The covalent modification of proteins with a suite of molecular tags, a process termed post-translational modification, is a powerful means to enhance the proteomic complexity of an organism far beyond that which is directly encoded by its genome. A particularly widespread form of modification involves the conjugation of the protein ubiquitin to specified substrates, which serves to regulate numerous cellular processes. The mechanism of ubiquitin conjugation, known as ubiquitylation, requires E3 ubiquitin ligases that specify and recruit substrate proteins for ubiquitin conjugation. Recent insights into the mechanisms of ubiquitylation demonstrate that E3 ligases can possess active regulatory properties beyond those of a simple assembly scaffold. This thesis describes the dimeric structure of the E3 ligase adaptor protein SPOP in complex with the N-terminal domain of Cul3 at 2.4 Å resolution. Here, it is demonstrated that SPOP forms large oligomers that can form heteromeric species with the closely related paralog SPOPL. In combination, SPOP and SPOPL form a molecular rheostat that can fine-tune E3 ubiquitin ligase activity by affecting the oligomeric state of the E3 complex. These results reveal a mechanism through which adaptor protein self-assembly may provide a graded level of regulation of the SPOP/Cul3 E3 ligase toward its multiple protein substrates.

The eukaryotic endoplasmic reticulum (ER) is a multifunctional organelle, primarily responsible for the folding and maturation of secretory proteins, as well as lipid metabolism, calcium homeostasis, ubiquitin-dependent signalling and cell fate decisions. ER-associated degradation (ERAD) oversees protein folding and delivers misfolded proteins for degradation by the proteasome via ubiquitin conjugation mediated by RING-type E3 ubiquitin ligases. An intact ERAD is crucial to cellular homeostasis, as unresolved protein imbalances cause ER stress that ultimately lead to apoptosis. The human ER accommodates at least 25 E3s, however our understanding is mostly limited to Hrd1 and AMFR/gp78, both of which have a defined function in ERAD. To understand the contribution of ER E3s to cellular and organelle homeostasis, this study used mass spectrometry of purified E3 complexes to identify cofactors and build interaction networks of ER-resident E3s. These findings will form the foundation for investigating the biological roles of these ubiquitin ligases. Transcriptional analysis highlighted the centrality of Hrd1 among all ER-resident E3s in response to protein misfolding in the ER. Additionally, the contribution of individual Hrd1 complex components to resolving proteotoxic stress was assessed using a misfolded antibody subunit (IgM heavy chain), rather than conventional pharmacological treatments. The ERAD components essential for substrate degradation and survival under proteotoxic stress were identified, highlighting the pivotal role of Hrd1, its cofactor SEL1L and the Derlin family members. Finally, it was demonstrated that autophagy induction in response to proteasome inhibition is key to relieve the burden of protein misfolding in the ER, as it sustained the survival of cells defective for ERAD. Importantly, this study proposes a potential involvement of Hrd1 in signalling from the ER to autophagy, suggesting potential crosstalk between the ERAD and autophagic pathways.

The MDM2 protein regulates the tumour suppressor protein p53, acting as its chaperone, regulating its translation and targeting p53 for degradation by the 26s proteasome via its E3 ligase activity. The E3 ligase activity of MDM2 is dependent on its C-terminal RING domain. E3 ligases containing a RING domain are traditionally thought to catalyse the transfer of ubiquitin from their conjugating enzyme (E2) partner to the target protein, in the final step of the ubiquitination cascade. Various E2 enzymes have been shown to interact with their partner E3 ligases, yet evidence for the interaction between MDM2 and its partner E2, UbcH5α has not yet been shown. It has been reported that the reason for this lack of evidence is that the interaction between the two is highly unstable. Here I show that MDM2 forms a stable isolatable interaction with UbcH5α, the C-terminal tail of MDM2 is not necessary for this interaction. Although RING E3 ligases were not previously thought to interact with ubiquitin, preliminary evidence is emerging that suggests that this interaction is possible indeed I show that MDM2 and ubiquitin form a stable complex. I demonstrate that UbcH5α and ubiquitin both interact with the RING of MDM2, specifically the 20 most C-terminal amino acids of MDM2. My results show that both these proteins can bind this region of the RING simultaneously. I also highlight specific residues including tyrosine 489 and arginine 479 important for UbcH5α and ubiquitin binding respectively and the negative affect that these mutations have on the E3 ligase activity of MDM2 towards p53. Furthermore I show by limited proteolysis and hydrogen deuterium exchange that UbcH5α can be allosterically activated by MDM2. A novel peptide phage display technique linked to next generation sequencing was developed to further confirm an allosteric change and demonstrates that UbcH5α has different binding specificity for peptides when in a free or ligand bound conformation. MDM2 is a popular target for cancer therapeutics due to its dysregulation throughout many cancer types, including 30% of soft tissue sarcomas. Dissecting the mechanism of MDM2 function is an important step in identifying specific drugable interfaces on MDM2 and its interacting partners so that effective therapeutics can be designed.

