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Il misfolding proteico e l’aggregazione di proteine sono problemi essenziali alla base di diversi disordini neurodegenerativi. Tali malattie sono caratterizzate dalla presenza di inclusioni di proteine misfoldate che possono interferire con la corretta proteostasi, portando alla perdita dell’attività neuronale cerebrale. Ciononostante, la formazione di aggregati proteici è un meccanismo messo in atto anche da cellule eucariote sane per proteggere il proprio proteoma. Tra tali siti di deposito, i Dendritic cells Aggresome-Like Induced Structures (DALISs) sono stati recentemente identificati in cellule dendritiche. Essi rappresentano dei depositi transienti per proteine difettive ubiquitinate ed antigeni, che si formano in risposta a diversi stress. La loro funzione non è ancora completamente compresa, specialmente dopo essere stati identificati anche in cellule non appartenenti al sistema immunitario. Ad oggi, nessuna indagine è stata condotta in altre cellule eucariote ma, fungendo da siti di deposito per proteine ubiquitinate, ci si aspetta di incontrarli anche in altri modelli cellulari. Il lievito Saccharomyces cerevisiae viene ampiamente utilizzato per la ricerca sugli aggregati cellulari, poiché presenta un ampio range di inclusioni dinamiche che sono stati dimostrate essere conservati tra diverse specie, nonostante il maggior grado di complessità nella composizione proteica e nelle funzionalità negli eucarioti multicellulari. Abbiamo identificato quattro diverse condizioni di stress in cui il lievito S. cerevisiae manifesta la formazione di corpi citoplasmatici: shock termico a 42°C, stress acuto da etanolo, deprivazione di azoto e carenza di glucosio. Abbiamo quindi sottoposto S. cerevisiae a questi stimoli di stress e investigato la formazione di inclusioni insolubili, concentrandoci sugli aggregati ubiquitinati. A questo fine, abbiamo deciso di includere anche il trattamento con puromicina come condizione di stress a cui esporre le cellule di lievito, poiché la puromicina è stata dimostrata indurre la formazione di DALIS in cellule dendritiche. Abbiamo dimostrato la formazione di aggregati ricchi di ubiquitina in cellule di lievito a seguito di stress acuto da etanolo, di deprivazione di azoto prolungata e di trattamento con puromicina. Abbiamo poi indagato la localizzazione cellulare degli aggregati ubiquitinati in cellule di S. cerevisiae grazie a microscopia a fluorescenza. Abbiamo osservato l’accumulo di aggregati ricchi di ubiquitina dalla forma rotonda, che sono disseminati nel cytosol e non colocalizzati con altre strutture cellulari. I corpi osservati rispecchiano le caratteristiche morfologiche riportate per i DALISs nelle cellule dendritiche. Abbiamo poi deciso di investigare come gli aggregati ricchi di ubiquitina indotti a seguito di stress acuto da etanolo vengano eliminati all’interno della cellula di lievito. Abbiamo dimostrato che l’accumulo di aggregati ricchi di ubiquitina è persistente nelle cellule mutanti atg1Δ rispetto alle cellule wild type, in cui il livello di proteine ubiquitinate decresce qualche ora dopo l’esposizione allo stress. Ciò suggerisce che il processo di autofagia è implicato nella rimozione degli aggregati ubiquitinati, poiché Atg1 è una chinasi che gioca un ruolo fondamentale nella regolazione di tale processo. D’altro canto, abbiamo osservato che le cellule mutanti cue5Δ mostravano un minor accumulo di ubiquitina durante l’intera durata del trattamento con etanolo, rispetto alle cellule wild type. La proteina di lievito Cue5 è una proteina che lega ubiquitina e che collabora con gli enzimi di coniugazione dell’ubiquitina Ucb4/Ubc5 e con l’ubiquitina ligasi Rsp5 per congiungere l’ubiquitina al suo substrato. Dato che abbiamo osservato una minor presenza di ubiquitina nelle cellule mutanti cue5Δ, suggeriamo che Cue5 possa avere un ruolo nell’ubiquitinazione stessa agli aggregati osservati in condizioni di stress acuto da etanolo. Protein misfolding and aggregation are major issues in neurodegenerative disorders. Such diseases are characterized by the presence of large inclusions of misfolded proteins which can interfere with the proper proteostasis, possibly leading to loss of neuronal activity. Nevertheless, the formation of protein aggregates is a conserved mechanism engaged also by healthy eukaryotic cells in response to external stimuli that alter their physiological environment. In this context, protein aggregates function as well-organized deposition sites which