES

Phosphodiesterase (PDE) inhibition reduces skeletal muscle atrophy, but the underlying molecular mechanism remains unclear. We used microdialysis to investigate the effects of different PDE inhibitors on interstitial tyrosine concentration as well as proteolytic activity and atrogenes expression in isolated rat muscle. Rolipram, a PDE-4-selective inhibitor, reduced the interstitial tyrosine concentration and rates of muscle protein degradation. The rolipram-induced muscle cAMP increase was accompanied by a decrease in ubiquitin proteasome system (UPS) activity and atrogin-1 mRNA, a ubiquitin-ligase involved in muscle atrophy. This effect was not associated with Akt phosphorylation but was partially blocked by a protein kinase A inhibitor. Fasting increased atrogin-1, MuRF-1 and LC3b expression, and these effects were markedly suppressed by rolipram. Our data suggest that activation of cAMP signaling by PDE-4 blockade leads to inhibition of UPS activity and atrogenes expression independently of Akt. These findings are important for identifying novel approaches to attenuate muscle atrophy. Muscle Nerve 44: 371-381, 2011

Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms

Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C. Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by > 60%, but also the level of myosin heavy chains (MHCs) by > 40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels. These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.