No description available.

A cotton fiber cDNA, GhRING1, and its corresponding gene have been cloned and characterized. The GhRING1 gene encodes a RING-type ubiquitin ligase (E3) containing 337 amino acids (aa). The GhRING1 protein contains a RING finger motif with conserved cysteine and histine residues at the C-terminus and is classified as a C3H2C3-type RING protein. Blast searches show that GhRING1 has the highest homology to At3g19950 from Arabidopsis. Real time RT-PCR analysis indicates that the GhRING1 gene is highly expressed in cotton fiber in a developmental manner. The transcript level of the GhRING1 gene reaches a maximum in elongating fibers at 15 DPA. In vitro auto-ubiquitination assays using wheat germ extract and a reconstitution system demonstrate that GhRING1 has the ubiquitin E3 ligase activity. A fiber specific lipid transfer protein 4 (FSltp4) is identified as the target substrate of GhRING1 by using a bacterial two-hybrid system. The binding of GhRING1 and FSltp4 is confirmed by using an in vitro pull down assay and a yeast two-hybrid system. The histochemical GUS assay was performed to analyze tissue specificity of the GhRING1 and At3g19950 promoters in transgenic Arabidopsis plants. The GUS assay shows that the promoter of At3g19950 is highly activated in leaves, roots, trichomes and also in anthers and stigma of flowers. In contrast, the GUS expression directed by the promoter of GhRING1 is only located at stipules and anthers and stigma of flowers. The GhRING1 is the first ubiquitin E3 gene isolated and studied from cotton. Based on the expression pattern of GhRING1, FSltp4, and GhUBC E2s and the identification of a fiber-specific target protein, FSltp4, we propose that protein ubiquitination occurs in fiber and the ubiquitin-proteasome pathway regulates fiber development.

La Neuropathie à Axones Géants (NAG) est une maladie neurodégénérative rare et fatale caractérisée par une détérioration du système nerveux central et périphérique, impliquant les fonctions motrices et sensorielles. La détérioration massive du système nerveux est accompagnée d'une désorganisation générale des Filaments Intermédiaires ce qui la différencie de nombreuses maladies neurodégénératives où seuls les neurofilaments(NFs) sont affectés. La protéine déficiente, la gigaxonine, est la sous-unité d'une ubiquitine ligase E3, responsable de la reconnaissance spécifique des substrats MAP1B, MAP1S et TBCB, seuls connus à ce jour.Dans le but d'étudier le rôle de la gigaxonine sur la survie neuronale, la désorganisation du cytosquelette et d'avoir un modèle animal suffisamment fort pour envisager des tests thérapeutiques, j'ai caractérisé un modèle murin de NAG. Pour ce faire, j'ai réalisé une étude comportementale des fonctions motrices et sensorielles ainsi qu'une étude histopathologique. Les souris NAG (129/SvJ) développent un phénotype moteur modéré dès 60 semaines alors que les souris NAG (C57BL/6) présentent un phénotype sensoriel dès 60 semaines. Les données histopathologiques ne présentent pas de mort neuronale mais les NFs sont sévèrement altérés. Les NFs sont plus abondant, leur diamètre est augmenté et leur orientation hétérogène, comme c'est observé chez les patients NAG.Nos résultats montrent que l'absence de gigaxonine induit un phénotype moteur et sensoriel modéré mais par contre reproduit la désorganisation massive des NFs observée chez les patients. Ce modèle va nous permettred'étudier le rôle de la gigaxonine, une ligase E3, sur l'organisation des NFs et ainsi comprendre les processus pathologiques impliqués dans d'autres maladies neurodégénératives caractérisée par une accumulation des NFs et un dysfonctionnement du système ubiquitine-protéasome comme les maladies d'Azheimer, de Parkinson etd'huntington ou la sclérose latérale amyotrophique. / Giant Axonal Neuropathy (GAN) is a rare and fatale neurodegenerative disorder characterized by a deterioration of the peripheral and central nervous system. The broad deterioration of the nervous system is accompanied with a general disorganization of the Intermediate Filaments which makes it different from other neurodegenerative disorders wherein only neurofilaments (NFs) are affected. The defective protein, gigaxonin, is the substrate adaptator of an