help the cell to protect its proteome in the waiting for the restoration of the physiological condition. Among the organized cytoplasmic deposition sites for proteins, Dendritic cell Aggresome-Like Induced Structures (DALISs) has recently been identified in dendritic cells. They represent transient deposits for ubiquitinated defective proteins and antigens, which are formed in response to several stresses. Their function is still not completely understood, especially since they were later characterized in non-immune cells as well. No investigation has ever been conducted in other eukaryotic cells, but since they act as deposition sites for ubiquitinated misfolded proteins, we can expect to identify them in other cellular models as well. The yeast Saccharomyces cerevisiae is widely used for the research on cellular aggregates, as it displays a range of different dynamic deposition sites that are demonstrated to be conserved between different species, despite the increased complexity in protein composition and function in multicellular eukaryotes. We identified four main stress conditions under which the yeast Saccharomyces cerevisiae manifests the formation of large cytoplasmic bodies, namely heat shock temperatures at 42°C, severe ethanol stress, nitrogen depletion and glucose starvation. Therefore, we subjected yeast cells to these stress conditions and investigated the formation of insoluble inclusions, with a particular focus on ubiquitin-rich protein aggregates. To this purpose, we decided to include puromycin treatment as a stress condition to which a puromycin-sensitive yeast strain should be exposed, as puromycin was demonstrated to induce the formation of DALISs in dendritic cells. We demonstrated the formation of ubiquitin-rich protein aggregates in S. cerevisiae cells following the exposure to severe ethanol stress, under prolonged nitrogen starvation, and following puromycin treatment. We further investigated the cellular localization of ubiquitinated aggregates of wild type S. cerevisiae cells by fluorescence microscopy. We observed that the accumulation of round-shaped ubiquitin-rich aggregates, which are disseminated in the cytosol and not colocalized with any cellular structure. Such structures reflect the characteristic morphology of DALIS reported in dendritic cells. We then decided to investigate how these ethanol-induced ubiquitin-rich protein aggregates are cleared inside the yeast cell. We demonstrated that the accumulation of ubiquitin-rich aggregates is persistent in atg1Δ mutant cells, whereas in wild type cells the level of ubiquitinated proteins decrease a few hours after the exposure to the stress. This suggests that autophagy is implied in the clearance of ubiquitinated aggregates, as Atg1 is a kinase which plays a crucial role in the regulation of this process. On the other hand, we observed that cue5Δ mutant cells displayed a minor accumulation of ubiquitin throughout the whole treatment with ethanol, compared to wild type cells. The yeast protein Cue5 is a ubiquitin-binding protein that collaborates with Ubc4/Ubc5 ubiquitin-conjugating enzymes and the Rsp5 ubiquitin ligase for the conjugation of ubiquitin to its substrate. Since we observed a lower presence of ubiquitin in cue5Δ mutant cells, we propose that Cue5 might have a role in the conjugation of ubiquitin itself to the aggregates under severe ethanol stress.

Protein ubiquitylation acts as a complex cell signaling mechanism since the formation of different mono- and polyubiquitin chains determines the substrate’s fate in the cell. E3 ligases define the specificity of this reaction by catalyzing the attachment of ubiquitin to the substrate protein. Thus, they represent an important regulatory component of this process. Large HERC ubiquitin ligases belong to the HECT E3 protein family and comprise HERC1 and HERC2 proteins. The physiological relevance of the Large HERCs is illustrated by their involvement in different pathologies, with a notable implication in cancer and neurological diseases. Understanding how cell signaling is altered in these different pathologies is important for uncovering novel therapeutic targets. To this end, this review summarizes the recent advances in how the Large HERCs regulate the MAPK signaling pathways. In addition, we emphasize the potential therapeutic strategies that could be followed to ameliorate the alterations in MAPK signaling caused by Large HERC deficiencies, focusing on the use of specific inhibitors and proteolysis-targeting chimeras.

