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El interés en el estudio del proceso de envejecimiento cerebral y los cambios comportamentales relacionados con la edad en caninos y felinos geriátricos ha aumentado en la última década (Mentzel, 2005a; Ingram & Williams, 2010). Las alteraciones del comportamiento canino que responden a cambios fisiopatológicos relacionados con la edad y que involucran distintas esferas conductuales y cognitivas, se engloban bajo la denominación de Síndrome de Disfunción Cognoscitiva (SDC) (Mentzel, 2005b). El cual fue descrito en 1997 y se observó en perros de más de 9 años (Ingram & Williams). El Síndrome de Disfunción Cognoscitivo (SDC) es también llamado Alzheimer canino por las similitudes histopatológicas cerebrales y conductuales relacionadas. En el presente estudio, los caninos geriátricos presentaron los mismos depósitos proteicos anormales presentes en la enfermedad de Alzheimer, como las placas seniles, los cuerpos ubiquitinados y la angiopatía amiloide, pero no presentaron los ovillos neurofibrilares, los cuales son los responsables de la demencia que se padece en la enfermedad. Las únicas herramientas de evaluación prácticas en clínica para esta patología son las encuestas y formularios relacionados con aspectos comportamentales. Además existen exámenes diagnósticos postmortem que permiten identificar de una manera eficaz la presencia de elementos anormales involucrados en la neuropatología, uno de ellos es la inmunohistoquímica. En este estudio se utilizaron los anticuerpos monoclonales de uso humano anti-b-amiloide, anti-proteína tau y anti-ubiquitina en muestras de cerebros de perros mayores de 10 años. El modelo canino hoy en día está constituyendo un significado indispensable para el estudio de los procesos de neurodegeneración porque ha permitido un acercamiento a la teoría del problema desde nuevas perspectivas (Dimakopoulos & Mayer, 2002).

Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code is transcribed to RNA and translated to proteins, but how proteins are degraded has remained a neglected research area. With the discovery of the lysosome by Christian de Duve it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis is largely non-lysosomal, but the mechanisms involved remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs.Entre los años 1950 y 1980 los científicos focalizaron sus estudios sobre la forma en que el código genético es transcripto al ARN y traducido a las proteínas, dejando de lado la forma en que éstas se degradan. Con el descubrimiento de los lisosomas por Christian de Duve se asumió que las proteínas se degradaban en el interior de esa organela. Sin embargo, varias líneas de trabajo independientes sugerían fuertemente que la proteólisis intracelular era en su mayor parte no lisosómica, aunque se desconocían sus mecanismos. El descubrimiento del sistema ubiquitina-proteosoma resolvió el enigma. Ahora sabemos que la degradación intracelular de proteínas participa en la regulación de un amplio espectro de procesos celulares como la división y el ciclo celular, la regulación de los factores de transcripción y el control de la calidad celular. No es sorpresa entonces que las aberraciones del sistema estén relacionadas con la patogénesis de enfermedades humanas como tumores y desórdenes neurodegenerativos, lo que llevó luego a un esfuerzo para desarrollar drogas basadas en este mecanismo.

Brain-derived neurotrophic factor (BDNF) regulates synaptic strengthening and memory consolidation, and altered BDNF expression is implicated in a number of neuropsychiatric and neurodegenerative disorders. BDNF potentiates N-methyl-D-aspartate receptor function through activation of Fyn and ERK1/2. STriatal-Enriched protein tyrosine Phosphatase (STEP) is also implicated in many of the same disorders as BDNF but, in contrast to BDNF, STEP opposes the development of synaptic strengthening. STEP-mediated dephosphorylation of the NMDA receptor subunit GluN2B promotes internalization of GluN2B-containing NMDA receptors, while dephosphorylation of the kinases Fyn, Pyk2, and ERK1/2 leads to their inactivation. Thus, STEP and BDNF have opposing functions. In this study, we demonstrate that manipulation of BDNF expression has a reciprocal effect on STEP61 levels. Reduced BDNF signaling leads to elevation of STEP61 both in BDNF(+/-) mice and after acute BDNF knockdown in cortical cultures. Moreover, a newly identified STEP inhibitor reverses the biochemical and motor abnormalities in BDNF(+/-) mice. In contrast, increased BDNF signaling upon treatment with a tropomyosin receptor kinase B agonist results in degradation of STEP61 and a subsequent increase in the tyrosine phosphorylation of STEP substrates in cultured neurons and in mouse frontal cortex. These findings indicate that BDNF-tropomyosin receptor kinase B signaling leads to degradation of STEP61 , while decreased BDNF expression results in increased STEP61 activity. A better understanding of the opposing interaction between STEP and BDNF in normal cognitive functions and in neuropsychiatric disorders will hopefully lead to better therapeutic strategies. Altered expression of BDNF and STEP61 has been implicated in several neurological disorders. BDNF and STEP61 are known to regulate synaptic strengthening, but in opposite directions. Here, we report that reduced BDNF signaling leads to elevation of STEP61 both in BDNF(+/-) mice and after acute BDNF knockdown in cortical cultures. In contrast, activation of TrkB receptor results in the degradation of STEP61 and reverses hyperlocomotor activity in BDNF(+/-) mice. Moreover, inhibition of STEP61 by TC-2153 is sufficient to enhance the Tyr phosphorylation of STEP substrates and also reverses hyperlocomotion in BDNF(+/-) mice. These findings give us a better understanding of the regulation of STEP61 by BDNF in normal cognitive functions and in neuropsychiatric disorders.

Dissertação de mestrado em Investigação Biomédica, apresentada à Faculdade de Medicina da Universidade de Coimbra Connexin43 (Cx43) is a membrane protein present in gap junctions (GJ) of several tissues, including the heart, where they permit the metabolic and electrical coupling vital for heart contraction. Previous studies carried out in our lab showed that degradation of Cx43 by macroautophagy requires the autophagy adaptor p62, that links the ubiquitinated protein to the nascent autophagosome, through the microtubule-associated protein-1 light chain 3 (LC3). Proteins that interact with LC3, like p62, contain a short hydrophobic LC3-interacting region (LIR). We identified an amino acid sequence in the N-terminus of Cx43 that is consistent with the accepted form for a LIR motif. The main goal of this study was to identify the LIR domain in Cx43 and establish the relevance of the motif for the degradation of Cx43. We hypothesized that the presence of this motif could mediate an alternative form of Cx43 degradation by autophagy that is independent of ubiquitin and p62. Using Cx43 mutanted in the putative LIR domain and through immunoprecipitation assays we evaluated the involvement of the LIR in mediating the direct interaction between Cx43 and LC3 and the influence of p62 in this interaction. Moreover, to investigate the stability of the Cx43 LIR mutants, we performed cycloheximide-chase assays to determine their half-life in several conditions. To evaluate the effect of the mutations in the subcellular distribution of the protein we used immunofluorescence confocal microscopy. Our results showed that Cx43 interacts with LC3 and that mutations in the LIR motif of Cx43 abrogate this interaction. In addition, we demonstrated that these mutants are more stable than wild type Cx43, suggesting that the LIR motif is required for Cx43 degradation. Altogether, the results obtained in this study establish a novel non-canonical pathway of degradation of Cx43 that implies the direct binding of Cx43 to LC3, without the need of ubiquitin and p62. A conexina 43 (Cx43) é uma proteína transmembranar presente nas gap junctions (GJ) de diversos tecidos, incluindo o coração onde permite o acoplamento metabólico e elétrico vital para o normal funcionamento do coração. Estudos prévios levados a cabo no nosso laboratório mostraram que a degradação da Cx43 por macroautofagia requer a proteína adaptadora p62, que liga proteínas ubiquitinadas ao autofagossoma, através da microtubuleassociated protein-1 light chain 3 (LC3). Proteinas que interagem com a LC3, como o p62, contém uma região hidrofóbica que interage com a LC3 (LIR). A Cx43 contém no seu Nterminal uma sequência de aminoácidos consistente com o estabelecido para um domínio LIR. O principal objectivo deste estudo é a caracterização do domínio LIR na Cx43 e estabelecer a relevância deste na degradação da Cx43 por macroautofagia. A presença deste domínio poderia então mediar a degradação da Cx43 por macroautofagia, de uma forma independente de ubiquitinação e do recrutamento do p62. Para tal, usamos construções de Cx43 mutadas neste possível domínio LIR e através de ensaios de imunoprecipitação avaliamos o envolvimento do LIR na interacção entre a Cx43 e a LC3 e a influência do p62 nesta interacção. Para comparar a estabilidade dos mutantes da Cx43 no dominio LIR, levamos a cabo ensaios de cycloheximide chase para determinar o tempo de meia vida da proteína, em diferentes condições. A avaliação da distribuição subcelular da proteina foi feita através de imunofluorescência. Os nossos resultados demostram que a Cx43 interage com a LC3 e que as muatações no domínio LIR levam a uma diminuição desta interacção. Além disso, estes mutantes mostram ser mais estáveis que a Cx43 não mutante, sugerindo que o motivo LIR é importante na degradação da Cx43. Em suma, os resultados obtidos neste estudo sugerem uma nova via não-canónica para a degradação da Cx43, que implica a ligação direta entre Cx43 e LC3, sem a necessidade de ubiquitinação prévia e do recrutamento do adaptador p62.

Made available in DSpace on 2016-09-27T13:39:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-09-14. Added 1 bitstream(s) on 2016-09-27T13:45:05Z : No. of bitstreams: 1 000869173.pdf: 2452278 bytes, checksum: 85fd789b99aa751120a5330cb5ab3a02 (MD5) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) O pacu (Piaractus mesopotamicus) apresenta excelentes características zootécnicas como rápido crescimento, parâmetro que está diretamente relacionado com o aumento da musculatura estriada esquelética. Pesquisas envolvendo a caracterização de genes musculares têm sido realizadas utilizando-se tecnologias moleculares, em espécies de peixes modelo como o Zebrafish que possui o genoma sequenciado. No pacu, espécie que não possui o genoma sequenciado, esta falta de informação genética dificulta a realização de pesquisas relacionadas com a identificação das vias de sinalização que regulam o desenvolvimento, o crescimento e a manutenção do fenótipo muscular. Além disso, a falta de informação promove uma limitação para o desenvolvimento de programas de melhoramento para esta espécie. Os principais objetivos do presente estudo foram: 1) aumentar os recursos genéticos disponíveis para a espécie, 2) comparar os transcriptomas dos tipos de músculo, vermelho e branco explorando as vias de sinalização que controlam os tipos de fibras musculares 3) investigar a expressão de deubiquitinas pertencente a família do sistema de Proteases ubiquitinas-específicas (USP) no músculo branco em resposta ao jejum e realimentação. Com o sequenciamento, obteve-se aproximadamente 0.6Tb de leituras a partir de bibliotecas do músculo esquelético vermelho e branco. Aproximadamente, 665 milhões de leituras foram utilizadas para a realização da montagem, resultando em 504.065 contigs com um comprimento médio de 1,334bp e N50 = 2,772bp; 47% do transcriptoma foi anotado com êxito, onde pode-se identificar cerca de 15.000 genes considerados únicos e aproximadamente 8000 sequências com região codificante completas. Dentre os genes identificados destacam-se 319 genes de vias metabólicas e 380 microssatélites. Novecentos e ciquenta e seis e 604 genes foram diferencialmente expressos entre o músculo vermelho e branco, respectivamente. Pacu (Piaractus mesopotamicus) has excellent husbandry features like rapid growth. This parameter is directly related to the increase of skeletal muscle. Researches involving the characterization of muscle genes have been performed using molecular technologies, in species of fish such as zebrafish model that has sequenced the genome. In pacu, a species that does not have the sequenced genome, this lack of genetic information makes it difficult to conduct research related to the identification of signaling pathways that regulate development, growth and maintenance of muscle phenotype. Moreover, the lack of information promotes a limitation for the development of breeding programs for this species. The main objectives of this study were: 1) to increase the available genetic resources for the species, 2) compare the transcriptomes of red and white muscle types, exploring the signaling pathways that control the types of muscle fibers 3) to investigate the expression of deubiquitins belonging to the family of ubiquitin-specific proteases system (USP) in muscle in response to fasting and feedback. With the sequencing, it was obtained approximately 0.6Tb readings from red skeletal muscle and white libraries. Approximately 665 million readings were used to perform the assembly, resulting in 504 065 contigs with an average length of N50 = 1,334bp and 2,772bp; 47% of the transcriptome was noted successfully, which can be identified about 15,000 genes considered as unique and approximately 8000 sequences coding complete region. Among the identified genes, we could observe 319 metabolic pathways genes and 380 microsatellite. Nine hundred fifty six and 604 genes were differentially expressed between red and white muscle, respectively. We identified 442 pairs of paralogs genes resulting from genomic duplication at the base source of teleost fish. It was possible to identify differential expression of paralogous genes include the types of fibers... CAPES: 1450/13-1 BEX FAPESP: 2012/02489-4

A fun??o neuronal saud?vel e a modifica??o sin?ptica precisam tanto da s?ntese quanto da degrada??o de prote?nas. Evid?ncias indicam que o turnover de prote?nas mediado pela atividade do proteassoma est? envolvido na plasticidade sin?ptica e na mem?ria de longa dura??o. No entanto, seu papel nas diferentes fases da mem?ria continua em discuss?o, e estudos anteriores n?o avaliaram a poss?vel necessidade da degrada??o de prote?nas na mem?ria de reconhecimento. Nesse trabalho, n?s mostramos que o inibidor de proteassoma, lactacistina, infundido na ?rea de CA1 do hipocampo em dois per?odos de tempo espec?ficos durante a consolida??o, prejudica a reten??o da mem?ria de reconhecimento em ratos. A administra??o da lactacistina n?o afetou a reconsolida??o da mem?ria. Estas descobertas fornecem a primeira evid?ncia para a import?ncia da atividade do proteassoma na mem?ria de reconhecimento, indicando que a degrada??o de prote?nas no hipocampo ? necess?ria durante duas janelas de tempo espec?ficas na consolida??o da mem?ria, contribuindo para a melhor compreens?o do papel do turnover de prote?na na forma??o da mem?ria. Altera??es dos n?veis de ferro cerebral t?m sido observadas em doen?as neurodegenerativas. J? foi demonstrado pelo nosso grupo de pesquisa, que a sobrecarga de ferro no per?odo neonatal resulta em graves e persistentes d?ficits de mem?ria na vida adulta. A degrada??o das prote?nas mediada pelo sistema ubiquitina-proteassoma desempenha um importante papel na regula??o de v?rios processos celulares e seu comprometimento tem sido implicado na patog?nese de doen?as neurodegenerativas. Ent?o, nesse estudo, tamb?m foram avaliados os efeitos da exposi??o de ferro no per?odo neonatal sobre a express?o das subunidades do proteassoma ?1, ?2, e ?5 e das prote?nas poliubiquitinadas em c?rebros de ratos aos 15 dias de vida e na idade adulta. Dois tipos de mem?ria dependente do hipocampo, aversiva e de reconhecimento, foram analisados em animais adultos. Confirmamos as evid?ncias anteriores, de que o ferro administrado no per?odo neonatal prejudica tanto a mem?ria aversiva quanto a mem?ria de reconhecimento. Os n?veis de prote?nas poliubiquitinadas encontraram-se elevados no hipocampo, mas n?o no c?rtex, de animais adultos tratados com ferro. A express?o g?nica das subunidades ?1 e ?5 foram afetadas pela idade no hipocampo, sendo maior nos primeiros est?gios de desenvolvimento, acompanhado por um aumento relacionado com a idade nos n?veis de prote?na poliubiquitinada em ratos adultos. No c?rtex, a express?o g?nica das tr?s subunidades do proteassoma foi significativamente mais elevada na idade adulta do que na idade neonatal. Em conjunto, esses resultados sugerem que a express?o das subunidades e a atividade do proteassoma s?o regulados de forma dependente da idade. Este, foi o primeiro estudo a investigar o sistema de degrada??o de prote?nas em c?rebros de ratos neonatos e tamb?m, o primeiro a estudar as subunidades do proteassoma comparando as idades neonatal e adulta. A exposi??o ao ferro no per?odo neonatal produz efeitos nocivos duradouros sobre o funcionamento do sistema ubiquitina-proteassoma, que pode estar relacionado com a defici?ncia de mem?ria induzida pelo ferro, fornecendo evid?ncias de que esse sistema pode ser considerado um alvo para o tratamento dos d?ficits de mem?ria. The healthy neuronal function and synaptic modification need both the synthesis and degradation of proteins. Evidence indicates that the protein turnover mediated by proteasome activity is involved in synaptic plasticity and long term memory. However, their role in different stages of memory is still under discussion, and previous studies did not evaluate the possible need of protein degradation in recognition memory. In this work, we have shown that proteasome inhibitor, lactacystin, infused in the CA1 area of the hippocampus at two specific time periods during consolidation affect the retention of the recognition memory in mice. The administration of lactacystin did not affect the reconsolidation of memory. These findings provide the first evidence for the importance of proteasome activity in memory recognition, indicating that the protein breakdown in the hippocampus is required during two specific time windows in memory consolidation, contributing to a better understanding of protein turnover's role in memory formation. Changes of brain iron levels have been observed in neurodegenerative diseases. It has been demonstrated by our research group, that iron overload in the neonatal period results in severe and persistent memory deficits in adulthood. Protein degradation mediated by the ubiquitin proteasome system plays an important role in regulating various cellular processes and their involvement has been implicated in pathogenesis of neurodegenerative diseases. So in this study was also evaluated the effects of iron exposure in the neonatal period on the expression of proteasome subunits ?1, ?2, and ?5 and polyubiquitinated proteins in brains of rats at 15 days of life and adulthood. Two types of hippocampal-dependent memory and recognition aversive were analyzed in adult animals. We confirm earlier evidence that iron administered in the neonatal period affect both the aversive memory and recognition memory. The levels of proteins polyubiquitinated found to be high in the hippocampus, but not in the cortex of adult animals treated with iron. The gene expression of the subunits ?1 and ?5 were affected by age in the hippocampus, which is higher in the early stages of development, accompanied by a related increase with age polyubiquitinated protein levels in adult rats. In the cortex, the gene expression of all three subunits of the proteasome was significantly higher in old adult than in neonatal age. Together, these results suggest that the expression of the subunits of the proteasome and are regulated activity of age-dependent manner. This was the first study to investigate the protein degradation system in the brain of rats newborns and also the first to study the proteasome subunits comparing neonatal and adult ages. Exposure to iron in the neonatal period produces long lasting harmful effects on the functioning of the ubiquitin-proteasome system, which may be related to deficiency of the iron induced memory, providing evidence that this system can be considered a target for the treatment of deficits memory.