E3 ubiquitin ligase, in charge of the specific recognition of MAP1B, MAP1S and TBCB. In order to study the role of gigaxonin on neuronal survival, the cytoskeleton disorganization and to have a relevant GAN animal model to evaluate efficacy of GAN treatments, I have characterized a GAN mouse model. I did a motor and sensory behavioural study and an histopathologic study. The GAN mice (129/SvJ) shown mild motordeficits starting at 60 weeks of age while sensory deficits were evidenced in C57BL/6 GAN mice. No apparent neurodegeneration was evidenced in GAN mice, but dysregulation of NFs was massive. NFs were more abundant, they shown the abnormal increased diameter and misorientation that are characteristics of the human pathology. Our results show that gigaxonin depletion induces mild motor and sensory deficits but recapitulates the severe NFs dysregulation seen in patients. Our model will allow us to study the role of the gigaxonin-E3 ligase in organizing NFs and understand the pathological processes engaged in other neurodegenerative disorders characterized by accumulation of NFs and dysfunction of the Ubiquitin Proteasome System, such as Amyotrophic Lateral Sclerosis, Huntington's, Alzheimer's and Parkinson's diseases.

DNA repair pathways have evolved to protect the genome from damage caused by intrinsic and extrinsic factors. Although numerous DNA repair mechanisms have been studied and reported, information regarding how they coordinate with the necessary changes in chromatin structure is scarce. Smc (structural maintenance of chromosomes) proteins are a conserved, essential family of proteins required for chromosome organization and accurate segregation. The budding yeast, Saccharomyces cerevisiae has three Smc-protein complexes: Smc1/3 complex (cohesin), Smc2/4 complex (condensin) and the Smc5/6 complex, required for sister chromatid cohesion, condensation and DNA repair, respectively. The chromatin associated Smc5/6 complex consists of Smc5, Smc6 and six non-smc elements (Nse1-Nse6). Smc5 and Smc6 are required for stability of repetitive chromosomal regions and sister chromatid recombination-mediated repair of double-strand breaks. Mms21/Nse2, a subunit of the Smc5/6 complex, is a SUMO E3-ligase, which conjugates SUMO (small ubiquitin-like modifier) to Smc5 and Yku70 (DNA repair protein) and its SUMO ligase activity protects the cells from extrinsic DNA damage. To address the role of Nse2 SUMO ligase in cellular events, we isolated mutants (nse2∆sl and nse2C221A) defective in the E3-ligase domain of Nse2 and found that these mutants are sensitive to genotoxic agents, for example MMS, UV or bleomycin, as expected. We found that cysteine 221 present in the SP-RING domain of Nse2 is required in the function of Nse2 in resisting genotoxic stress. We found that nse2∆sl cultures are slow growing and show increased abundance of cells having 2N DNA content (indicative of a G2-M cell cycle delay or arrest) relative to wild type cells. The DNA damage checkpoint pathway is activated to a limited extent in unchallenged nse2∆sl mutant cells indicating that cells lacking the SUMO ligase activity of Nse2 incur spontaneous DNA damage. Furthermore nse2∆sl cells are exquisitely sensitive to caffeine, an agent known to override the DNA damage checkpoint in a number of organisms by inhibiting the DNA damage checkpoint transducer ATR (Homo sapiens), Mec1 (Saccharomyces cerevisiae) and Rad3 (Schizosaccharomyces pombe). In order to investigate the importance of the DNA damage checkpoint pathway for nse2∆sl cells, we employed a genetic approach. We found that nse2∆sl exhibits synthetic sick interaction with mec1∆ but not tel1∆ (defective in Mec1 or Tel1 PI kinases) or mrc1∆ (defective in Mrc1 or mediator of replication checkpoint 1) indicating that the DNA damage induced Mec1 dependent checkpoint pathway is selectively required but the replication stress checkpoint pathway is dispensable for optimal growth of unchallenged nse2∆sl cells. In order to further investigate the role of Nse2 in S phase events, we used camptothecin (CPT), a drug that induces S phase specific double strand breaks. CPT inhibits topoisomerase I by trapping the covalent Top1-DNA intermediate. Collision of a DNA replication fork with such a complex