HERC2 gene encodes an E3 ubiquitin ligase involved in several cellular processes by regulating the ubiquitylation of different protein substrates. Biallelic pathogenic sequence variants in the HERC2 gene are associated with HERC2 Angelman-like syndrome. In pathogenic HERC2 variants, complete absence or marked reduction in HERC2 protein levels are observed. The most common pathological variant, c.1781C > T (p.Pro594Leu), encodes an unstable HERC2 protein. A better understanding of how pathologic HERC2 variants affect intracellular signalling may aid definition of potential new therapies for these disorders. For this purpose, we studied patient-derived cells with the HERC2 Pro594Leu variant. We observed alteration of mitogen-activated protein kinase signalling pathways, reflected by increased levels of C-RAF protein and p38 phosphorylation. HERC2 knockdown experiments reproduced the same effects in other human and mouse cells. Moreover, we demonstrated that HERC2 and RAF proteins form molecular complexes, pull-down and proteomic experiments showed that HERC2 regulates C-RAF ubiquitylation and we found out that the p38 activation due to HERC2 depletion occurs in a RAF/MKK3-dependent manner. The displayed cellular response was that patient-derived and other human cells with HERC2 deficiency showed higher resistance to oxidative stress with an increase in the master regulator of the antioxidant response NRF2 and its target genes. This resistance was independent of p53 and abolished by RAF or p38 inhibitors. Altogether, these findings identify the activation of C-RAF/MKK3/p38 signalling pathway in HERC2 Angelman-like syndrome and highlight the inhibition of RAF activity as a potential therapeutic option for individuals affected with these rare diseases. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This study was funded by the following grants: JLR (Agencia Estatal de Investigación: PID2020-120344RB-I00/MCIN/AEI/10.13039/501100011033) and FV (PDC2021-121776-I00 and PID2020-117278 GB-I00 from MCIN/AEI/10.13039/501100011033 and FEDER “Una manera de hacer Europa” “NextGenerationEU”/PRTR. And Grant 202038-30 from “La Marató de TV3”). As well, this article is based upon work from COST Action ProteoCure CA20113, supported by COST (European Cooperation in Science and Technology). JSG and AMM received FPU Fellowships (FPU17/02413 and FPU18/06325, respectively) from the Spanish Ministry of Universities.

Orientador: Licio Augusto Velloso Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: A proteína PGC1? é um co-ativador de transcrição gênica que desempenha papel importante na regulação de uma série de fenômenos metabólicos que compreendem desde o controle da termogênese e mitocondriogênese até a regulação da secreção de insulina e a produção hepática de glicose. Como vários dos fenômenos biológicos controlados direta ou indiretamente pela PGC1? tem importância vital, a regulação dos níveis de PGC1? nos tecidos deve ser finamente ajustada. Nos últimos anos, inúmeros estudos exploraram os mecanismos envolvidos com o controle da expressão gênica e tradução da PGC1?. Entretanto, apenas alguns poucos estudos avaliaram a degradação da mesma. Um dos mais importantes mecanismos envolvidos com a regulação funcional e da meia-vida de proteínas é a ubiquitinação, que pode direcionar proteínas alvo ao proteassoma para degradação ou a outras modificações pós-traducionais. O objetivo do presente estudo foi avaliar a participação de uma proteína com atividade deubiquitinase e ubiquitina ligase, a A20, na manutenção da homeostase do tecido adiposo de animais submetidos à dieta rica em gordura e voluntários humanos magros e obesos antes e após cirurgia de redução de peso. Foram utilizados o tecido adiposo branco visceral e subcutâneo e o tecido adiposo marrom de camundongos Swiss machos submetidos a 16 semanas de dieta hiperlipídica e o tecido adiposo subcutâneo de voluntários magros e obesos antes e após a cirurgia bariátrica. Esses tecidos foram avaliados quanto ao conteúdo protéico e expressão gênica da proteína A20, e sua associação com a PGC1? por imunoprecipitação e imunofluorescência, bem como a ubiquitinação desta última. Os resultados obtidos a partir do tecido adiposo de humanos mostram uma diminuição na expressão da proteína A20 nos pacientes antes e após a cirurgia bariátrica com relação aos voluntários magros. A PGC1? aparece mais ubiquitinada nos pacientes obesos em relação e a associação entre A20 e PGC1? parece aumentar com o ganho de peso na mesma proporção que o conteúdo protéico de PGC1? parece diminuir. No tecido adiposo subcutâneo de camundongos obesos, observamos uma diminuição de PGC1? bem como redução da marcação por cadeias de poliubiquitina desta proteína, associado a um aumento de A20 e aumento da associação de A20 com PGC1?. Camundongos obesos foram também tratados com um oligonucleotídeo antisense (ASO) para A20, resultando na redução de sua expressão gênica. Os animais tratados apresentaram uma piora na tolerância à glicose no teste de GTT o que ocorreu concomitantemente a redução de PGC1?. Nossos resultados indicam que, no tecido adiposo, a A20 se associa a PGC1? e a redução da sua expressão resulta em redução da expressão da PGC1? o que é acompanhando de