[cat] Les mutacions del gen PARK2 estan associades amb l‘aparició de parkinsonisme juvenil autosòmic recessiu (AR-JP). A través de diferents mecanismes aquestes mutacions comporten la pèrdua de la funció E3 lligasa de la proteïna parkina, amb el que promouen l’acumulació tòxica de proteïnes que resulta en una lenta però progressiva degeneració i mort neuronal. En aquest treball, s’ha descrit la proteïna RTP801/REDD1, proapoptòtica i reguladora de la cinases de supervivència mTOR i Akt, com a substrat de la parkina. L’E3 lligasa parkina interacciona amb la proteïna proapoptòtica RTP801 i en catalitza la poliubiquitinització K48, facilitant així la seva degradació proteasomal, i contribuint a mantenir-ne els nivells proteics baixos. En efecte, s’ha observat que la pèrdua de funció de la parkina provoca una acumulació de l’RTP801 en models cel·lulars i en ratolins genoanul·lats per parkina (PaKO). L’augment en els nivells de la proteïna RTP801 també és remarcable en seccions post mortem de la substància negra pars compacta (SNpc) i en fibroblasts de pacients amb mutacions patològiques del gen PARK2. Per altra banda, l’expressió ectòpica de la parkina disminueix els nivells proteics de l’RTP801 i exerceix un efecte protector vers la toxicitat dependent de l’elevació d’aquesta, ja sigui per la sobreexpressió ectòpica de l’RTP801 o pel tractament amb la neurotoxina parkinsoniana 6-OHDA. Aquest efecte és dependent de la poliubiquitinització i degradació de l’RTP801, com ho demostra l’absència de protecció vers el mutant no ubiquitinitzable RTP801 K-R. De manera consistent, la parkina mutant inactiva falla en produir aquest efecte protector, mentre que la depleció de la parkina produeix una exacerbació dels efectes deleteris de l’RTP801 i contribueix a la inactivació de la via de supervivència cel·lular mTOR/Akt. Per tot plegat, es proposa que la parkina és capaç de prevenir l’acumulació tòxica de la proteïna proapoptòtica RTP801 en un context parkinsonià. En conjunt, aquests resultats indiquen que l’RTP801 es un nou substrat de l’activitat E3 lligasa de la parkina, y que podria estar contribuint a la neurodegeneració causada por la pèrdua de l’expressió o activitat de la parkina. L’intercanvi d’informació entre neurones és un procés vital que té lloc en els contactes sinàptics, o sinapsis. En aquest treball s’ha demostrat que l’RTP801 es troba especialment expressat en les espines sinàptiques en conjunció amb la densitat postsinàptica dels contactes excitadors. A nivell funcional, s’ha vist que l’RTP801 ectòpica provoca un deteriorament de la transmissió sinàptica excitadora, causat per la disminució en la intensitat de l’expressió de diverses proteïnes sinàptiques. També s’ha determinat que l’efecte deleteri de la toxina 6-OHDA sobre el sistema de neurotransmissió glutamatèrgica és dependent de l’elevació dels nivells sinàptics de l’RTP801. Inesperadament, la manca de parkina tan sols produeix una lleuger increment dels nivells sinàptics de l’RTP801, tal com es dedueix dels experiments realitzats en cultius corticals de rata, i en ratolins PaKO, on inesperadament la proteïna RTP801 es situa preferentment en el compartiment presinàptic. Al contrari que en el teixit humà estriatal post mortem, on l’RTP801 és postsinàptic. La pèrdua d’innervació dopaminèrgica a l’estriat parkinsonià promou un efecte distròfic en les dendrites de les neurones espinoses mitjanes estriatals. En aquest treball s’ha descrit que les mostres estriatals post mortem provinents de pacients parkinsonians presenten una major acumulació de l’RTP801 tant en els homogenats com en les sinapsis, així com una reducció de l’activació de la via mTOR/Akt. Aquestes observacions obren la porta a una possible implicació de l’RTP801 en la disfunció sinàptica estriatal característica de la malaltia de Parkinson., [eng] Mutations in the PARK2 gene are associated with an Autosomal Recessive form of Juvenile Parkinsonism (AR-JP). These mutations affect parkin solubility and impair its E3 ligase activity leading to a toxic accumulation of proteins within susceptible neurons that results in a slow but progressive neuronal degeneration and cell death. Here, we report that RTP801/REDD1, a pro-apoptotic negative regulator of survival kinases mTOR and Akt, is one of such parkin substrates. We observed that parkin knockdown elevated RTP801 in sympathetic neurons and neuronal PC12 cells while ectopic parkin enhanced RTP801 poly-ubiquitination and proteasomal degradation. In parkin knockout mouse brains and in human fibroblasts from AR-JP patients with parkin mutations, RTP801 levels were elevated. Moreover, in human postmortem Parkinson’s disease (PD) brains with mutated parkin, nigral neurons were highly positive for RTP801. Further consistent with the idea that RTP801 is a substrate for parkin, the two endogenous proteins interacted in reciprocal co-immunoprecipitates of cell lysates. A potential physiological role for parkin-mediated RTP801 degradation is indicated by observations that parkin protects neuronal cells from death caused by RTP801 over-expression by mediating its degradation, while parkin knockdown exacerbates such death. Similarly, parkin knockdown enhanced RTP801 induction in neuronal cells exposed to the PD mimetic 6-OHDA and increased sensitivity to this toxin. This response to parkin loss-of-function appeared to be mediated by RTP801 since it was abolished by RTP801 knockdown. Taken together these results indicate that RTP801 is a novel parkin substrate that may contribute to neurodegeneration caused by loss of parkin expression or activity. In this work it has also been investigated the role of RTP801 in synaptic plasticity. We have observed high RTP801 expression in the postsynaptic compartment in rat striatum, cortex, hippocampus and ventral midbrain. The synaptic sublocalization of RTP801 in human striatum is also clearly postsynaptic. At a functional level, ectopic RTP801 dampens the excitatory synaptic transmission by decreasing the levels of several synaptic markers. It has also been shown that the deleterious postsynaptic effect of 6- OHDA is dependent on RTP801 induction in cortical cultures. Finally, we described that RTP801 accumulates in the synaptosomes isolated from striatal postmortem tissue from PD and AR-JP patients, where we have also described an inactivation of the mTOR/Akt, which may contribute to the striatal synaptic dysfunction in PD.

Orientadora : Profª Drª Adriana Frohlich Mercadante Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 20/03/2015 Inclui referências : f. 62-68 Área de concentração Resumo: A proteína prion celular, ou PrPC, é uma glicoproteína extracelular, ancorada na membrana por uma molécula de glicofosfatidilinositol (GPI). A conversão de PrPC para uma isoforma mal dobrada e infecciosa, PrPSc, é responsável pelo desenvolvimento das doenças espongiformes transmissíveis (TSEs), ou doenças priônicas. Apesar de extensa pesquisa e do fato de PrPC ser conservada entre as espécies, não se chegou a um consenso sobre sua função fisiológica. Recentemente nosso grupo identificou a interação entre PrPC e Stub1 (STIP1 homology and U-Box containing protein 1), também denominada CHIP (C-terminus of HSC70-interacting protein) - uma co-chaperona citoplasmática que apresenta atividade ubiquitina E3-ligase, através do sistema de duplo-híbrido em leveduras. Stub1/CHIP age fazendo a triagem de proteínas para o dobramento ou degradação. Entretanto, a função biológica dessa interação ainda não estava elucidada. Primeiramente, o objetivo desse trabalho foi confirmar a interação entre PrPC e Stub1/CHIP através de técnicas de pull-down e co-imunoprecipitação. A interação foi confirmada tanto em ensaios com proteínas superexpressas, através da transfecção de células HEK293T com plasmídeos com capacidade de expressar PrPC e Stub1/CHIP, quanto endógenas, a partir de amostras de encéfalo e epitélio olfatório de camundongo, corroborando com os dados achados pelo sistema de duplohíbrido. A segunda etapa desse trabalho teve como objetivo caracterizar a interação de Stub1/CHIP e PrPC. Focou-se no envolvimento de Stub1/CHIP na estabilidade/degradação de PrPC, supondo-se que Stub1/CHIP participaria no controle de qualidade de PrPC. Dessa forma, ensaios de degradação e ubiquitinação foram realizados em células transfectadas com plasmídeos com capacidade de expressar PrPC e Stub1/CHIP. Nossos resultados indicam que Stub1/CHIP está envolvida na degradação de PrPC e sugerem que a via de degradação envolvida seja a dependente de ubiquitina-proteossomo. Esses trabalho indica um importante papel de Stub1/CHIP na estabilidade/degradação de PrPC, sendo um possível alvo de estratégias terapêuticas no estudo das TSEs. Palavras-chave: PrPC. Stub1/CHIP. Ubiquitina-proteossomo. Prion. Controle de qualidade de proteínas. Abstract: The cellular prion protein (PrPc) is a glycoprotein bound to cell membrane via a glycosylphosphatidylinositol (GPI) anchor. A conversion of PrPC to its misfolded and infectious isoform (PrPSc) is responsible for the development of transmissible spongiform encephalopathies (TSEs), or prion diseases. Despite intensive research and the fact that PrPC is conserved across different species, a consensus about its physiological function has not yet been reached. Recently, our group identified the interaction between PrPC and Stub1 (STIP1 homology and U-Box containing protein 1), also called CHIP (C-terminus of HSC70-interacting protein) through yeast twohybrid screen, a cytoplasmic co-chaperone which presents as ubiquitin E3-ligase activity. Stub1/CHIP acts screening proteins to their folding or degradation. Nevertheless, the biological function of this interaction was not yet elucidated. The first objective of this work was to confirm the interaction between PrPC and Stub1/CHIP through pull-down and co-immunopreciptation assays. This interaction was confirmed in assays from super expressed proteins, through transfection of HEK293T cells with plasmids expressing PrPC and Stub1/CHIP, and from endogenous proteins assays, through mouse brain and olfactory epithelium sampling, corroborating the data from yeast-two-hybrid screen. The second part of this study aimed to characterize the interaction between Stub1/CHIP and PrPC. We focused on the involvement of Stub1/CHIP in the stability/degradation of PrPC, assuming that Stub1/CHIP would participate in PrPC quality control. In this way, degradation and ubiquitination assays were performed in cells transfected with plasmids expressing PrPC and Stub1/CHIP. Our results indicate that Stub1/CHIP is involved in the degradation of PrPC. This work indicates an important role of Stub1/CHIP in the stability/degradation of PrPC, which could become a target for therapeutic strategies in the study of the TSEs. Keywords: PrPC. Stub1/CHIP. Ubiquitin-proteasome. Prion. Protein quality control.

© 2015 Authors; published by Portland Press Limited. Despite being a common mechanism in eukaryotes, the process by which protein monoubiquitination is produced and regulated in vivo is not completely understood. We present here the analysis of the process of monoubiquitination of the proteasomal subunit Rpn10 (regulatory particle non-ATPase 10), involved in the recruitment of polyubiquitinated substrates. Rpn10 is monoubiquitinated in vivo by the Nedd4 (neural precursor cell expressed developmentally down-regulated 4) enzyme Rsp5 (reverses SPT-phenotype protein 5) and this modification impairs the interaction of Rpn10 with substrates, having a regulatory effect on proteasome function. Remarkably, a disordered region near the ubiquitin-interacting motif of Rpn10 plays a role in the restriction of the polyubiquitin extension activity of Rsp5. Mutations in this disordered region promote ubiquitin chain extension of Rpn10. Thus, our work sheds light on the molecular basis and the functional relevance of a type of monoubiquitination that is driven by the substrate. Moreover, we uncover a putative role for disordered regions in modulating ubiquitin-protein ligation.

Protein monoubiquitination is a post-translational modification that consists of the conjugation of a single ubiquitin molecule to a target protein residue. Monoubiquitination regulates protein activity and localization and is involved in DNA repair, histone regulation, and receptor endocytosis. Although the mechanisms by which monoubiquitination regulates protein function are still not well understood, there are some insights into the mechanisms that promote this modification. In this work, we discuss some of the principles of the processes that produce monoubiquitination in vivo; i.e., how ubiquitin conjugating enzymes, ubiquitin ligases or other co-factors can produce direct substrates monoubiquitination, and how ubiquitin specific proteases can indirectly convert polyubiquitinated to monoubiquitinated proteins. La monoubicuitinación es una modificación post-traduccional que consiste en la conjugación de una única molécula de ubicuitina a un sustrato. Se ha descrito que la monoubicuitinación controla la localización y la función de proteínas implicadas en procesos tales como la reparación del ADN, la regulación de histonas, la expresión génica y la endocitosis. A pesar de que todavía existen muchas incógnitas respecto a los mecanismos por los cuales la monoubicuitinación regula la función proteica, sí que se conocen algunos de los mecanismos que promueven la monoubicuitinación de sustratos. En este trabajo se discutirán algunos de los principios de los procesos que generan la monoubicuitinación in vivo a través de enzimas conjugadoras de ubicuitina (E2), de ubicuitina ligasas (E3), de las proteasas desubicuitinasas y de otros co-factores. La monoubicuitinació és una modificació post-traduccional que consisteix en la conjugació d’una sola molècula d’ubicuitina a un substrat. S’ha descrit que la monoubicuitinació controla la localització i la funció de proteïnes implicades en processos tals com la reparació de l’ADN, la regulació d’histones, l’expressió gènica i l’endocitosi. Tot i que encara hi ha moltes incògnites respecte als mecanismes pels quals la monoubicuitinació regula la funció proteica, sí que es coneixen alguns dels mecanismes que promouen la monoubicuitinació de substrats. En aquesta revisió s’hi discutiran alguns dels principis dels processos que generen la monoubicuitinació in vivo a través d’enzims conjugadors d’ubicuitina (E2), d’enzims ubicuitina lligasa (E3), de les proteases desubicuitinases i d’altres co-factors.