results in double-strand and single-strand breaks in DNA. We found that nse2∆sl is CPT-sensitive and that nse2∆sl top1-8 has a synthetic sick phenotype. Thus, our chemical and genetic interaction studies suggest that the SUMO ligase activity of Nse2 may be required when Top1 function is compromised. Interestingly, human and yeast Top1 proteins are known to be sumoylated. Our findings suggest that MMS-induced enhancement of Top1 sumoylation in budding yeast is partially dependent on SUMO ligase activity of Nse2. Since both sumoylation and Top1 play a role in telomere maintenance, we also examined the telomere length in single as well as double mutants and found that there is slight telomere lengthening in nse2∆sl top1-8 double mutant. To gain further insight into the genetic interaction between Nse2 and other proteins which affect DNA topology, we also investigated genetic interaction of Nse2 with other topoisomerases. We found that top3-2 nse2∆sl exhibited a synthetic sick phenotype but nse2∆sl top2-4 showed partial rescue of temperature sensitivity. In order to investigate whether chromosome integrity is compromised in nse2∆sl cells we employed a YAC (yeast artificial chromosome) based assay to examine GCRs (gross chromosomal rearrangements). We found elevated levels of GCR in nse2∆sl cells compared to wild type cells. Furthermore, deletion of DNA Topoisomerase1 in nse2∆sl background selectively destabilizes a longer YAC relative to shorter YACs. We also examined the effect of varying origin number on YAC stability in nse2∆sl as well as top1∆ and nse2∆sl top1∆ cells. We found that a YAC having fewer origins is not destabilized in nse2∆sl and top1∆ single mutants but is destabilized in the nse2∆sl top1∆ double mutant. Since Nse2 is a non-SMC member of the Smc5/6 complex, we also investigated the effect of varying origin number on YAC stability in smc6-56 and smc656 top1∆ mutants. We found that the stability of a YAC is modestly compromised in the smc6-56 mutant but its derivative having fewer origins is not further destabilized, rather it seems to be stabilized. In order to gain molecular insights into the involvement of the SUMO ligase activity of Nse2 in maintenance of chromosome integrity, we examined sumoylation of specific substrates following a candidate approach. Smc5 and Yku70 are known targets of Nse2dependent sumoylation. We found that Smc6 is also sumoylated and that the MMS-induced enhancement of Smc6 sumoylation in budding yeast is partially dependent on Nse2. To understand the functional significance of Smc5 sumoylation, we mutated lysine residues of all the four predicted sumoylation sites ψKXE/D, individually as well as all four together. We found that all the single as well as quadruple mutants were weakly sensitive to MMS suggesting that these putative sumoylation sites of Smc5 may contribute towards countering MMS-induced DNA damage. Interestingly, we found that Smc5 sumoylation is enhanced when treated with MMS (methyl methane sulfonate) but not significantly with HU (hydroxyurea) and CPT (camptothecin). We also generated putative ATP-binding defective mutants in Smc5. Previous studies suggest that the ATPase motif is required for the essential function of some Smc proteins (for example, Smc1 and Smc6). We found that smc5K75E and smc5K75Q, having a mutation in the lysine residue of the conserved GXGKS motif present in the Walker A type box at the Nterminus exhibited a null phenotype implying that this conserved lysine residue is required for essential function of Smc5. In this study, employing genetic and biochemical methods, we have characterized the Nse2 SUMO ligase defective mutant and analyzed its role in the unperturbed mitotic cell cycle and in genome maintenance. We have also employed genetic methods to study the involvement of both Nse2 and DNA Topoisomerase I in maintaining genomic stability. Lastly, we have addressed the functional significance of Lysine residues of putative sumoylation sites and the conserved ATP-binding motif of Smc5 by mutational analysis. In conclusion, our study highlights an important role for the SUMO ligase activity of Nse2 in maintaining genomic stability and suggests that sumoylation of Smc5 may be important for resisting MMS-induced genotoxic stress.