uma piora no controle homeostático da glicose Abstract: Peroxisome proliferator-activated receptor ? coactivator 1 alpha (PGC-1?) plays an important role in whole body metabolism and, particularly in glucose homeostasis. Its expression is tightly regulated and, small variations in tissue levels can have a major impact in a number of physiological and pathological conditions. Recent studies have shown that the ubiquitin/proteasome system plays a role in the control of PGC-1? degradation. Here we evaluated the interaction of PGC-1? with the protein A20, which plays a dual-role in the control of the ubiquitin/proteasome system acting as a deubiquitinase and as an E3 ligase. We employed immunoprecipitation, quantitative real-time PCR and immunofluorescence staining to evaluate PGC-1?, A20, PPAR? and ubiquitin in the adipose tissue of humans and mice. Our results show that, in distinct sites of the adipose tissue A20 binds to PGC-1?. At least in the subcutaneous fat of humans and mice the levels of PGC-1? decrease during obesity, while its physical association with A20 increases. The inhibition of A20 leads to a reduction of PGC-1? and PPAR? expression, suggesting that A20 acts as a protective factor against PGC-1? disposal. Thus, we provide evidence that mechanisms regulating PGC-1? ubiquitination are potentially involved in the control of the function of this transcriptional co-activator Mestrado Fisiopatologia Médica Mestra em Ciências

Orientadores: Lício Augusto Velloso, Marciane Milanski Ferreira Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Dentre os fatores ambientais que contribuem para o desenvolvimento de obesidade, o consumo de dietas ricas em ácidos graxos saturados desempenha o papel mais importante. Estudos recentes realizados por vários grupos, inclusive o nosso, revelam que ácidos graxos saturados presentes na dieta levam ao desenvolvimento de resistência hipotalâmica à ação dos hormônios leptina e insulina, fenômeno este fundamental para que ocorra a quebra no equilíbrio entre ingestão e gasto calórico. Até o momento caracterizaram-se dois mecanismos moleculares potencialmente envolvidos na iniciação do processo que resulta na disfunção hipotalâmica na obesidade, a ativação de TLR4 e a indução de estresse de retículo endoplasmático, ambos levando a uma resposta inflamatória local e, eventualmente, a apoptose neuronal. Estudos recentes têm revelado que frente a situações que oferecem risco de dano celular, ativa-se um mecanismo de controle de tráfico e degradação protéica chamado sistema ubiquitina-proteassoma (UPS). O acúmulo de agregados protéicos positivos para ubiquitina pode gerar toxicidade celular e regular a plasticidade neuronal. Também a modulação de componentes do UPS pode gerar neurodegeneração hipotalâmica e fenótipo obeso em animais experimentais. Neste estudo aventamos a hipótese que durante períodos prolongados de obesidade a ativação anômala do UPS contribuiria para a perpetuação do quadro de obesidade. De fato, os resultados obtidos revelam que roedores com predisposição para a obesidade induzida por dieta mantém, a princípio, a capacidade de regular adequadamente a UPS no hipotálamo. Com o passar do tempo esta capacidade é perdida resultando numa maior dificuldade para perda de peso frente à redução do aporte calórico. Roedores com mutações que os protegem da inflamação, não apresentam distúrbio funcional do UPS quando expostos a dieta rica em ácidos graxos e, são também protegidos da obesidade. Portanto, o defeito funcional do UPS no hipotálamo no curso de obesidade prolongada, constitui-se num fator importante contribuindo para a refratariedade ao tratamento e perpetuação da doença Abstract: The consumption of high-fat diets, especially those rich in saturated fatty acids, plays the most important role in the development of obesity. Recent studies by several groups, including ours, have shown that dietary long-chain saturated fatty acids lead to the development of hypothalamic resistance to leptin and insulin, an important condition contributing for breaking of the balance between caloric intake and energy expenditure. Two molecular mechanisms are currently known to play a triggering role in this process; activation of TLR4 and endoplasmic reticulum stress, both leading to local inflammation and eventually apoptosis of neurons. The ubiquitin-proteasome system (UPS) plays an important role in the control of protein recycling in the cell. The accumulation of ubiquitin-positive protein aggregates can cause cell toxicity and regulate neuronal plasticity. Also the modulation or differential activation of UPS can produce hypothalamic neurodegeneration and obese phenotype in experimental animals. Here, we hypothesized that under prolonged diet-induced obesity, a defect in the UPS in the hypothalamus could contribute for the defective control of energy homeostasis leading to the refractoriness of obesity to caloric restriction. In fact in an obesity-prone rodent strain, prolonged, but not short-term obesity was accompanied by functional abnormality of the UPS in the hypothalamus. In mutants protected from inflammation, resistance to diet-induced obesity was accompanied by stability of the UPS in the hypothalamus. Thus, defect of the UPS in the hypothalamus, during prolonged obesity is an important factor contributing the refractoriness of obesity to caloric restriction Doutorado Biologia Estrutural, Celular, Molecular e do Desenvolvimento Doutora em Fisiopatologia Médica