Cellular homeostasis depends, partially, on the proteolysis regulated by the Ubiquitin-Proteasome System (UPS). This proteolysis includes the degradation of misfolded, damaged or unnecessary proteins for the cell and comprises two phases. In the first stage, molecules of Ubiquitin are attached covalently to a target substrate at the amide group (ε-NH2) of some lysine residues. This process depends on the enzymatic activity of three enzymes, E1, E2 and E3. Afterwards, the proteasome takes care of the degradation of the substrate. The proteasome is a barrel-shaped multiprotein complex of 2,5 MDa present in the nucleus and cytosol of eukaryotic and Archaebacteria cells. The canonical degradation signal is a chain of ubiquitins, which is built via isopeptide bonds between the glycine G76 at the C-terminus of the molecule and lysine K48 of the next Ubiquitin.

Proteins that fail to fold in the endoplasmic reticulum (ER) or cannot find a pattern for assembly are often disposed of by a process named ER-associated degradation (ERAD), which involves transport of the substrate protein across the ER membrane (dislocation) followed by rapid proteasome-mediated proteolysis. Different ERAD substrates have been shown to be ubiquitinated during or soon after dislocation, and an active ubiquitination machinery has been found to be required for the dislocation of certain defective proteins. We have previously shown that, when expressed in tobacco (Nicotiana tabacum) protoplasts, the A chain of the heterodimeric toxin ricin is degraded by a pathway that closely resembles ERAD but is characterized by an unusual uncoupling between the dislocation and the degradation steps. Since lysine (Lys) residues are a major target for ubiquitination, we have investigated the effects of changing the Lys content on the retrotranslocation and degradation of ricin A chain in tobacco protoplasts. Here we show that modulating the number of Lys residues does not affect recognition events within the ER lumen nor the transport of the protein from this compartment to the cytosol. Rather, the introduced modifications have a clear impact on the degradation of the dislocated protein. While the substitution of the two Lys residues present in ricin A chain with arginine slowed down degradation, the introduction of four extra lysyl residues had an opposite effect and converted the ricin A chain to a standard ERAD substrate that is disposed via a process in which dislocation and degradation steps are tightly coupled.

High-performance liquid chromatography purification followed by mass spectrometry analyses highlighted that human senile cataractous lens includes a 8182 Da species which is absent in the normal lens, whereas a 8566/8583 Da species is present in both lenses. Western blot analysis identified both species as ubiquitin. The species at lower molecular weight is a shorter form due to the cleavage of the C-terminal residues 73-76. As it is the last amino acid of ubiquitin which is involved in the protein degradation mechanism, we suggest that this structure modification compromises the function of ubiquitin and consequently the physiologically occurring degradation of the lens proteins. © 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved. High-performance liquid chromatography purification followed by mass spectrometry analyses highlighted that human senile cataractous lens includes a 8182 Da species which is absent in the normal lens, whereas a 8566/8583 Da species is present in both lenses. Western blot analysis identified both species as ubiquitin. The species at lower molecular weight is a shorter form due to the cleavage of the C-terminal residues 73-76. As it is the last amino acid of ubiquitin which is involved in the protein degradation mechanism, we suggest that this structure modification compromises the function of ubiquitin and consequently the physiologically occurring degradation of the lens proteins. © 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Nedd4L is a HECT-type E3 ubiquitin ligase (it covalently binds ubiquitin molecules before transferring them to the final substrate). Ubiquitination is a posttranslational modification (PTM) that labels proteins for a variety of fates, the most relevant one being proteasome-mediated degradation. Nedd4L is responsible for the regulation of the turnover of the sodium channel ß-ENaC as well as Smad2/3, mediator proteins of the signalling pathway activated by TGF-ß-like cytokines. It also targets the TGF-ß receptor itself. Defects in its function have been related to hereditary hypertension (Liddle’s syndrome), and could be relevant in certain sorts of cancer and metastasis. CDK8/9 and GSK3-ß are two kinases that regulate the phosphorylation of the Smads, enabling them to carry out their function in cooperation with transcription factors and other partner proteins. At the same time, they label the Smads for their recognition by ubiquitin ligases. This provides the cell with a mechanism to give a transient response to the cytokines of the TGF-ß type. In order to identify the residues and the phosphorylation patterns that are relevant for the interactions of the Smads with both the transcription factors and the ubiquitin ligases, we have prepared a set of phosphopeptides corresponding to the sequences of Smad1 and Smad3. Like all other members of the Nedd4 family, Nedd4L has a multi-domain architecture of the type C2-WW-HECT. Several ligases of the family exist in a latent conformation established through inter-domain contacts that occlude the catalytic site in the HECT domain, involving either the C2 domain (Smurf1, Smurf2, WWP2, Nedd4, Nedd4L) or the central segment where the WW domains are located (Itch). Certain cellular events displace these contacts, inducing the transition to the active conformation. In the case of Nedd4L, increases of the intracellular levels of Ca2+ activate the ligase. The hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) delivers into the cytosol the inositol 1,4,5-triphosphate (IP3), a second messenger that mobilizes the intracellular Ca2+ reserves. The C2 domain of Nedd4L interacts both with Ca2+ ions and with IP3. Using a structural and biophysical approach based on Nuclear Magnetic Resonance (NMR) we have described the specific interactions between the HECT and C2 domains that inhibit the catalytic function. Ca2+ binds the C2 domain with high affinity using the same binding surface and compromises these contacts. In addition, it mediates the interaction with IP3. These results provide the structural fundament for the activation and the relocation to the plasma membrane of Nedd4L mediated by Ca2+. The HECT domain has a highly conserved PY site (HECT-PY). The PY motifs are the sequences recognized by WW domains. Central to this recognition is the coordination of the tyrosine residue in the PY motif by the WW domain. In the crystallographic structure of the Nedd4L HECT domain the tyrosine residue of the HECT-PY motif appears buried in the hydrophobic core and not accessible for binding. It has been shown that the WW domains of Nedd4L recognize the HECT-PY motif of the ligase only after the unfolding of the HECT domain. We raised the hypothesis that the recognition of the HECT-PY motif by one of Nedd4L WW domains may play a role in the auto-ubiquitination mechanism of the ligase. Our data confirm that only when the fold of the HECT domain is partially damaged, the PY site is accessible for being recognized by the WW domains. We present the NMR solution structure of the complex between the WW3 domain and the HECT-PY motif. The site is protected in functional Nedd4L molecules, which are able to recognize it in damaged molecules and label them with ubiquitin for degradation., Nedd4L és una E3 ubiquitín lligasa responsable de la regulació de la vida mitja del canal de sodi ß-ENaC i de Smad2/3, proteïnes mediadores de la ruta de senyalització activada per citocines TGF-ß. Defectes en la seva funció han estat relacionats amb la hipertensió hereditària (Síndrome de Liddle), i podrien ser rellevants en determinats tipus de càncer i metàstasi. CDK8/9 i GSK3-ß són dues quinases que regulen l’estat de fosforilació de les Smads, habilitant-les per dur a terme llur funció en cooperació amb factors de transcripció al mateix temps que les marquen per ser reconegudes per ubiquitín lligases. Amb l’objectiu d’identificar els residus i els patrons de fosforilació rellevants hem preparat un set de fosfopèptids que corresponen a les seqüències de Smad1/3. Nedd4L presenta una arquitectura multi-domini C2-WW-HECT. Diverses lligases de la família de Nedd4 existeixen en una conformació latent en què contactes inter-domini oclouen el lloc catalític en el domini HECT, involucrant bé el domini C2 (Smurf1/2, WWP2, Nedd4, Nedd4L) o la zona central amb els dominis WW (Itch). Certs esdeveniments cel•lulars desplacen aquests contactes, induint la transició a la conformació activa. L’increment dels nivells intracel•lulars de Ca2+ activa Nedd4L. La hidròlisi del fosfolípid de membrana PIP2 allibera l’IP3 provocant aquest increment. El domini C2 de Nedd4L interacciona tant amb el Ca2+ com amb l’IP3. Utilitzant l’RMN hem descrit els contactes HECT-C2 en la conformació latent i hem observat que el Ca2+ s’uneix al domini C2 amb alta afinitat utilitzant el mateix lloc d’unió, a més d’afavorir la interacció amb l’IP3. Així, hem aportat el fonament estructural per a l’activació i re­localització a la membrana cel•lular de Nedd4L. El domini HECT presenta un lloc PY altament conservat (HECT-PY). Els motius PY són reconeguts pels dominis WW. Proposem que el reconeixement del motiu HECT-PY per part d’un dels dominis WW de Nedd4L estigui implicat en l’auto-ubiquitinació. Hem observat que només quan el plegament del domini HECT està compromès, el lloc PY és accessible. Presentem l’estructura per RMN del complex WW3-HECT-PY. El motiu està protegit en molècules funcionals de Nedd4L, capaces de reconèixer-lo en molècules danyades i ubiquitinar-les.

This paper presents a combined approach with two aims. The first is to analyze the reported sequence of the enzyme ubiquitin carboxyl-terminal hydrolase 14 of Giardia intestinalis (UBP6) through computational methods to find components related with its hypothetical function. The second is to determine if the protein-coding gene is expressed in G. intestinalis and, if such is the case, also determine its transcription pattern along the life cycle of the parasite. It was established that the protein belongs to the family of Cys-dependent deubiquitinases and more specifically to ubiquitin specific proteases (USPs). Moreover, the catalytic center with the complete triad as well as typical features of the USP motif were also identified. Since the computational findings suggest that the enzyme could be functional, reverse transcription coupled to PCR was used as a first approach to establish if in fact the coding gene is expressed in the parasite. Interestingly, it was found not only that the gene is expressed, but also that there is a transcription variation along the life cycle of the parasite. These two findings are the starting point for further studies since they tentatively suggest that this enzyme could be involved in the protein turnover that occurs during parasite encystation. Although preliminary, this study is the first report concerning the study of a specific deubiquitinating enzyme in the parasite G. intestinalis. En este trabajo se presenta una estrategia combinada que buscaba, primero, analizar por métodos computacionales la secuencia de la enzima ubiquitina carboxilo-terminal hidrolasa 14 de Giardia intestinalis (UBP6) reportada para buscar componentes relacionados con su función hipotética y segundo, determinar si el gen que codifica para la proteína se expresa en G. intestinalis y si lo hace, cómo es su patrón de transcripción a lo largo del ciclo de vida del parásito. Se encontró que la proteína pertenece a la familia de deubiquitinasas dependientes de cisteína y más específicamente a las proteasas específicas para ubiquitina (USPs por ubiquitin specific proteases). También se identificaron el centro catalítico con la triada completa así como características típicas del motivo USP. Teniendo en cuenta que los resultados computacionales sugieren que la enzima puede ser funcional, se usó la técnica de transcripción reversa acoplada a PCR como un primer acercamiento para establecer si el gen codificante se expresa en el parásito. De manera interesante, se determinó no solo que el gen se expresa sino que existe una variación de su transcripción a lo largo del ciclo de vida del parásito. Estos hallazgos son el punto de partida para posteriores estudios ya que sugieren de manera preliminar que esta enzima podría estar involucrada en el recambio de proteínas que ocurre en el parásito durante el proceso de enquistación. Aunque preliminar, este estudio es el primer reporte acerca de una enzima deubiquitinadora específica en el parásito G. intestinalis. Este artigo apresenta uma abordagem combinada com dois objetivos. A primeira é analisar a sequência informou da enzima ubiquitina carboxil-terminal hidrolase 14 de Giardia intestinalis (UBP6) através de métodos computacionais para encontrar os componentes relacionados com a sua função hipotética. A segunda é para determinar se o gene de codificação da proteína é expressa em G. intestinalis e, se for o caso, também determinar o seu padrão de transcrição ao longo do ciclo de vida do parasita. Foi estabelecido que a proteína pertence à família de deubiquitinases Cys-dependentes e mais especificamente para proteases específicas de ubiquitina (USPs por ubiquitin specific proteases). Além disso, o centro catalítico com a tríade completo, bem como as características típicas do motivo USP também foram identificados. Uma vez que os resultados computacionais sugerem que a enzima poderia ser funcional, a transcrição reversa acoplada a PCR foi utilizado como uma primeira abordagem para determinar se, de facto, o gene codificante é expressa no parasita. Curiosamente, verificou-se não só que o gene é expresso, mas também que há uma variação de transcrição ao longo do ciclo de vida do parasita. Estes dois elementos são o ponto de partida para estudos posteriores, uma vez que tentativas sugerem que esta enzima pode estar envolvida no refill de proteínas que ocorre durante o parasita encistamento. Embora preliminares, este estudo é o primeiro relatório relativo ao estudo de uma enzima deubiquitinadora específica no parasita intestinalis.

La rápida y específica degradación de proteínas por el sistema proteosomaubiquitinaha emergido como un importante mecanismo de regulación en eucariotas.Algunos de los procesos básicos celulares regulados por proteólisis dependiente deubiquitina incluyen la progresión del ciclo celular, iniciación de la transcripción otransducción de señales. Las enzimas clave en el proceso de ubiquitinación son lasligasas de ubiquitina E3, puesto que son las que actúan como moduladores para elreconocimiento del sustrato y además son las que transfieren la ubiquitina activada adicho sustrato. Las proteínas Skp1, Cul1 y la proteína F-box (SCF) son el grupo deligasas E3 mejor conocido. Dada la importancia de dicho proceso, resultó de graninterés investigar el papel de las proteínas pertenecientes a la maquinaria derenovación proteica, en la patogénesis de Fusarium oxysporum f. sp. lycopersici sobreplantas de tomate.En este trabajo nos hemos centrado en el estudio de una proteína F-box, Fbp1,ortóloga de GRR1 en S. cerevisiae. Para estudiar el papel del gen fbp1 en la virulenciade F. oxysporum se ha llevado a cabo la interrupción dirigida del gen en la estirpesilvestre del hongo. La infección de plantas de tomate inoculadas con los mutantesdeficientes en el gen mostró un retraso en el proceso de infección, lo que indica queFbp1, aunque no es esencial para la patogenicidad, es necesaria para la completavirulencia de F.oxysporum. El mutante Δfbp1 presentó un retraso significativo en todaslas fuentes de carbono y nitrógeno analizadas, además de un patrón de crecimientoondulado de las hifas líder y una tinción del Spk muy abundante y deslocalizada.Mutantes de F. oxysporum en el gen fbp1 mostraron niveles reducidos de fosforilaciónde Fmk1 y compartían características fenotípicas con los mutantes Δ fmk1, tales comodefectos en el crecimiento invasivo, en la colonización del tejido vivo de la planta y enla capacidad de fusión vegetativa. Por otro lado, los bajos niveles de fosforilación de laMAPK Mpk1 que exhibió el mutante nulo fbp1 junto con la sensibilidad a compuestoscomo SDS y CFW, sugieren que Fbp1 contribuye al mantenimiento de la integridadcelular.Realizamos un análisis proteómico, en condiciones de crecimiento invasivo, paraconocer las proteínas diferencialmente expresadas en la cepa mutante Δ fbp1 y poderasí identificar posibles dianas del complejo SCFFbp1. De las 41 proteínas identificadas,17 de ellas estaban sobre-expresadas en el mutantΔe fbp1 y relacionadas confunciones susceptibles de sufrir ubiquitinación: transporte, proteolisis, respuesta aestrés y organización de los componentes celulares..., Fast and specific degradation proteins by the ubiquitin-proteasome system haverecently emerged as a major regulatory mechanism of cellular function in eukaryotes.Some basic cellular processes regulated by ubiquitin proteasome system are cell cycle,signal transduction and transcription. The key enzymes in the ubiquitination processare the E3 ubiquitin ligases, because the function as the susbtrate recognition moduleof the system and are capable of transferring the activated ubiquitin to the substrate.Spk1, Cul1 and F-box protein (SCF) E3 ligases are amoung the best-understood ligases.Given the importance of this process, it was of great interest investigating the role ofproteins belonging to the protein turnover machinery and their contribution toFusarium oxysporum pathogenicity.In this study, we have focused in the study of an F-box protein called Fbp1 whichencodes the orthologe of S. cerevisiae GRR1 gene. In order to investigate the role Fbp1in pathogenicity of F. oxysporum we performed a targeted disruption of fbp1 gene.Disease progression on tomato plant inoculated with Δfbp1 mutant showed reducedvirulence suggesting that Fbp1 is essential for full virulence in F.oxysporum. The Δfbp1mutant showed reduced hyphal growth on all carbon and nitrogen sources analysed.In the mutant strain, leader hyphae displayed a zig-zag growth phenotype and anabundant and delocalized staining Spk. F. oxysporum strains lacking Δ fbp1 had areduced phosphorylation levels of Fmk1 and shared characteristic phenotypes withΔfmk1mutants, including defects in hyphal growth under nutrient-limited conditions,invasion of the underlying substrate and colonization of living plant tissue.Interestingly, low phosphorylation levels of Mpk1 and hypersensitivity to cell walltargeting compounds like CFW and SDS showed Δ fbp1 mutant, suggesting that Fbp1contributes to cell wall integrity via a MAPK Mpk1 pathway.In order to know differentially expressed proteins in Δfbp1 mutant invasiongrowthconditions, we carried out a proteomic approach to identify the target proteinsof SCFFbp1 complex. 17 of the 41 proteins differentially expressed in the present workwere up-regulated in the Δfbp1 mutant and they were related with processsusceptible to be ubiquininated: transport, proteolysis, response to stress and cellularcomponent organization.One of the most abundant protein in the Δfbp1 mutant was the FOXG_0146gene, a 14-3-3 protein annotated in the Fusarium database like Bmh2. 14-3-3 proteinsare dimeric and highly conserved proteins which are involved in a many cellular...