L’Hypertension Hyperkaliémique Familiale (FHHt) est une forme rare d’hypertension artérielle associée à une hyperkaliémie et une acidose métabolique hyperchlorémique. Ces troubles sont corrigés par les diurétiques thiazidiques qui inhibent le co-transporteur Na+-Cl- NCC exprimé dans le néphron distal. Cette sensibilité aux diurétiques thiazidiques a laissé́ supposer que la FHHt est causée par une activation de NCC. En 2001, des mutations, de type gain-de-fonction, responsables de la FHHt ont été découvertes dans les gènes codant les sérine-thréonine kinases WNK1 et WNK4 [With No (K) lysine]. Des études in vitro ont montré que WNK1 et WNK4 stimulent NCC de façon indirecte, via la phosphorylation et l'activation de la kinase SPAK (Ste20 like Proline-Alanine rich Kinase). In vivo, l’activation de SPAK joue un rôle central dans le développement de la FHHt due aux mutations de WNK4. L'implication de SPAK n'a pas été définie dans le cas des mutations de WNK1. Nous avons donc croisé les souris WNK1+/FHHt, porteuses d'une mutation FHHt de WNK1, avec les souris SPAK243A/243A, porteuses d’une mutation abolissant l’activation de SPAK par les WNK. L’ensemble des phénotypes observés chez les souris WNK1+/FHHt est corrigé chez les souris WNK1+/FHHt:SPAK243A/243A démontrant ainsi le rôle central de SPAK dans la FHHt causée par les mutations WNK1. En 2012, de nouvelles mutations ont été identifiées dans les gènes codant CUL3 et KLHL3, deux composants d’un complexe ubiquitine ligase. Comme les mutations de WNK1 et WNK4, ces mutations entraînent une augmentation de l’expression de WNK1 et de WNK4. De façon inattendue, les patients FHHt porteurs d'une mutation CUL3 présentent un phénotype plus sévère que les autres. Des études suggèrent que ces mutations perturbent la fonction non seulement du néphron distal mais également des artères. Afin de vérifier cette hypothèse, nous avons généré et comparé deux modèles de souris : les souris pgk-Cul3Δ9, exprimant la mutation Cul3 de façon ubiquitaire, comme les patients, et les souris sm22-Cul3Δ9, exprimant la mutation uniquement dans les cellules musculaires vasculaires lisses. Les deux modèles sont hypertendus, mais les souris pgk-Cul3Δ9 le sont significativement plus que les souris sm22-Cul3Δ9, ce qui prouve que l’hypertension causée par les mutations Cul3 résulte du cumul d’une atteinte rénale et vasculaire. Récemment, de nouvelles mutations faux-sens d'un domaine dit acide de WNK1 ont été identifiées dans un petit nombre de patients atteints de FHHt. Ce domaine est nécessaire à la liaison à KLHL3 et donc à l’ubiquitination des WNK. Notre étude montre que, in vitro, ces mutations entraînent une accumulation d'une seule isoforme de WNK1. Chez la souris, la mutation du domaine acide provoque le même phénotype que les patients, ainsi qu’une augmentation de la phosphorylation de SPAK et du co-transporteur NCC. Cette étude a donc permis de démontrer le rôle essentiel de ce domaine dans la régulation de l'abondance de WNK1 dans le néphron distal in vivo. En conclusion, mon travail a permis une meilleure compréhension du rôle joué par SPAK, WNK1 et CUL3 dans le développement de la FHHt et, plus largement, de la physiopathologie de la FHHt. / Familial Hyperkalemic Hypertension (FHHt) is a rare form of hypertension associated with hyperkalemia and hyperchloremic metabolic acidosis. These disorders are all corrected by thiazide diuretics that inhibit the Na+-Cl- NCC cotransporter expressed in the distal nephron. The sensitivity of FHHt patients to thiazide diuretics strongly suggested that FHHt is caused by NCC activation. In 2001, gain-of function-mutations were identified in the genes encoding the serine-threonine kinases WNK1 and WNK4 [With No (K) lysine] in a subst of FHHt patients. In vitro studies demonstrate that WNK1 and WNK4 indirectly stimulate NCC, through the phosphorylation and activation of SPAK (Ste20 like Proline-Alanine rich Kinase). In vivo, SPAK activation plays a central role in the pathogenesis of FHHt caused by WNK4 mutations. However, the implication of SPAK has never been shown for the WNK1 mutations. Thus, we crossed WNK1+/FHHt mice, bearing the WNK1-FHHt mutation, with SPAK243A/243A mice bearing a mutation abolishing SPAK activation by the WNKs. All FHHt phenotypes observed in WNK1+/FHHt were corrected in WNK1+/FHHt:SPAK243A/243A mice demonstrating the central role of SPAK in FHHt caused by WNK1 mutations. In 2012, new mutations have been identified in CUL3 and KLHL3 genes. The products of these genes are both part of a ubiquitin ligase complex. As WNK1 and WNK4 mutations, these mutations lead to an increased expression of L-WNK1 and/or WNK4. Surprisingly, patients with CUL3 mutations display a more severe phenotype. Previous studies have suggested that CUL3 could be involved not only in the regulation of ion transport in the distal nephron but also in the regulation of vascular tone. To verify this hypothesis, we generated and compared two mouse models: pgk-Cul3Δ9 mouse model bearing, as patients, an ubiquitous Cul3 mutation, and sm22-Cul3Δ9 mouse model that express the Cul3 mutation only in vascular smooth muscle cells. Both models are hypertensive, but pgk-Cul3Δ9 mice display a more severe hypertension than sm22-Cul3Δ9 mice. It demonstrates that the hypertension caused by Cul3 mutations results from both renal and vascular disorders. Recently, new missense mutations have been identified in WNK1 acidic motif in a small number of FHHt patients. This acidic motif is necessary for the liaison to KLHL3 and therefore for WNK ubiquitination. Our study shows that, in vitro, these mutations lead to the accumulation of only one isoform of WNK1. In mice, the mutation of WNK1 acidic motif leads to an increased phosphorylation of SPAK and NCC. Therefore, it demonstrates the essential role of the acidic motif in the regulation of WNK1 abundance in vivo in the distal nephron. This work contributes to a better comprehension of the role played by SPAK, WNK1 and CUL3 in FHHt and more generally in the regulation of blood pressure and Na+/K+ homeostasis.

E3-Ubiquitin-Ligasen übertragen Ubiquitin auf die von ihnen gebundenen Substratproteine. Durch diese Ubiquitinierung werden Proteine für den kontrollierten Abbau im Ubiquitin-Proteasom-System markiert. Dieser Prozess beeinflusst aber auch die Aktivität verschiedener Signalwege, die Lokalisation von Proteinen oder die strukturelle Integrität zellulärer Komponenten. MuRF1, MuRF2 und MuRF3 sind E3-Ubiquitin-Ligasen, die hauptsächlich in quergestreifter Muskulatur exprimiert werden. Von MuRF1 ist bereits bekannt, dass es u. a. über die Ubiquitinierung von Myosinen und deren anschließender Degradation an der Entwicklung der Herz- und Skelettmuskelatrophie beteiligt ist. Da das Wissen über MuRF2 und MuRF3 in diesem Zusammenhang noch begrenzt ist, sollte die Auswirkung der kombinierten Keimbahndeletion von MuRF2 und MuRF3 in einem Mausmodell untersucht werden. Der Doppelknockout (DKO) von MuRF2 und MuRF3 führte zu Veränderungen der Morphologie und der Funktionsfähigkeit der Skelett- und Herzmuskulatur. In Skelettmuskelfasern kam es zur Ablagerung myosinhaltiger Proteinaggregate, zu einer Zunahme an langsam kontrahierenden Muskelfasern sowie zum Auftreten von Myozyten mit zentral gelegenen Nuclei als Anzeichen von Regenerationsprozessen. Isolierte Skelettmuskeln von DKO-Mäusen entwickelten eine geringere maximale spezifische Kraft als Muskeln aus Kontrolltieren. Ihre Herzen waren morphologisch unauffällig. Dennoch waren die Kontraktion des linken Ventrikels und das Schlagvolumen reduziert. Darüber hinaus zeigten isolierte Kardiomyozyten Beeinträchtigungen der Kontraktionsfähigkeit und der Kalziumströme in vitro. Eine massenspektrometrische Untersuchung ergab, dass in den Muskeln der MuRF2/3-DKO-Mäuse im Vergleich zu den Kontrollmäusen 12 Proteine in erhöhter Menge vorhanden waren. Eine Anreicherung von MAPKAP-K3, einem dieser Proteine, und von MAPKAP-K2 konnte im Western Blot von Proteinlysaten aus Skelettmuskeln und dem Herz der MuRF2/3-DKO-Mäuse detektiert werden. / E3 ubiquitin