Made available in DSpace on 2015-01-26T13:21:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-05Bitstream added on 2015-01-26T13:30:54Z : No. of bitstreams: 1 000799339.pdf: 643539 bytes, checksum: 8e393b7ddf87588d52857005898757c9 (MD5) A literatura aponta que no diabetes mellitus, a expressão gênica de proteínas envolvidas no UPS (ubiquitine-proteasome system) encontra-se acima dos níveis basais, ocasionando degradação de proteínas miofibrilares. Sabe-se que a enzima de conjugação E214kDa e ubiquitina ligase E3 - MuRF1 (Muscle Ring Finger-1) podem ser consideradas como biomarcadores de tal via. Os modelos de animais diabéticos apresentam maior expressão de NFkB (fator nuclear kappa B), o qual tem papel importante na regulação da transcrição do gene do GLUT4. O exercício físico configura-se como método terapêutico no tratamento do diabetes mellitus. A contração muscular melhora a captação de glicose na musculatura esquelética, translocando o GLUT4 via proteína cinase ativada por 5´-AMP (AMPK). Objetivo: Avaliar o efeito de seis semanas de treinamento de salto sobre conteúdo proteico de GLUT4, e expressão gênica (mRNA) dos genes Slc2a4 (proteína GLUT4), UBb (Ubiquitina), E214kDa, Trim63 (proteína MuRF1), NFkB p105 e Prkaa2 (proteína AMPKα2) no músculo gastrocnêmio de ratos diabéticos... The literature suggests that in diabetes mellitus, the gene expression of proteins involved in UPS (ubiquitine - proteasome system) lies above baseline levels, causing degradation of myofibrillar proteins. It is known that E214kDa conjugating enzyme and ubiquitin ligase E3 - MuRF1 (Muscle Ring Finger -1) may be regarded as biomarkers of this pathway. The models of diabetic animals shows increased expression of NFkB (Nuclear Factor kappa B), which plays an important role in the regulation of GLUT4 gene transcription. Exercise appears as a therapeutic method in the treatment of diabetes mellitus. Muscle contraction improves glucose uptake in skeletal muscle GLUT4 translocating protein kinase pathway activated by 5' - AMP (AMPK). Objective: To evaluate the effect of six weeks of jumping on GLUT4 protein content of training, and gene expression (mRNA) of Slc2a4 (GLUT4 protein), UBB (Ubiquitin), E214kDa, Trim63 (MuRF1 protein) , NFkB p105 gene and Prkaa2 (AMPKα2) protein in the gastrocnemius muscle of diabetic rats...

UbcH10 is a component of the Ubiquitin Conjugation Enzymes (Ubc; E2) involved in the ubiquitination cascade controlling the cell cycle progression, whereby ubiquitin, activated by E1, is transferred through E2 to the target protein with the involvement of E3 enzymes. In this work we propose the first three dimensional model of the tetrameric complex formed by the human UbA1 (E1), two ubiquitin molecules and UbcH10 (E2), leading to the transthiolation reaction. The 3D model was built up by using an experimentally guided incremental docking strategy that combined homology modeling, protein-protein docking and refinement by means of molecular dynamics simulations. The structural features of the in silico model allowed us to identify the regions that mediate the recognition between the interacting proteins, revealing the active role of the ubiquitin crosslinked to E1 in the complex formation. Finally, the role of these regions involved in the E1-E2 binding was validated by designing short peptides that specifically interfere with the binding of UbcH10, thus supporting the reliability of the proposed model and representing valuable scaffolds for the design of peptidomimetic compounds that can bind selectively to Ubcs and inhibit the ubiquitylation process in pathological disorders.

Tesis (Doctor en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2014 Las modificaciones postraducción de proteínas son importantes para la regulación de la fisiología de la célula ya que contribuyen significativamente a la diversidad estructural y funcional de las proteínas. Una de estas modificaciones es la arginilación, la cual consiste en la unión covalente de un residuo de arginina en el extremo NH2 de proteínas aceptoras, específicamente sobre ácido aspártico, ácido glutámico, o cisteína. Esta reacción es catalizada por la enzima arginil-ARNt transferasa (Ate1) tanto en el citosol como en el núcleo celular. En nuestro laboratorio se ha establecido previamente que calreticulina (CRT), una proteína mayoritariamente residente del retículo endoplásmico (RE), se retrotransloca al citoplasma donde es uno de los sustratos de la arginilación. Además, se demostró que en condiciones de estrés que conducen a una disminución de los niveles intracelulares de Ca2+, CRT arginilada (R-CRT) se asocia a gránulos de estrés (GSs) que son complejos de ARNm y cuya función se correlaciona con una reducción selectiva de la traducción proteica. En el presente trabajo se describe la función de la arginilación de CRT en relación a la tasa de recambio ("turnover") de CRT citoplasmática, la cual modularía la estabilidad de la proteína. Además, se establece la vía de degradación de R-CRT en relación al rol que desempeña la arginilación. En este estudio se demuestra que tanto R-CRT como CRT se degradan por la vía proteosomal, aunque R-CRT tiene menor susceptibilidad que CRT a ser degradada por el proteosoma. Se determinó que la vida media de R-CRT es el doble que la de CRT citoplasmática sin arginilar. Cuando se sobre expresaron ambas proteínas en células knockout para la enzima Ate1 (células ATE1–/–), se confirmó que R-CRT es degradada de manera menos eficaz que CRT por el proteosoma. También, se encontró que CRT es degradada por una vía independiente de ubiquitina (Ub), mientras que la degradación de R-CRT es vía dependiente de Ub. En nuestro laboratorio recientemente se demostró que la arginilación de CRT promueve un alto grado de dimerización. En este sentido, la sobre expresión de una mutante de CRT (C146A-CRT-EGFP) que impide la formación de homodímeros, muestra niveles menores de R-CRT respecto de los encontrados cuando se sobre expresa CRT de tipo salvaje (wt), indicando que la dimerización de RCRT es en parte responsable de su estabilización. Por último, se encontró que tanto la expresión citoplasmática de R-CRT como la inhibición del proteosoma incrementan los niveles de R-CRT en la superficie celular. En su conjunto, estos resultados indican que una vez que CRT alcanza el citoplasma, la misma puede ser degradada por el proteosoma a través de una vía independiente de Ub o puede ser modificada por la enzima Ate1 formando R-CRT, la cual también es susceptible a la degradación proteosomal pero por vía dependiente de Ub, mostrando una vida media más larga que la CRT no modificada. Por otra parte, la estabilización de CRT por medio de su arginilación y dimerización, le permitiría a CRT participar en diferentes funciones en el citoplasma e incluso en otros compartimientos subcelulares a los que podría acceder desde el citoplasma. Fil: Goitea, Víctor Enrique. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina. Fil: Hallak, Marta Elena. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina. Fil: Hallak, Marta Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Fil: Alvarez, Cecilia Inés. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Fil: Alvarez, Cecilia Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Fil: Pérez, Mariela Fernanda. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacología; Argentina. Fil: Pérez, Mariela Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina. Fil: Maccioni, Hugo José. Fernando Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina. Fil: Maccioni, Hugo José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Fil: Rossi, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Medicina Trasnacional; Argentina.

The tumor suppressor p53 is a transcription factor that coordinates the cellular response to several kinds of stress. p53 inactivation is an important step in tumor progression. Oligomerization of p53 is critical for its posttranslational modification and its ability to regulate the transcription of target genes necessary to inhibit tumor growth. Here we report that the HECT E3 ubiquitin ligase HERC2 interacts with p53. This interaction involves the CPH domain of HERC2 (a conserved domain within Cul7, PARC, and HERC2 proteins) and the last 43 amino acid residues of p53. Through this interaction, HERC2 regulates p53 activity. RNA interference experiments showed how HERC2 depletion reduces the transcriptional activity of p53 without affecting its stability. This regulation of p53 activity by HERC2 is independent of proteasome or MDM2 activity. Under these conditions, up-regulation of cell growth and increased focus formation were observed, showing the functional relevance of the HERC2-p53 interaction. This interaction was maintained after DNA damage caused by the chemotherapeutic drug bleomycin. In these stressed cells, p53 phosphorylation was not impaired by HERC2 knockdown. Interestingly, p53 mutations that affect its tetramerization domain disrupted the HERC2-p53 interaction, suggesting a role for HERC2 in p53 oligomerization. This regulatory role was shown using cross-linking assays. Thus, the inhibition of p53 activity after HERC2 depletion can be attributed to a reduction in p53 oligomerization. Ectopic expression of HERC2 (residues 2292-2923) confirmed these observations. Together, these results identify HERC2 as a novel regulator of p53 signaling.

UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains. In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon–intron structure. We also show that the human UEV1 gene is fused with the previously unknown gene Kua. In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins. In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua–UEV transcript, encoding a two-domain protein. Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs. Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua–UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua–UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua–UEV protein confers new biological properties to this regulator of variant polyubiquitination./n/n[Kua cDNAs isolated by RT-PCR and described in this paper have been deposited in the GenBank data library under accession nos. AF1155120 (H. sapiens) and AF152361 (D. melanogaster). Genomic clones containing UEV genes: S. cerevisiae, YGL087c (accession no. Z72609); S. pombe, c338 (accession no. AL023781); P. falciparum, MAL3P2 (accession no. AL034558); A. thaliana, F26F24 (accession no. AC005292); C. elegans, F39B2 (accession no. Z92834); D. melanogaster, AC014908; and H. sapiens, 1185N5 (accession no. AL034423). Accession numbers for Kua cDNAs in GenBank dbEST: M. musculus, AA7853; T. cruzi, AI612534. Other Kua-containing sequences: A. thaliana genomic clones F10M23 (accession no. AL035440), F19K23 (accession no. AC000375), and T20K9 (accession no. AC004786).

SCF (Skp1/Cul1/F‐box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F‐box protein, FBXO28 that controls MYC‐dependent transcription by non‐proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2‐mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F‐box mutant unable to support MYC ubiquitylation results in an impairment of MYC‐driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK‐FBXO28‐MYC axis as a potential molecular drug target in MYC‐driven cancers, including breast cancer.

El marcaje de proteínas con ubiquitina, conocido como ubiquitinación, cumple diferentes funciones que incluyen la regulación de varios procesos celulares, tales como: la degradación de proteínas por medio del proteosoma, la reparación del ADN, la señalización mediada por receptores de membrana, y la endocitosis, entre otras (1). Las moléculas de ubiquitina pueden ser removidas de sus sustratos gracias a la acción de un gran grupo de proteasas, llamadas enzimas deubiquitinizantes (DUBs) (2). Las DUBs son esenciales para la manutención de la homeostasis de la ubiquitina y para la regulación del estado de ubiquitinación de diferentes sustratos. El gran número y la diversidad de DUBs descritas refleja tanto su especificidad como su utilización para regular un amplio espectro de sustratos y vías celulares. Aunque muchas DUBs han sido estudiadas a profundidad, actualmente se desconocen los sustratos y las funciones biológicas de la mayoría de ellas. En este trabajo se investigaron las funciones de las DUBs: USP19, USP4 y UCH-L1. Utilizando varias técnicas de biología molecular y celular se encontró que: i) USP19 es regulada por las ubiquitin ligasas SIAH1 y SIAH2 ii) USP19 es importante para regular HIF-1α, un factor de transcripción clave en la respuesta celular a hipoxia, iii) USP4 interactúa con el proteosoma, iv) La quimera mCherry-UCH-L1 reproduce parcialmente los fenotipos que nuestro grupo ha descrito previamente al usar otros constructos de la misma enzima, y v) UCH-L1 promueve la internalización de la bacteria Yersinia pseudotuberculosis. The conjugation of ubiquitin to proteins, known as ubiquitination, has different cellular functions; they include targeting proteins for degradation by the proteasome, regulation of DNA damage repair signaling, membrane receptor signaling and endocytosis (1). The ubiquitin moieties can be de-conjugated from their substrates or other ubiquitin moieties by a large group of proteases named deubiquitinating enzymes (DUBs) (2). DUBs are essential for the maintenance of the ubiquitin homeostasis in the cell and regulation of the ubiquitination status of the different substrates. The diversity of these proteases hints on their specificity for certain targets and participation in particular cellular pathways. Although several DUBs have been thoroughly studied, at present the targets and physiological roles of most of them remain unknown. Here, we studied the functional roles of the ubiquitin specific protease 19 (USP19), USP4 and the ubiquitin C-terminal hydrolase (UCH-L1), using several cellular and molecular techniques. We found that, i) USP19 can be regulated by SIAH ubiquitin ligases, ii) USP19 is important for controlling the key regulator of response to hypoxia, HIF-1α, iii) USP4 is a proteasome-interacting DUB, iv) an mCherry-UCH-L1 chimera reproduces only partially previous phenotypes described for UCH-L1, and v) UCH-L1 promotes Yersinia pseudotuberculosis internalization. ERACOL Universidad del Rosario Karolinska Institutet

Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together to control sterol biosynthesis at different levels and thereby allowing independent regulation of multiple products of the mevalonate pathway. DOI: http://dx.doi.org/10.7554/eLife.00953.001, eLife digest All cells are enclosed by a membrane that is made up of fatty molecules called lipids and is studded with proteins. This membrane allows cells to detect and react to outside events. Since external conditions, such as temperature, can vary dramatically, membranes need to be able to adjust their properties. For example, lipids become more fluid as the temperature rises, so membranes respond to heat stress by incorporating molecules called sterols to increase their rigidity. In fact, sterols have profound effects on membrane properties and are essential to regulate a number of cellular processes. But high levels of sterols can become toxic, so it is essential that they are carefully controlled. Sterols, such as ergosterol in yeast or cholesterol in mammals, are synthesized in a tightly regulated multi-step process; some of the early steps in sterol production also make common building blocks for other key molecules in the cell. A mechanism to control sterol levels is the regulated destruction of an enzyme that carries out an early step of their synthesis. This occurs via one branch of the ER-associated degradation (ERAD) pathway, which also destroys non-functional proteins. Now, Foresti et al. have found that sterol synthesis is also regulated by another branch of the ERAD pathway. This second control point, which occurs later in the biosynthetic process, allows cells to regulate sterol levels independent of the other products of the pathway that are derived from the same preliminary compounds. In yeast, the two ERAD branches are directed by Hrd1 and Doa10. These are both ubiquitin ligases—proteins that attach a tag called ubiquitin to other proteins, thus labeling them for recycling by the proteasome (essentially a waste-disposal complex in the cell). To identify the proteins that are tagged by Doa10, Foresti et al. compared protein levels in strains lacking Doa10 with those in wild type yeast. Unexpectedly, the enzyme Erg1, which helps to synthesize ergosterol, was more abundant in cells lacking Doa10. Foresti et al. found that Doa10 tagged Erg1 for destruction when levels of the building blocks of ergosterol rose inside the cell. These ergosterol intermediates are toxic to yeast, which converts them into less harmful molecules known as sterol esters using the proteins Are1/2. When the DOA10 or ARE1/2 genes were deleted, these intermediates were more abundant; strikingly, they became even more prevalent when all three genes were knocked out in the same strain. In contrast, blocking the other ERAD branch by deleting HRD1 did not cause ergosterol intermediates to accumulate, nor did it exacerbate the effects of ARE1/2 knockout. When combined with previous findings, these results provide evidence that the different branches of the ERAD pathway regulate ergosterol synthesis at distinct steps. The same mechanism is observed in human cells when high levels of cholesterol are detected. By identifying parallel routes to control sterol levels, this work reinforces the importance of membrane integrity to life. DOI: http://dx.doi.org/10.7554/eLife.00953.002

Dissertação de mestrado em Investigação Biomédica (Oncobiologia), apresentada à Faculdade de Medicina da Universidade de Coimbra. Fundação para a Ciência e Tecnologia (FCT) no âmbito do projecto "Unravelling the molecular events of gap junction remodelling in ischemic heart" - PTDC/SAU-ORG/119296/2010 e PEST-C/SAU/UI3282/2011-COMPETE

Intercambiar la especifidad de las isoformas de SUMO1 y SUMO2/3 para SENP6/SENP7 SENP6 y SENP7 son los miembros más divergentes de la familia de proteasas SENP y los únicos miembros que llevan cuatro inserciónes o “loops” localizadas en su dominio catalítico. Al sobreponer el dominio catalítico de SENP7 sobre el complejo SENP2-SUMO podemos ver una posible interfaz entre el Loop1 de SENP7 y SUMO. También identificamos diferentes residuos de SUMO1 y SUMO2 que podrían formar parte de la interfaz. Diseñamos una serie de mutantes de SUMO1 y SUMO2 donde se intercambian entre ellos los residuos que forman parte de la interfaz. De esta manera fuimos capaces de intercambiar la actividad proteolítica de SENP6 y SENP7 hacia estos substratos. El Loop1 de SENP6 y SENP7 es responsable de la especificidad de la interfaz entre los SUMOs y las SENP6/7 Diseñamos una serie de mutantes del Loop1 de SENP7 dentro de la posible interfaz entre el Loop1 de SENP7 y SUMO para determinar los papeles estructurales y funcionales de los residuos que están dentro de esta región. Los mutantes de SENP6/7 sin el Loop1 perdían gran parte de su actividad, esta se podía recuperar un poco al reemplazar las cuatro prolinas del Loop1 por glicinas. De esta manera probamos que el Loop1 tiene un papel por lo menos estructural en el reconocimiento de SUMO. También identificamos el residuo Lys691 del Loop1 de SENP7 como elemento indispensable en la actividad de la enzima ya que al mutar este residuo por un aspártico disminuye la actividad para sustratos con SUMO2. Por otra parte el residuo Asp71 de SUMO2 es uno de los residuos propuestos para el reconocimiento de SUMO2/3. Cuando mutamos este residuo a lisina, vemos una bajada en la actividad de SENP7. Este hecho sugiere que Asp71 de SUMO2 está interaccionando con Loop1 y quizas con la Lys691, y que la bajada en la actividad es causada por una repulsión de cargas. Por otro lado con el objectivo de estudiar el efecto de Loop1 en la desconjugación de especies SUMOyladas, insertamos los ocho residuos de SENP6 Loop en el dominio catalítico de SENP2. Esta inserción provoca un aumento de la actividad sustancial de SENP2 contra diSUMO en comparación con la forma wild type. Complejos con substratos Para ver si SENP6 es capaz de formar algún complejo estable, producimos cantidades en miligramos de los mutantes inactivos de Δ3SENP6C1030S y de Δ2Δ3SENP6C1030S. Incubamos cada proteasa con los precursors de SUMO, con RanGAP1-SUMO2 y con diSUMO2. De todos los substratos que probamos, diSUMO2 fue el único que podía formar un complejo estable con Δ3SENP6CS y Δ2Δ3SENP6CS por copurificación en una columna de gel fitración. Con Δ2SENP6CS no pudimos formar ningún complejo. Con esta información llegamos a la conclusión que el Loop3 de SENP6 reduce, quizá por razones de entropía, la capacidad de SENP6 de formar un complejo estable con diSUMO2. Caracterización del Loop3 de SENP6 Para descifrar el papel que este loop tiene en el contexto de la proteasa, producimos y purificamos los 185 residuos de Loop3 de SENP6. Análysis 1-H 1-D RMN mostraba una falta de estructura terciaria dentro del loop, pero proteólisis limitada y espectroscopia de masas mostraban un fragmento estable de cerca de 11kDa. Dicroísmo circular y FTIR tambien sugieren la presencia de algunos elementos de estructura secundaria. Globalmente, la caracterización biofísica del Loop3 de SENP6 muestra que el loop es una proteína desestructurada., Swapping the SUMO Isoform Specificity of SENP6/7 SENP6 and SENP7 are the most divergent members in the SENP family of proteases and they are the only members that bear four loop insertions dispersed throughout their catalytic domains. The superposition of the SENP7 catalytic domain with the SENP2-SUMO complex revealed a tentative SENP7 Loop1-SUMO interface and upon further inspection, distinct residues on SUMO1 and SUMO2 were identified at the interface. A series of mutants were constructed bearing characteristics of both SUMOs and by swapping the residues from SUMO1 to SUMO2 and vice versus we were able to both decrease and increase the activity of the SENP6 and SENP7 toward these substrates. Loop1 SENP6/7 Is Responsible For SUMO Interface Specificity In addition to mutations on SUMO we constructed a series of mutations on SENP7-Loop1 within the tentative SENP7-Loop1-SUMO interface to determine the structural and functional roles of the residues that reside within this region. We were able to recover some of the activity lost by the removal of Loop1 by replacing the four prolines of Loop1 with glycines proving that Loop1 plays at least a structural role in SUMO recognition. We also identified Lys691 of Loop1 in SENP7 as indispensible to the activity of the enzyme. D71 is one of the residues proposed to confer SUMO2/3 specificity in the previous swapping experiments. We mutated this residue in diSUMO2 and saw a decrease in activity of SENP7. This could be explained by our theory that this region on SUMO is interacting with Loop1, more specifically K691, and the decrease in activity was caused by a charge clash between SUMO2 and SENP7 Loop1. To further show the utility of Loop1 in deconjugation of multi-SUMOylated species we inserted the eight residues of SENP6 Loop1 into SENP2. We saw an overall increase in activity of SENP2 against diSUMO2 but not against any other substrate tested. Complexes With Substrates In order to see if SENP6 was able to form any stable complexes in solution, we produced milligram amounts of Δ3SENP6CS and Δ2Δ3SENP6CS (the two constructs of the protein that showed both good yields in protein production and high performance in activity assay). We incubated each protease with SUMO precursors, RanGAP1-SUMO2 and diSUMO2 substrates. Of all the substrates tested, only diSUMO was able to form a stable complex with Δ3SENP6CS and Δ2Δ3SENP6CS. Δ2SENP6CS was also tested but there was no indication of any complex formation, leading to the hypothesis that SENP6 Loop3 was impeding, perhaps entropically, the ability of SENP6 to form a stable complex with diSUMO2. SENP6 Loop3 Characterization Loop3 takes up roughly 40% and 20% of the catalytic domains of SENP6 and SENP7 respectively. In our loop deletion experiments we saw an overall increase in the activity when Loop3 was not present and removal of Loop3 proved vital to the ability of the enzyme to form a stable complex with diSUMO2. In order to try to decipher what role this loop plays in the context of the protease, we isolated the 184 insert from SENP6 and produced and purified the protein. 1-H 1-D NMR pointed to an overall lack of tertiary structure within the loop but limited proteolysis and mass spectrometry analyses showed a stable fragment of around 11kDa. Further circular dichroism and Fourier Transform Infrared Spectroscopy suggested the presence of some secondary structural elements but overall characterization of SENP6 Loop3 showed a mainly unstructured loop.

Background: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the dysfunctional Cl - channel in Cystic Fibrosis, undergoes complex biosynthesis at the endoplasmic reticulum involving several molecular chaperones including Hsp70 and many co-chaperones. Bcl-2associated athanogenes (BAGs) constitute a protein family sharing an Hsc70-binding domain. BAG-1 possesses an ubiquitin-like domain (Ub-LD) responsible for proteasomal association and for promoting substrate release from Hsc70/Hsp70 in vitro by accelerating the chaperone ATP/ADP exchange rate. Methods: Herein, we studied the in vivo effect of BAG-1 on the turnover and processing of wild type (wt)- and F508del-CFTR, the most frequent mutation in CF patients. Results: Results show that BAG-1 associates with both wt- and F508del-CFTR (in higher yields with the latter) through its Ub-LD and independently of Hsc70. Moreover, the immature form of F508del-CFTR (but not of wt-CFTR) is stabilized by BAG-1 overexpression, albeit in a cell-type specific way, without detectable maturation. Data also show that BAG-1 and the proteasome inhibitor ALLN are not additive on stabilizing F508del-CFTR and this effect depends on BAG-1 Ub-LD. Moreover, under BAG-1 overexpression, a reduction in ubiquitinylated-CFTR occurs suggesting that BAG-1 competes with Ub. Conclusion: Overall, data are compatible with a mechanism in which BAG-1 stabilizes F508del-CFTR by direct binding, probably competing out ubiquitin to partially avoid its proteasomal degradation.

La caña de azúcar (Saccharum spp.) pertenece a la familia Poaceae y representa uno de los cultivos de mayor importancia económica debido a su alta acumulación de sacarosa, contabilizando cerca del 70% de la producción mundial, alta eficiencia en producción de biomasa, producción de biocombustibles, de primera y segunda generación, y cogeneración de energía. Se cultiva en áreas tropicales y subtropicales del mundo. En Colombia el cultivo de caña para la producción de azúcar ocupa un área de 218.311 ha produciendo en promedio 114.6 toneladas de caña por hectárea. El proceso de transformación genética en plantas permite introducir genes asociados a características que mejoren la calidad de un cultivo. Los promotores hacen parte de un gen y tiene la función de acoplar la maquinaria transcripcional para asegurar su expresión. Actualmente el proceso de transformación genética en caña de azúcar no dispone de un promotor obtenido a partir del genoma de la caña, estable a través del tiempo. Por lo tanto el objetivo de éste estudio fue aislar un promotor propio del genoma de la caña y evaluarlo en la inducción del gen reportero uidA (Gus). A partir del ADN de la variedad CC 01-678 se amplificaron dos fragmentos con tamaños de 1282 pares de bases (pb) (AY699) y 1303 pb (AY700), correspondientes al promotor del gen sacarosa sintetasa (SuSy). Por otro lado el promotor Ubiquitina de maíz, con un tamaño de 2000 pb, se usó como control y se obtuvo por medio de PCR a partir del plásmido pUbi/1SD. Tres construcciones genéticas denominadas pCAY699, pC-AY700 y pC-Ubi se obtuvieron reemplazando el promotor CaMV35S presente en el vector pCambia 1305.2 por los dos fragmentos aislados del gen SuSy así como por el promotor Ubiquitina. El análisis histoquímico de expresión transitoria del gen reportero uidA, dirigida por los diferentes promotores, se realizó utilizando callo embriogénico de la variedad CC 84-75. La mayor expresión se observó con el promotor Ubiquitina, contabilizando en promedio 406 ± 9.77 puntos azules, mientras que con el promotor CaMV35S y AY700 sólo se observaron 46 ± 3.48 y 4 ± 1.33 respectivamente //Abstract: / Sugarcane (Saccharum spp.) belongs to the family Poaceae and is one of the most economically important crops because of its high accumulation of sucrose, accounting for about 70% of global output, high efficiency in biomass production, production biofuels, of first and second generation, and energy cogeneration. It is cultivated in tropical and subtropical countries around the world. In Colombia, the cultivation of sugarcane for sugar production occupies an area of 218,311 ha, produced an average of 114.6 tonnes of cane per hectare. The process of genetic transformation in plants allows the introduction of genes associated with characteristics that improve the quality of a crop. The promoters are part of a gene and have the function of coupling the transcriptional machinery to ensure its expression. Currently the genetic transformation of sugarcane does not have a promoter obtained from the sugarcane genome, stable over time. Therefore, the objective of this study was to isolate a promoter from the sugarcane genome and evaluate the induction of the reporter gene uidA (Gus). From the DNA of the sugarcane variety CC 01-678, two fragments with sizes of 1282 base pairs (bp) (AY699) and 1303 bp (AY700) were amplified from the sucrose synthase gene promoter (SuSy). Furthermore, maize Ubiquitin promoter with a size of 2000 bp, was used as control and was obtained by PCR from plasmid pUbi/1SD. Three genetic constructs called pC-AY699, pC-AY700 and pC-Ubi, were obtained by replacing the CaMV35S promoter present in the vector pCambia 1305.2 by each of the two fragments isolated from the SuSy gene as well as the ubiquitin promoter. Histochemical analysis of transient expression of uidA reporter gene, directed by the different promoters, was performed using embryogenic callus of variety CC 84-75. The highest expression was observed using the ubiquitin promoter, accounting on average 406 ± 9.77 blue dots, while using the CaMV35S and AY700 only 46 ± 3.48 and 4 ± 1.33 blue dots was observed respectively. Maestría

L'estudi a nivell molecular de la ruta de senyalització activada per TGF-B té un impacte notable en el panorama actual donada la seva implicació en processos autoimmunitaris i carcinogènics. D'altra banda, l'elucidació a nivell estructural dels mecanismes moleculars que permeten a les ubiquitin lligases de tipus E3 marcar específicament llurs dianes per a la degradació proteosòmica - entre d'altres - resulta fonamental donada la seva importància pel que fa al control sobre el turnover proteic a nivell intracel•lular. En aquest projecte es pretén elucidar els mecanismes d'activació i catlàlisi de les ubiquitin lligases E3 tipus HECT Smurf1 i Nedd4L al mateix temps que se n'estudia la implicació en la regulació dels agents missatgers de la ruta del TGF-B. Així, la tasca es divideix en tres sub-projectes els quals se centren en a) l'estudi de la interacció d'aquestes lligases amb llurs dianes; b) l'elucidació del mecanisme d'activació i c) del de catàlisi d'aquests enzims. Per tal d'assolir aquests objectius ens servim principalment dels avantatges que ens aporta la Ressonància Magnètica Nuclear i altres tècniques biofísiques, principalment la ITC. Durant el temps que he gaudit de la beca FI, m'he centrat principalment en la preparació de pèptids per SPPS, llur purificació per HPLC i caracterització per MS i RMN. Aquests pèptids representen diferents patrons de fosforilació de certes dianes de les lligases esmentades, de manera que han estat emprats per a l'estudi d'interaccions proteïna-proteïna per ITC i RMN. M'he iniciat doncs en l'ús d'aquestes tècniques. D'altra banda, també he preparat mostres protèiques mitjançant l'ús de sistemes d'expressió bacterians basats en E.coli, incloent l'amplificació i clonació del gen que codifica per la proteïna d'interés així com la seva expressió, purificació i caracterització per MS i RMN. The study at a molecular level of the TGF-B-activated signalling pathway has got a relevant impact within the current panorama given its implications in a variety of autoimmune and carcinogenic disfunctions. On the other side, the elucidation at a structural level of the molecular mechanisms allowing the type E3 ubiquitin ligases to specifically label their targets for proteosomal degradation - among others - is crucial given its relevance regarding the control of the intracell protein turnover. The current study intends to elucidate the activation and catalysis mechanisms of the HECT-type E3 ubiquitin ligases Smurf1 and Nedd4L while their implication in the regulation of the TGF-B related messenger agents is studied as well. Therefore, the work is divided into three sub-projects fucused on a) the study of the interaction of these ligases with their targets; b) the elucidation of the activation and c) catalysis mechanisms of these enzymes. In order to do that we use mainly the advantages provided by Nuclear Magnetic Resonance and other biophysical techniques such as ITC. During the time I've been a FI fellow I have mainly focused in the preparation of peptides by SPPS, their purification using HPLC and characterization by MS and NMR. These peptides represent a variety of phosphorilation patterns of certain ligases' targets, and so they have been used for the study of protein-protein interactions by ITC and NMR. I have therefore been introduced in the use of these techniques. On the other side, I have also prepared protin samples using bacterial expression systems based on E.coli, including the amplification and clonning of the gene codifying the protein of interest and its expression, purification and characterization by MS and NMR.