ligases attach the small modifier ubiquitin to their substrate proteins. This ubiquitin-tag not only marks proteins for the proteasome dependent degradation, but also influences the activity of signalling pathways, the localisation of proteins or the structural integrity of cellular components. MuRF1, MuRF2, and MuRF3 are E3 ubiquitin ligases predominantly expressed in striated muscles. MuRF1 is involved in cardiac and skeletal muscle atrophy by mediating proteasome-dependent degradation of myosins. The knowledge about MuRF2 and MuRF3 in this context is limited. Therefore, a mouse model was used to analyse the impact of the combined deletion of MuRF2 and MuRF3. The double knockout (DKO) of MuRF2 and MuRF3 influenced the structure and function of skeletal and cardiac muscle. Skeletal muscle fibres exhibited myosin-containing protein aggregates, a fibre-type shift towards slow fibres, and myoycytes with central nuclei which is an indication of regeneration. Maximal force development was reduced in isolated hindlimb muscles M. soleus and M. extensor digitorum longus of MuRF2/3-DKO mice. Hearts were morphologically normal. No protein aggregates or signs of fibrosis were detected. However, heart performance was impaired. The contractibility of the left ventricle and the ejection fraction were reduced. Isolated cardiomyocytes showed a diminished contractibility. Furthermore, their speed of contraction and relaxation was reduced and they had impaired calcium transients. Mass spectrometric analysis of muscle lysates identified 12 enriched proteins in MuRF2/3-DKO muscles. Western Blot analysis confirmed that MAPKAP-K3, one of these proteins, and MAPKAP-K2 were enriched in lysates of skeletal muscles and left ventricles of MuRF2/3-DKO mice. Further investigations will show how MAPKAP-K2- and MAPKAP-K3-signalling pathways are involved in the development of the MuRF2/3-DKO-phenotype.

Adenovirus humanos são responsáveis por infecção respiratória aguda (IRA) em crianças e adultos, sendo os das espécies B e C mais frequêntes. Com o objetivo de estudar a variabilidade genética de HAdVs, 3087 amostras de aspirado de nasofaringe foram colhidas de crianças, em São Paulo, de 1995 à 2006. A PCR direcionada ao gene VA-RNA detectou 677 adenovírus (22%). O sequenciamento dos genes hexon, fibra e região E3 foram utilizados para determinar os sorotipos e estudar sua variabilidade genética. Dos 677 adenovírus, 69% são da espécie B, 23% da C e 0,7% da E. Variabilidades genéticas foram observadas em todas as regiões estudadas, por meio de mutações, evidenciadas por substituições, recombinações e deleções. Os genes que apresentaram maior variabilidade foram VA-RNA e E3 ORF7.7. Genomas virais de DNA, como dos adenovírus, podem se manter estáveis em condições diversas, contudo, a pressão sofrida por esses genomas, através da resposta imunológica do hospedeiro, fazem com que seu mecanismos evolutivos entrem em operação e variabilidades genéticas sejam observadas. / Human adenoviruses (HAdV) cause acute respiratory disease (ARD) in children and adults, being the adenovirus from species B and C the most frequently detected. With the aim of study the genetic variability of HAdV, 3087 nasopharyngeal aspirate were collected from children in São Paulo, from 1995 to 2006. PCR assay directed to adenovirus VA-RNA gene detected 677 HAdV (22%). Sequencing of the hexon and fiber genes and the E3 region were done to determine the serotypes and study genetic variability. Among the 677 adenoviruses detected 69 % were classified as species B, 23% as species C and 0,7 % as species E. Genetic variability was observed in all studied region, specially at the 7.7Orf of the E3 region and the VA-RNA gene. Genetic modifications were observed as recombination, substitutions and deletions. It is known that viral DNA genomes, as adenovirus, remain genetically stable under a variety of conditions, however, the pressure of the host on these viruses make that their evolutionary mechanisms come to operation and genetic variability are observed.