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2012 Submitted by Lurdes Saramago (lurdes.saramago@fc.ul.pt) on 2013-05-03T17:48:21Z No. of bitstreams: 1 ulfc102758_tm_sara_canato.pdf: 2478570 bytes, checksum: cd707f6b88f697ce503d5066448f7ee8 (MD5) Made available in DSpace on 2013-05-03T17:51:29Z (GMT). No. of bitstreams: 1 ulfc102758_tm_sara_canato.pdf: 2478570 bytes, checksum: cd707f6b88f697ce503d5066448f7ee8 (MD5) Previous issue date: 2012

Heterozygous mutations in the melanocortin-4 receptor (MC4R) gene represent the most frequent cause of monogenic obesity in humans. MC4R mutation analysis in a cohort of 77 children with morbid obesity identified previously unreported heterozygous mutations (P272L, N74I) in two patients inherited from their obese mothers. A rare polymorphism (I251L, allelic frequency: 1/100) reported to protect against obesity was found in another obese patient. When expressed in neuronal cells, the cell surface abundance of wild-type MC4R and of the N74I and I251L variants and the cAMP generated by these receptors in response to exposure to the agonist, α-MSH, were not different. Conversely, MC4R P272L was retained in the endoplasmic reticulum and had reduced cell surface expression and signaling (by ≈3-fold). The chemical chaperone PBA, which promotes protein folding of wild-type MC4R, had minimal effects on the distribution and signaling of the P272L variant. In contrast, incubation with UBE-41, a specific inhibitor of ubiquitin activating enzyme E1, inhibited ubiquitination of MC4R P272L and increased its cell surface expression and signaling to similar levels as wild-type MC4R. UBE41 had much less profound effects on MC4R I316S, another obesity-linked MC4R variant trapped in the ER. These data suggest that P272L is retained in the ER by a propensity to be ubiquitinated in the face of correct folding, which is only minimally shared by MC4R I316S. Thus, studies that combine clinical screening of obese patients and investigation of the functional defects of the obesity-linked MC4R variants can identify specific ways to correct these defects and are the first steps towards personalized medicine, This work has been funded by Fondo de Investigación Sanitaria (PI09/91060, PI10/02512, PI01/00747), CIBERobn Instituto de Salud Carlos III (ISCIII), Fundación Mutua Madrileña (AP2561/2008), Fundación Endocrinología y Nutrición, the National Institutes of Health (R01DK080424 to GB), and the Arkansas Tobacco Settlement (to GB). CS-J and GAM-M were recipients of fellowships from ISCIII (FI08/00365 and CM05/00100, respectively)

Universidade Federal de Minas Gerais The quadriceps muscle weakness and atrophy after the anterior cruciate ligament (ACL) rupture, is present before and after its reconstruction, and are important limitations that restrict the return of subjects to activities of daily living and sports. The combination of reduced synthesis and increased levels of protein degradation results in a rapid loss of muscle protein. The rapid muscle proteolysis occurs in several animal models and in different conditions in humans, and are mainly associated to the ubiquitn proteasome system (UPS). Neuromuscular mechanisms are associated with atrophy and weakness of the quadriceps muscle after ACL rupture and reconstruction, but the molecular pathways involved in this process are unknown. The identification of these pathways may facilitate the establishment of therapeutic strategies to alleviate the muscle deficit. Thus, the objective of this thesis was to evaluate the effect of ACL transection of rats in the expression of genes of UPS (MuRF-1, Atrogina-1), as well as the expression of myostatin (involved in control of muscle mass) by the Real Time PCR (polymerase chain reaction) technique. It was evaluated the content of ubiquitinated proteins by Western blotting and the cross-sectional area of muscle fibers (CSA) of the vastus medialis (VM), rectus femoris (RF) and vastus lateralis (VL) of the quadriceps. Elevated Atrogin-1, MuRF-1 and myostatin mRNA levels were observed after ACL transection in all muscles assessed, as the reduction of CSA in VM and VL muscles. The results of this thesis contributed to the understanding of the mechanisms of quadriceps muscle atrophy after ACL transection. A atrofia e fraqueza do quadríceps após a ruptura do ligamento cruzado anterior (LCA), presentes antes e após sua reconstrução, são importantes limitações que restringem o retorno dos indivíduos às atividades da vida diária e desportivas. A rápida proteólise muscular que ocorre nos diversos modelos animais e em diferentes condições em humanos se deve, principalmente, ao sistema ubiquitna proteassoma (UPS). Mecanismos neuromusculares são associados à atrofia e fraqueza do quadríceps após a ruptura e reconstrução do LCA, porém as vias moleculares envolvidas nesse processo são desconhecidas. A identificação dessas vias pode favorecer o estabelecimento de estratégias terapêuticas para amenizar o déficit muscular. Assim, o objetivo dessa tese foi avaliar o efeito da transecção do LCA de ratos na expressão dos genes pertencentes ao UPS (MuRF-1, a Atrogina-1), e também da Miostatina (envolvida no controle da massa muscular) pela técnica de PCR (reação em cadeia da polimerase) em tempo real no quadriceps. Avaliou-se ainda o conteúdo de proteínas ubiquitinadas por Western Blotting e a área de secção transversa das fibras musculares (AST) dos músculos Vasto Medial (VM), Reto Femoral (RF) e Vasto Lateral (VL). Níveis elevados de Atrogina-1, MuRF-1 e Miostatina ocorreram após transecção do LCA em todos os músculos avaliados assim como a redução da AST nos músculos VM e VL. Os resultados desta tese contribuiram para a compreensão dos mecanismos de atrofia do quadríceps após a transecção do LCA.

Previous studies have demonstrated the existence and expression of genes essential to the process of protein ubiquitination in Giardia intestinalis, indicating that the ubiquitin-proteasome system may be involved in the degradation of proteins during its life cycle of the parasite. In this study, purification of ubiquitin was conducted from protein extracts of G. intestinalis trophozoites. Then, an anti-ubiquitin specific antibody was obtained to standardize an assay for the detection and evaluation of ubiquitination patterns. Finally, HSP90 was identified as an ubiquitinated protein in this protozoan. This post-translational modification could have regulatory effects associated with the functionality of the protein or its turnover to regulate key molecular events during the parasite’s life cycle. Estudios previos han demostrado la existencia y expresión de genes esenciales para el proceso de ubiquitinación de proteínas en Giardia intestinalis, indicando que el sistema ubiquitina-proteosoma puede estar involucrado en el proceso de degradación de proteínas de este parásito durante su ciclo de vida. En el presente trabajo se realizó la purificación de ubiquitina a partir de extractos proteicos de trofozoítos de G. intestinalis, se produjo un anticuerpo anti-ubiquitina específico que permitió la estandarización de un ensayo para la detección y evaluación de los patrones de ubiquitinación, y se identificó la HSP90 como una proteína ubiquitinada en este protozoario. Esta modificación post-transduccional puede tener efectos regulatorios asociados con la funcionalidad de la proteína o con el recambio para regular eventos moleculares claves durante el ciclo de vida del parásito. Estudos anteriores demonstraram a existência e expressão de genes essenciais para o processo de ubiquitinação de proteínas em Giardia intestinalis, indicando que o sistema ubiquitina-proteassoma podem estar envolvidos na degradação de proteínas do parasita durante seu ciclo de vida. Neste trabalho, foi realizada a purificação da proteína ubiquitina de extratos de trofozoítos de G. intestinalis, foi produzido um antiicorpo anti-ubiquitina específico que permitiu a padronização de um ensaio para a detecção e avaliação de padrões de ubiquitinação e foi identificado a HSP90 como uma proteína ubiquitinada no protozoário. Esta modificação pós-transducional pode ter efeitos regulamentares associados com a funcionalidade das proteínas ou com o a substituição para regular eventos moleculares importantes durante o ciclo de vida do parasita.

Estudiamos la ubiquitinación de proteínas en el eucariota tempranamente divergente Giardia intestinalis con el propósito de hacer inferencias sobre los papeles tempranos o primitivos de la Ub y la ubiquitinación en el linaje eucariota. Encontramos que el gen de la enzima activadora de Ub – E1 es esencial y es regulado durante la enquistación, además interesantemente la proteína se localiza en la pared de los quistes. En cuanto a la expresión de la Ub y los patrones de proteínas ubiquitinadas determinamos que hay diferencias entre los patrones de proteínas ubiquitinadas citoplasmáticas y nucleares. Durante la enquistación la ubiquitinación se ve drásticamente afectada, observándose una disminución considerable en la cantidad de proteínas ubiquitinadas. La localización de especies de Ub mostró presencia de proteínas mono y poli-ubiquitinadas en el citoplasma, y una fuerte señal de proteínas mono-ubiquitinadas en los núcleos, además señales en la periferia sugieren asociación de ubiquitinación con vacuolas periféricas similares a lisosomas (PVs), y localización en la pared del quiste sugiere una asociación con esta matriza. Identificamos como posibles proteínas efectoras (proteínas de unión a Ub - UBPs) a la enzima E1, dos proteínas de la subunidad reguladora del proteosoma (Rpn2 y Rpn6), dos enzimas DUBs (Ubp14 y Doa4) y dos chaperonas (Bip y Hsp90-NT). Identificamos proteínas ubiquitinadas en trofozoítos y durante la enquistación. De acuerdo con las proteínas identificadas proponemos que la ubiquitinación en G.intestinalis participa en una amplia gama de procesos celulares entre ellos: transcripción y traducción, regulación de la expresión génica a través de la modificación de histonas, endocitosis y tráfico intracelular de proteínas, control del ciclo celular, dinámica del citoesqueleto, plegamiento de proteínas y ERAD, y en la biosíntesis de la pared del quiste entre otros. Nuestros resultados sugieren que la ubiquitinación es indispensable en el desarrollo y diferenciación de G.intestinalis. Además que más que en procesos asociados con degradación proteosomal la ubiquitinación tiene un papel en procesos no degradativos indispensables en el desarrollo de este eucariota primitivo. / Abstract. Here we study the protein Ubiquitination in the early-branching eukaryote Giardia intestinalis as a model to do inferences about the primitive or early roles of Ubiquitin and ubiquitination in the eukaryote linage. We found that ubiquitin activating enzyme (E1) gene is esential and is regulated during encystation, besides the protein localize in the cyst wall. About ubiquitina expresión and ubiquitinated proteins pattern we found that are diferences in the citiplasmatic and nuclear patterns. Ubiquitination is regulated during encystation, we observed a drastically decrease in ubiquitinated proteins when this diferentation process is induced. Localization of ubiquitin species show presence of mono- and poli-ubiquitinated proteins in citoplasm, and strong signal of mono-ubiquitinated proteins in nucleus, a peipheral localization suggets asociation between poli-ubiquitinated proteins and lysosoma-like peripheral vacuoles (PVs), morover a relationship with the cyst wall. We identify ubiquitin binding proteins: the E1 enzyme, the proteasome subunits Rpn2 and Rpn6, the DUBs Ubp14 and Doa4, and the chaperons Bip and Hsp90-NT. We identify ubiquitinated proteins in trophozoites and during encystation, these proteins suggest that ubiquitination is involven in several cellular processes in G.intestinalis: transcription and translation, gene expression regulation, endocytosis, cell cycle control, cytoskeleton dynamics, folding of proteins and ERAD, and the cyst wall bioshyntesis. Our results suggest that ubiquitination is important in G.intestinalis’s development and diferentiation. Moreover, the ubiquitination roles in non-degradative process are the indispensable roles in G.intestinalis’s biology. Doctorado

El interés en el estudio del proceso de envejecimiento cerebral y los cambios comportamentales relacionados con la edad en caninos y felinos geriátricos ha aumentado en la última década (Mentzel, 2005a; Ingram & Williams, 2010). Las alteraciones del comportamiento canino que responden a cambios fisiopatológicos relacionados con la edad y que involucran distintas esferas conductuales y cognitivas, se engloban bajo la denominación de Síndrome de Disfunción Cognoscitiva (SDC) (Mentzel, 2005b). El cual fue descrito en 1997 y se observó en perros de más de 9 años (Ingram & Williams). El Síndrome de Disfunción Cognoscitivo (SDC) es también llamado Alzheimer canino por las similitudes histopatológicas cerebrales y conductuales relacionadas. En el presente estudio, los caninos geriátricos presentaron los mismos depósitos proteicos anormales presentes en la enfermedad de Alzheimer, como las placas seniles, los cuerpos ubiquitinados y la angiopatía amiloide, pero no presentaron los ovillos neurofibrilares, los cuales son los responsables de la demencia que se padece en la enfermedad. Las únicas herramientas de evaluación prácticas en clínica para esta patología son las encuestas y formularios relacionados con aspectos comportamentales. Además existen exámenes diagnósticos postmortem que permiten identificar de una manera eficaz la presencia de elementos anormales involucrados en la neuropatología, uno de ellos es la inmunohistoquímica. En este estudio se utilizaron los anticuerpos monoclonales de uso humano anti-b-amiloide, anti-proteína tau y anti-ubiquitina en muestras de cerebros de perros mayores de 10 años. El modelo canino hoy en día está constituyendo un significado indispensable para el estudio de los procesos de neurodegeneración porque ha permitido un acercamiento a la teoría del problema desde nuevas perspectivas (Dimakopoulos & Mayer, 2002). The interest in the study of the process of cerebral aging and the changes in the behaviour related to age in geriatric canine and feline has increased in the last decade (Mentzel, 2005a; Ingram & Williams, 2010). The alterations of the canine behaviour that respond to physiopathological changes related to the age and which involve different conducts and cognitive spheres are included under the denomination of Canine Cognitive Dysfunction Syndrome (CDS) called canine Alzheimer, considering the cerebral and histopathologic behavioural similarities. In the present study the geriatric patients displayed the same abnormal protein deposits present in Alzheimer disease, such as senile plaques ubiquitinate bodies and the amyloid angiopathy. However they did not display the balls of neurofibrillary tangles which are characteristic of the disease. The only practical tools of evaluation in clinic for this pathology are the surveys and forms related to behavioural aspects. In addition there are post mortem exams such as those in inmunohistochemistry, that allow an effective identification of abnormal elements present in the neuropathology. In this study the monoclonal antibodies were used anti-b-amyloid, anti-protein tau and anti-ubiquitin in samples of brains in dogs over 10 years of age. The canine model constitutes an indispensable meaning for the study of the neurodegeneration processes because it has allowed an approach to the theory of the problem from a new perspective (Dimakopoulos & Mayer, 2002).

A conjugação de SUMO aos substatos alvo ocorre através de um mecanismo análogo ao da ubiquitina, pela ação sequencial de três classes de enzimas: E1 ativadora (Aos1-Uba2), E2 conjugadora (Ubc9) e E3 ligases (PIAS, Polycomb2 e RanBP2). SUMO é sintetizada como um precursor inativo, que requer processamento por proteases específicas (SENPs). A conjugação de SUMO é uma modificação reversível e transitória. As mesmas enzimas que convertem SUMO em sua forma madura também catalisam a clivagem dos seus substratos. A reconstituição da via de sumorilação em Schistosoma mansoni, baseado em busca por homologia utilizando banco de dados disponíveis, mostra a existência de 9 genes envolvidos com esta via: dois genes para SUMO (Smsmt3b/c), um para E1 (Smaos1-uba2), um para E2 (Smubc9), dois para E3 (Smpias e Smranbp2) e dois para SENPs (Smsenp1/7). Neste trabalho, os níveis de expressão destes genes foram quantificados utilizando o método de qRT-PCR e RNA total obtidos a partir de cercária, verme adulto e esquistossômulos de 3,5 horas a 7 dias de cultivo in vitro (MTS). Os resultados evidenciaram um padrão de expressão gênica diferencial para todos os genes analisados. Vale ressaltar que os níveis de transcritos foram 3 vezes maiores em MTS-3,5h quando comparado com cercária e verme adulto. Os níveis de SmUbc9 foram analisados por Western blot, utilizando anticorpos policlonais específicos para a proteína de S. mansoni produzidos utilizando a proteína recombinante. Os resultados sugerem níveis equivalentes da SmUbc9 nos estágios analisados. O perfil de substratos sumorilados foi determinado utilizando a técnica de Western blot usando anti-SUMO. Foi observado um perfil estágio-específico de conjugados sumorilados e um aumento nos estágios iniciais do desenvolvimento dos esquistossômulos. Estes dados demonstram que os constituintes da via de sumorilação são diferencialmente expressos, sugerindo um perfil estágio-específico durante a transição cercária-esquistossômulo. Considerando que esta transição não é controlada por uma síntese real de proteínas, esses resultados em conjunto, sugerem que a via de modificação pós-traducional dependente de SUMO pode regular processos biológicos importantes durante esse período do ciclo biológico do parasito. Attachment of SUMO to target proteins occur through by similar mechanism to ubiquitination, with sequential action of three enzymes: E1 activating (Aos1-Uba2), E2 conjugation (Ubc9) and E3 ligases (PIAS, Polycomb2 and RanBP2). SUMO is synthesized as inactive precursors that require processing by specific proteases (SENPs). SUMO attachment is a reversible and transient modification. The same enzymes that convert SUMO to its mature form also catalyse the cleavage from their substrates. Reconstitution of SUMOylation pathway in Schistosoma mansoni, based on homology search using avaible databases, shown the existence of nine genes related with SUMOylation: two genes to SUMO (Smsmt3b/c), one to E1 (Smaos1-uba2), one to E2 (Smubc9), two to E3 (Smpias e Smranbp2) and two to SENPs (Smsenp1/7). In this work, the expression levels of these genes was quantified by qRT-PCR and total RNA obtained from cercariae, adult worm and mechanically transformed schistosomula cultivated from 3.5 h to 7 days in vitro (MTS). The results revealed differential gene expression profile to all analyzed genes. We also observed that the transcripts levels were 3 times higher in MTS-3,5h when compared with cercariae and adult worm. The SmUbc9 levels were analysed by Western blot, using specific polyclonal antibodies to S. mansoni protein produced using recombinant protein. The results suggest equivalent levels of SmUbc9 in the same evolutive stages analysed. The SUMOylated substrates profile was detected by Western blot using anti-SUMO. We observed a stage-specific profile of SUMOylated conjugated and an accumulation in initial stages of schistosomula development. These data demonstrate that SUMOylation pathway components are differentially expressed in S. mansoni, suggesting a stage-specific profile during cercariae-schistosomula transition. Whereas this transition is not controlled by real protein synthesis whole results suggest that posttranslational modification SUMO dependent could regulate important biological process during this period of parasite life-cycle

Germ-line mutations in the breast cancer susceptibility gene BRCA1 strongly increase the risk of developing breast and ovarian cancer in women. Different hypothesis have been proposed to explain this tissue specificity. One of the most argued hypothesis is the one that proposes a link between BRCA1 and ovarian hormones' action. Much data have been published in the last years pointing to an important role of progesterone receptor (PR) in inducing normal mammary development and also breast cancer formation. This study aimed to search for a functional relationship between BRCA1 and PR in breast cancer cells. We have found that BRCA1 inhibits the transcriptional activity of PR. We have investigated in more detail the mechanism of this effect. BRCA1 and PR interact in vivo in a ligand-independent fashion. Most importantly, BRCA1 alters the ligand-independent and dependent degradation of PR protein through its ubiquitination and this might have a direct effect on the level of PR recruitment on regulated promoters. BRCA1 is recruited to the hormone-responsive regions of PR-target genes and affects the presence of histone deacetylase activity and the level of monoubiquitinated histone H2A, linking BRCA1 action with chromatin status. These findings support a connection between BRCA1, the principal tumour suppressor responsible for familial breast cancer, and the progesterone receptor transcriptional activity. This relationship can be hypothesized to be reflected in the BRCA1-related breast tumourigenesis., Mutaciones germinales en el gen breast cancer susceptibility gene BRCA1 aumentan altamente el riesgo de padecer cáncer de mama y ovario en mujeres. Se han propuesto diferentes hipótesis para explicar esta especificidad de tejido. Una de las hipótesis más argumentadas es la que propone una relación entre BRCA1 y la acción de las hormonas ováricas. En los últimos años se han publicado numerosos datos señalando al papel esencial del receptor de progesterona (PR) en la inducción del desarrollo normal de la mama y en la formación del cáncer de mama. Este estudio pretendía buscar una relación funcional entre BRCA1 y PR en células de cáncer de mama. Hemos demostrado que BRCA1 inhibe la actividad transcripcional de PR. Hemos investigado en más detalle el mecanismo de este efecto. BRCA1 y PR interaccionan in vivo de una manera independiente de ligando. Y lo que es más, BRCA1 altera la degradación independiente y dependiente de ligando de PR a través de su ubiquitinización y esto podría tener un efecto directo en el nivel de reclutamiento de PR en promotores regulados. BRCA1 es reclutado a las regiones de respuesta a hormona de genes diana de PR y afecta la presencia de actividad histona desacetilasa y el nivel de histona H2A monoubiquitinada, estableciendo un enlace entre la acción de BRCA1 y el estado de la cromatina. Estos hallazgos apoyan una conexión entre BRCA1, el principal supresor de tumor responsable del cáncer de mama hereditario, y la actividad transcripcional del receptor de progesterona. Se puede hipotetizar que esta relación se ve reflejada en el proceso de tumorigénesis BRCA1-dependiente.

O sequenciamento do genoma de Leishmania major, Trypanosoma cruzi e T. brucei mostrou que cada genoma contém entre 8300-1200 genes que codificam proteínas, dos quais aproximadamente 6500 são comuns entre estas espécies. Neste estudo nós focamos a busca nos bancos de dados de tripanossomatídeos por proteínas envolvidas com o metabolismo de ubiquitina. Existem basicamente nove subfamílias distintas de enzimas envolvidas com esta via, denominadas DUBs: ubiquitina C-terminal hidrolases (UCHs), proteases específicas ao processamento de ubiquitina (USPs), proteases relacionadas à doença de Machado-Joseph (MJDs), proteases de tumor ovariano, também chamadas otubaínas (OTUs), proteases com motivo JAMM, sentrinas, autofaginas, peptidases semelhantes à papaína de vírus de RNA de fita dupla e eucariotas (PPPDEs), e metaloproteases Wss1p-símile (WLMs). Neste trabalho nós utilizamos ferramentas de bioinformática para identificar esse repertório de enzimas nos bancos de dados públicos de genomas das espécies L. infantum, L. major, L. braziliensis, T. brucei e T. cruzi. Nossa pesquisa in silico obteve 163 possíveis entradas que codificam para DUBs nestas espécies quando comparadas por BLASTx à proteínas ortólogas conhecidas no GenBank. Destas, 84 pertenciam à subfamília USP, 10 à JAMM, 10 à UCH, 15 à OTU, 5 à sentrina, 24 à PPPDE, 10 à autofagina, 5 à WLM, e não foram encontradas sequências correspondentes à MJD. No genoma do T. cruzi foi também observado a presença de genes duplicados, onde 63 entradas puderam ser resumidas, com no mínimo 95% de similaridade, à 34 entradas. No entanto, em cada genoma de Leishmania estudado foram encontrados em média 32 entradas para DUBs, e sua maioria possuindo ortólogos no mesmo gênero. Análises comparativas dos genes responsáveis pela remoção de ubiquitina mostraram diferenças significativas nos tamanhos das suas sequências de nucleotídeos bem como em suas composições de aminoácidos, especialmente nas regiões não cobertas por domínios conservados, sugerindo que há manutenção da função, mas diferente especificidade aos substratos. Além disso, a expressão relativa dos genes TcUSP7, -10, -14, -15 e TcUCH-L3 determinadas por RT-qPCR mostraram perfis similares de expressão em formas epimastigotas das cepas Berenice-62, Berenice-78, Colombiana e Y do T. cruzi. No entanto, os níveis de TcUSP15 foram menores quando comparados às outras cepas analisadas neste estudo, enquanto os níveis de TcUCH-L3 foram os maiores. Finalmente, nós determinamos a atividade DUB utilizando o substrato CBZ-Gly-Gly- Arg-7-amido-4-metilcumarina em extratos nucleares e citosólicos e a conjugação de proteínas com ubiquitina e SUMO nos mesmos extratos utilizando western blot e anticorpos específicos para ubiquitina e SUMO. Nossos resultados mostraram que a atividade é predominantemente citosólica. A técnica de western blotting mostrou que os conjugados ubiquitinados são preferencialmente encontrados no citoplasma e os sumorilados no núcleo. Esses resultados evidenciam a complexidade e diversidade das DUBs em tripanossomatídeos e abre a possibilidade de explorar a relevância das interações na regulação das vias mediadas por ubiquitina nesses parasitos. The genome sequences of Leishmania major, Trypanosoma brucei and T. cruzi revealed that each genome contains 8300-12000 protein-coding genes, of which approximately 6500 are common to their genomes. In this study we focused the mining the trypanosomatid databases looking for proteins involved in ubiquitin metabolism. There are basically nine distinct subfamilies of enzymes involved in this pathway, denominated DUBs: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific processing proteases (USPs), Machado-Joseph disease proteases (MJDs), ovarian tumour proteases (OTUs), JAMM motif proteases, sentrins, autophagins, Permuted Papain fold Peptidases of DsRNA viruses and Eukaryotes (PPPDEs), and Wss1p-like metalloproteases (WLMs). In this work we used bioinformatic approaches to identity these set of enzymes in L. infantum, L. major, L. braziliensis, T. brucei and T. cruzi public genome databases. Our in silico search retrieved 163 putative entries coding to DUBs in these species among databases when compared to known GenBank ortholog proteins through BLASTx. Of these, 84 belong to USP subfamily, 23 to JAMM, 10 to UCH, 15 to OTU, 5 to sentrin, 24 to PPPDE, 10 to autophagin, 5 to WLM, and where not found entries to MJD. In T. cruzi genome was also observed the presence of duplicated genes, where the 63 entries might be resumed, with at least 95% of similarity, in 34 sequences. However, in each Leishmania genome studied were found on average 32 entries for DUBs, and most of them have its orthologues into the same genus. Comparative analysis of genes responsible for ubiquitin removal showed significant difference in their respective nucleotide sequence lengths as well as amino acid composition, especially with regards to regions outer conserved domains, suggesting maintaining of function, but different substrate specificity. In addition, the relative gene expression of TcUSP7, -10, -14, -15 and TcUCH-L3 determined by RT- qPCR showed similar profile in epimastigotes forms between Berenice-62, Berenice-78, Colombian and Y strains of T. cruzi. However, the levels of TcUSP15 were lowest when compared to strains analyzed in this study, while the levels of TcUCH-L3 where highest. Finally, we determinate the DUB activity using the substrate CBZ-Gly-Gly- Arg-7-amido-4-methylcoumarin in nuclear and citosolic extracts and the ubiquitin and SUMO protein conjugation in the same extracts using western blot and specific antibody to ubiquitin and SUMO. Our results showed that the activity were predominantly citosolic. The western blotting showed that preferentially conjugate ubiquitin was found in the cytoplasm and sumoylation in the nucleus. These results evidence the complexity and diversity of DUBs in tripanosomatids and open the possibility to explore the relevance of their interactions in regulation of ubiquitin- mediated pathways in these parasites.

Clínicamente, la enfermedad de Lafora es una forma de epilepsia mioclónica progresiva, autosómica recesiva. Se desarrolla debido a la mutación del gen que codifica para la laforina y/o la malina, las cuales están involucradas en la regulación del metabolismo del glicógeno. Los corpúsculos de Lafora que identifican la enfermedad están compuestos por agregados densos de poliglicano que se encuentran en inclusiones tanto en el cerebro, como en otros tejidos. Describimos una mujer de 18 años con una historia familiar de epilepsia mioclónica; ella desarrolló crisis mioclónicas que se correlacionaban con anormalidades electroencefalográficas, que empeoraron progresivamente, asociadas a un deterioro cognitivo. El diagnóstico fue confirmado con la presencia de corpúsculos de Lafora encontrados en las glándulas sudoríparas de una biopsia de piel, con tinciones de PAS e inmunohistoquímicas positivos para ubiquitina, lo que permitió distinguir estos corpúsculos de otras inclusiones PAS positivas. Este es el primer caso descrito de enfermedad de Lafora en Chile.

Clínicamente, la enfermedad de Lafora es una forma de epilepsia mioclónica progresiva, autosómica recesiva. Se desarrolla debido a la mutación del gen que codifica para la laforina y/o la malina, las cuales están involucradas en la regulación del metabolismo del glicógeno. Los corpúsculos de Lafora que identifican la enfermedad están compuestos por agregados densos de poliglicano que se encuentran en inclusiones tanto en el cerebro, como en otros tejidos. Describimos una mujer de 18 años con una historia familiar de epilepsia mioclónica; ella desarrolló crisis mioclónicas que se correlacionaban con anormalidades electroencefalográficas, que empeoraron progresivamente, asociadas a un deterioro cognitivo. El diagnóstico fue confirmado con la presencia de corpúsculos de Lafora encontrados en las glándulas sudoríparas de una biopsia de piel, con tinciones de PAS e inmunohistoquímicas positivos para ubiquitina, lo que permitió distinguir estos corpúsculos de otras inclusiones PAS positivas. Este es el primer caso descrito de enfermedad de Lafora en Chile. Clinically Lafora disease (LD) is an autosomal recessive form of progressive myoclonus epilepsy (PME). It develops due to mutations in gene that encodes laforin or malin, which may be involved in regulating the glycogen metabolism. Pathognomonic Lafora bodies are composed by dense aggregates of polyglucosans inclusions, found in brain and other organs. We describe 18 year oldfemale withfamily history ofmyodonic epilepsy. She developed worsening myodonic seizures with correlative electroencephalogram abnormalities and a progressive mental deterioration. Diagnosis was confirmed with periodic acid-schiff (PAS) positive lafora bodies and ubiquitin-positive inmunohistochemical stain, which helped to distinguish from other PAS positive inclusions. This is the first case describe of Lafora disease in Chile.

[cat] La degradació de proteïnes mitjançant el sistema de les ubiquitines i el proteasoma (UPS) és un procés altament regulat, en el qual diverses famílies enzimàtiques s'encarreguen de la correcte conjugació i deconjugació de molècules d'ubiquitina a un determinat substrat. Una d'aquestes famílies és la dels enzims deubiquitinitzants (DUBs), de la qual se n'han descrit, en humans, més de 90 membres. USP25 (21q11.2) codifica per un enzim deubiquitinitzant i produeix tres isoformes per splicing alternatiu. Una d'elles, generada per la inclusió de l'exó 19a entre el 18 i el 19b, presenta una expressió restringida a múscul esquelètic i cor (USP25m). Mitjançant un assaig de dos híbrids en llevat varem determinar que USP25m interacciona amb una proteïna d'unió a la miosina (MyBPC1), amb l'actina 1 (ACTA1) i amb la filamina C (FLNC). Totes aquestes proteïnes són sarcomèriques, regulen la diferenciació i el manteniment de les cèl.lules musculars i han estat implicades en diverses patologies humanes. Assajos bioquímics realitzats per aprofundir en la rellevància funcional d'aquestes interaccions ens mostren que els nivells de MyBPC1 estan regulats pel proteasoma, i que USP25m és capaç d'abolir seva degradació. En canvi, ACTA1 i FLNC són proteïnes estables, suggerint que la seva interacció amb USP25m està relacionada amb mecanismes reguladors de l'estructura i la funció del sarcòmer, i no amb la seva degradació. USP25 conté tres dominis d'unió a ubiqüitina implicats en el reconeixement de substrats i pot ser modificada per diversos tipus de modificació posttraduccional com la ubiqüitinació, la sumoïlació, la fosforilació i l'acetilació. El fet que sigui una proteïna multimodificada suggereix que la seva activitat enzimàtica ha d'estar finament regulada. Per últim, la sobreexpressió d'USP25 és capaç d'induïr una aturada en el cicle cel·lular, per mecanismes encara desconeguts., [eng] USP25 is a deubiquitinating enzyme (DUB) that generates a muscle-specific isoform, USP25m, by alternative splicing. USP25m interacts with several sarcomeric proteins, but similarly to most DUBs, the contribution of different protein domains to enzymatic activity and substrate recognition remains to be ascertained. In silico analysis revealed the position of the catalytic signatures and predicted three ubiquitin binding domains (UBDs): one ubiquitin-associated domain (UBA) and 2 ubiquitin-interacting motifs (UIMs). By generating serial and combinatorial deletions of these domains, we analyzed their contribution to USP25m catalytic activity, substrate recognition, subcellular localization, and post-translational modifications, such as ubiquitination and sumoylation. Our results indicate that ablation of these motifs do not abrogate USP25 deubiquitinating activity, neither promoted any significant alteration in subcellular localization of the enzyme nor ubiquitin. However, USP25 UBDs contribute to specific substrate recognition, as interaction with each substrate requires a different combination or number of UBDs, in an additive or synergistic manner. USP25m is sumoylated in vitro and ubiquitinated, although the UBDs are not required for these post-translational modifications. In addition, USP25m is phosphorylated in Tyr, Ser/Thr residues and acetylated. The physiological relevance of all these modifications remains to be ascertained. USP25 overexpression causes cell cycle arrest, most probably at the G1/S transition.

Orientador : Marco Aurélio Krieger Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduaçâo em Biologia Celular e Molecular. Defesa: Curitiba, 2005 Inclui bibliografia Resumo: Os mecanismos envolvidos no processo de diferenciação celular do T. cruzi das formas epimastigotas (não infectantes) para as formas tripomastigotas metacíclicas (infectantes), tem sido motivo de estudo de nosso grupo há muitos anos. Com o intuito de compreender os diversos mecanismos envolvidos neste processo, temos efetuado uma busca sistemática de genes diferencialmente expressos utilizando diferentes metodologias. Em tripanosomas o controle do mecanismo da expressão gênica é feito em sua maioria a nível pós-transcricional ou pós-traducional (Elias, 2001). Consequentemente estes parasitas podem se adaptar rapidamente às transições entre o inseto vetor e o hospedeiro fazendo uma reprogramação na expressão de seus genes. Durante as mudanças adaptativas na morfologia, fisiologia e comportamento muitas proteínas são selecionadas e outras são degradadas. A regulação da diferenciação celular pode ser feita pela degradação ou não de proteínas específicas importantes. A fim de investigar a existência da via proteolítica proteossomo-ubiquitina, bem como a expressão dos seus componentes utilizamos neste trabalho uma ferramenta da genômica funcional conhecida como microarranjo de DNA. O microarranjo utilizado possui uma cobertura de 45% para os genes dessa via proteolítica. Os resultados das análises das hibridações com RNA polissomal e RNA total de epimastigotas, durante o início da metaciclogênese, sugerem que os genes da via sofrem uma regulação complexa de sua expressão. Alguns componentes apresentam perfis comuns de expressão o que abre novas perspectivas para investigação de possíveis interações entre eles Abstract: The mechanisms involved in the cellular differentiation of T. cruzi epimastigote (non-infectious forms) into metacyclic trypomastigotes (infectious forms) have been studied by our group for many years. In order to understand the several mechanisms involved in this process a systematic search for differentially expressed genes was made using different technologies. In trypanosomes, the gene expression control mechanism is mainly post-transcriptional or post-translational (Elias, 2001). Consequently, parasites can rapidly adapt to the vector-host transition by changes in its genes expression. Many proteins are selected and others are degraded during the adaptative morphology, physiology and behavioral changes. Cellular differentiation regulation may be done by specific proteins degradation. In order to investigate the existence of the ubiquitina-proteasome pathway, in genomic fashion, DNA microarray technology was used. A microarray covering 45% for genes involved in this pathway was used. Results of the polysomal RNA and total RNA hybridization analysis, during the start of metacyclogenesis, indicated complex gene expression regulation. Some components in this pathway show a common pattern and opens new perspectives to investigation their putative interactions.

La degradación de proteínas mediada por el sistema ubiquitina/proteasoma es un mecanismo de control de la abundancia de proteínas específicas dentro de la célula. Muchas proteínas son marcadas para su degradación por una familia de E3 ubiquitin-ligasas multiméricas, denominadas complejos SCF. Estos complejos están compuestos por tres polipéptidos constantes – Skp1, Cdc53/Cul1, Rbx1/Hrt1 – y un cuarto componente variable, la proteína adaptadora con motivo F-box. Utilizando el sistema del doble híbrido de levaduras, como así también ensayos de interacción in vitro, hemos identificado por primera vez a los componentes del núcleo invariante de los complejos SCF en la mosca de la fruta Drosophila melanogaster. Mediante experimentos de complementación funcional en S.cerevisiae demostramos que la proteína Rbx1 de Drosophila puede restaurar la viabilidad de mutantes hrt1 de levaduras, indicando que los complejos SCF están funcionalmente conservados en la evolución. Slimb es una proteína de Drosophila con motivo F-box, que cumple distintas funciones en el desarrollo y la fisiología celular. En esta tesis, hemos analizado la función de Slimb durante la ovogénesis de Drosophila. Demostramos que la pérdida de función de Slimb en la línea germinal provoca una reducción en los niveles de dE2F2 y Dp, lo cual se correlaciona con alteraciones en la regulación de los ciclos mitóticos durante la formación del cisto, con anomalías en la endorreplicación del genoma de las células nodrizas y con defectos en el vertido del contenido del citoplasma de las células nodrizas dentro del ovocito. Nuestros resultados sugieren que Slimb regula indirectamente los niveles de E2F2 y Dp, controlando de esta manera el desarrollo del cisto. Por otro lado, la generación de clones mutantes slimb en la línea somática del ovario dio lugar a la aparición de alteraciones en la morfología del germario y a defectos en la encapsulación del cisto por parte del epitelio folicular. Asimismo, ovarios con clones somáticos slimb exhibieron retrasos en la diferenciación del epitelio folicular, lo cual se correlacionó con la aparición de células polares ectópicas, falta de tallos interfoliculares y deslocalización del ovocito dentro del folículo. Encontramos también que en la ovogénesis media, Slimb se necesita en el epitelio folicular para especificar a las células foliculares que darán origen a los apéndices dorsales. La hiperactivación débil de la vía de Dpp dio lugar a la aparición de fenotipos semejantes a los de pérdida de función de slimb, los cuales fueron totalmente suprimidos cuando se sobre-expresó la proteína Slimb simultáneamente con Dpp. Significativamente, en ovarios con clones slimb se observó la expresión ectópica del gen Broad-Complex y del reportero transcripcional dad-LacZ, como así también una acumulación anormal de la proteína co-Smad, Medea. Proponemos que Slimb normalmente regula negativamente a la vía de Dpp, destinando a degradación a la proteína Medea. Finalmente, proporcionamos evidencias de que Slimb interacciona genéticamente con componentes y con reguladores de los complejos SCF, sugiriendo que Slimb funciona como subunidad adaptadora de este complejo en el ovario de Drosophila. Ubiquitin/proteasome mediated proteolysis is a commonly employed mechanism for the control of protein abundance in the cell. Many proteins are targeted for degradation by a family of multimeric E3 ubiquitin ligases, termed SCF complexes. These complexes are composed of three constant proteins- Skp1, Cdc53/Cul1, Rbx1/Hrt1- and a variable adaptor subunit, the Fbox protein. By utilizing the yeast two hybrid system and in vitro interactions we have identified for the first time the components and overall structure of the basic SCF complex in Drosophila melanogaster. A functional complementation test performed in a S.cerevisiae showed that Drosophila Rbx1 can restore the viability of yeast hrt1 mutant cells, indicating that the complex is functionally conserved throughout evolution. Slimb (Slmb) is a Drosophila F-Box protein that fulfills several roles in development and cell physiology. We have analyzed here its participation in egg chamber development by generating mutant clones in the ovary. slmb loss of function in the germline provoked a reduction in dE2F2 and dDP levels, which correlated with misregulation of mitotic cycles during cyst formation, abnormal nurse cell endoreplication and impairment of dumping of the nurse cell content into the oocyte. We suggest that Slmb indirectly downregulates E2F2 and Dp levels, thereby controlling mitotic and endocycle replication during cyst development, as well as the processess of nurse cell dumping and apoptosis. slmb mutant clones in the somatic line caused alterations in the morphology of the germarium and defects in encapsulation of the cyst. Furthermore, slmb somatic clones exhibited a delay in follicle cell differentiation, which correlated with the occurrence of ectopic polar cells, lack of interfollicular stalks and mislocalization of the oocyte. We found that, at midoogenesis, Slmb was required in the follicular epithelium to specify the shape and position of two patches of cells that will give rise to dorsal appendages. Mild overactivation of the Dpp pathway provoked similar phenotypes that could be antagonized by simultaneous overexpression of Slmb and interestingly, ectopic expression of the Broad-Complex gene and of a dad-LacZ reporter element, as well as over-accumulation of the co-Smad protein Medea, were recorded in slmb somatic clones. We propose that Slimb normally downregulates the Dpp pathway in follicle cells by targeting Medea for proteasomal degradation. Finally, we provide evidences that Slmb genetically interacts with components and regulators of the SCF complex, suggesting that Slmb functions as a subunit of an SCF complex in the ovary. Fil: Muzzopappa, Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

13 páginas, 17 figuras, 1 tabla., [EN] With the aim of setting new molecular markers of certain function in the olive, several genes expressed during olive fruit ripening have been isolated and characterized, also analyzing temporal an spatial expression of such genes. Among other, the following marker genes have been analyzed: Δ9- stearoyl-ACP-desaturase, a key enzyme for fatty acid biosynthesis; a putative plastidic glucose translocator (pGlcT) expressed in non-photosynthetic tissues like the mature olive fruit; and finally, polyubiquitin and cullin, components of the ubiquitin-mediated protein degradation system. In a parallel way, protein extracts have been isolated from olive reach, in order to analyze their protein profiles by SDS-PAGE. The major protein component of such extracts corresponds to a family of storage proteins of the 11Stype. After raising a rabbit antiserum to these proteins, a spatial and temporal analysis of them was performed during seed development and germination. Finally, oleosin purification from olive seeds has been accomplished. The biological meaning attributed to each of these markers, forecast their future usage in olive improvement programmes. However, more information particularly concerning the knowledge of these genes and their expression is needed, and therefore, basic research on olive should be carried through, [ES] Con objeto de establecer nuevos marcadores moleculares en el olivo con función determinada, se han aislado y caracterizado varios genes que se expresan durante la maduración del fruto del olivo, y se ha realizado un análisis temporal y espacial de la expresión de estos genes. Entre otros, se han analizado los siguientes marcadores: la Δ9- estearoyl-ACP- desaturasa, enzima clave en la biosíntesis de ácidos grasos; un posible translocador plastídico de glucosa (pGlcT), que se expresa en tejidos no fotosintéticos como es el fruto maduro del olivo; y finalmente, también se han identificado y caracterizado poliubiquitina y culina, componentes del sistema de degradación proteica mediado por ubiquitina. Paralelamente, se han preparado extractos crudos a partir de semillas de olivo con objeto de analizar sus perfiles proteicos mediante SDS-PAGE,. Los componentes mayoritarios de estos extractos corresponden a una familia de proteínas de almacenamiento tipo 11S. Después de obtener un suero específico para este tipo de proteínas, se hizo un seguimiento espacial y temporal de las mismas durante el desarrollo y germinación de las semillas. Por último, también se ha llevado a cabo la purificación de oleosinas a partir de semillas. El significado biológico atribuido a cada uno de estos marcadores, hace preveer que en un futuro puedan ser utilizados en programas de mejora del olivo. Pero antes se requiere una mayor información y profundizar en el conocimiento de estos genes y su expresión, por lo que es necesario continuar las investigaciones a nivel básico en el olivo., Este trabajo fue financiado por el proyecto INIA-CAO99-003.

Huntington's disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cells, suggest that an impairment of the ubiquitin-proteasome system (UPS) may be key in HD pathogenesis. To test whether proteasome activity is impaired in vivo, we performed enzymatic assays for the three peptidase activities of the proteasome in brain extracts from the HD94 conditional mouse model of HD. We found no inhibition of any of the activities, suggesting that if UPS impairment happens in vivo, it is not at the level of the proteasome catalytic core. Intriguingly, the chymotrypsin- and trypsin-like activities increased selectively in the affected and aggregate-containing regions: cortex and striatum. Western blot analysis revealed no difference in total proteasome content whereas an increase in the interferon-inducible subunits of the immunoproteasome, LMP2 and LMP7, was observed. These subunits confer to the proteasome catalytic properties that are optimal for MHC-I peptide presentation. Immunohistochemistry in control mouse brain revealed LMP2 and LMP7 mainly in neurons. Accordingly, their increase in HD94 mice predominantly took place in neurons, and 5% of the ubiquitin-positive cortical aggregates were also LMP2-positive. Ultrastructural analysis of neurons with high level of immunoproteasome subunits revealed signs of neurodegeneration like nuclear indentation or fragmentation and dark cell appearance. The neuronal induction of LMP2 and LMP7 and the associated signs of neurodegeneration were also found in HD postmortem brains. Our results indicate that LMP2 and LMP7 participate in normal neuronal physiology and suggest a role in HD neurodegeneration., This work was supported by grants from Comunidad Autónoma de Madrid, Asociación Española de Corea de Huntington (ACHE), Fundación “La Caixa”, Spanish Comisión Interministerial de Ciencia y Tecnología, and by an institutional grant from Fundación Ramón Areces. E.M.-A. was the recipient of predoctoral fellowships from ACHE and Fundación Ferrer. M.D.-H. is the recipient of a postdoctoral fellowship from Comunidad Autónoma de Madrid.