Agradecimentos: LNBio/CNPEM; Brazilian National Council for Scientific and Technological Development (CNPq)National Council for Scientific and Technological Development (CNPq) [306879/2014-0, 310536/2014-6, 422790/2016-8]; Sao Paulo Research Foundation (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2012/50981-5, 2013/08028-1, 2015/06281-7]; NIH/NIGMSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of General Medical Sciences (NIGMS) [R01 GM088055] Ubiquitin-conjugating enzymes (E2) enable protein ubiquitination by conjugating ubiquitin to their catalytic cysteine for subsequent transfer to a target lysine side chain. Deprotonation of the incoming lysine enables its nucleophilicity, but determinants of lysine activation remain poorly understood. We report a novel pathogenic mutation in the E2 UBE2A, identified in two brothers with mild intellectual disability. The pathogenic Q93E mutation yields UBE2A with impaired aminolysis activity but no loss of the ability to be conjugated with ubiquitin. Importantly, the low intrinsic reactivity of UBE2A Q93E was not overcome by a cognate ubiquitin E3 ligase, RAD18, with the UBE2A target PCNA. However, UBE2A Q93E was reactive at high pH or with a lowpK a amine as the nucleophile, thus providing the first evidence of reversion of a defective UBE2A mutation. We propose that Q93E substitution perturbs the UBE2A catalytic microenvironment essential for lysine deprotonation during ubiquitin transfer, thus generating an enzyme that is disabled but not dead FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Fechado

The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention., This work was funded by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Exactas y Naturales (FCEyN), Universidad de Buenos Aires (UBA) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina. (PICT 2005 N° 33631). Additional support was received from Boehringer-Ingelheim research fellowship. The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from the Wellcome Trust [104111].

A pathogenic GGGCCC hexanucleotide expansion in the first intron/promoter region of the C9orf72 gene is the most common mutation associated with amyotrophic lateral sclerosis (ALS). The C9orf72 gene product forms a complex with SMCR8 (Smith-Magenis Syndrome Chromosome Region, Candidate 8) and WDR41 (WD Repeat domain 41) proteins. Recent studies have indicated roles for the complex in autophagy regulation, vesicle trafficking, and immune response in transgenic mice, however a direct connection with ALS etiology remains unclear. With the aim of increasing understanding of the multi-functional C9orf72-SMCR8-WDR41 complex, we determined by mass spectrometry analysis the proteins that directly associate with SMCR8. SMCR8 protein binds many components of the ubiquitin-proteasome system, and we demonstrate its poly-ubiquitination without obvious degradation. Evidence is also presented for localization of endogenous SMCR8 protein to cytoplasmic stress granules. However, in several cell lines we failed to reproduce previous observations that C9orf72 protein enters these granules. SMCR8 protein associates with many products of genes associated with various Mendelian neurological disorders in addition to ALS, implicating SMCR8-containing complexes in a range of neuropathologies. We reinforce previous observations that SMCR8 and C9orf72 protein levels are positively linked, and now show in vivo that SMCR8 protein levels are greatly reduced in brain tissues of C9orf72 gene expansion carrier individuals. While further study is required, these data suggest that SMCR8 protein level might prove a useful biomarker for the C9orf72 expansion in ALS., United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Neurological Disorders & Stroke (NINDS) 1R03NS087290-01, United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) R21HD083915-01A1, ALS Therapy Alliance 2013-F-067, European Research Council (ERC) ERC-STG-2012-309433, MINECO-FEDER SAF2017-89745-R, Wellcome Trust-University of Edinburgh Institutional Strategic Support Fund (ISFF2), United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Center for Advancing Translational Sciences (NCATS) UL1 TR003098

Tesis (Doctor en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2020 La endocitosis es un proceso crucial para todas las células ya que les permite incorporar material desde el espacio extracelular y controlar la disponibilidad de proteínas transmembrana en la membrana plasmática (MP). Después de ser internalizadas por endocitosis las proteínas cargo se transportan a los endosomas y luego a los lisosomas/vacuolas donde son degradadas o pueden ser recicladas de nuevo a la MP siguiendo la vía de transporte retrógrado desde los endosomas al aparato de Golgi, donde son incorporadas nuevamente a la vía secretora. En la levadura, la endocitosis seguida de reciclado a la MP resulta en una distribución polarizada de las proteínas , que se concentran en la membrana de la célula hija. De acuerdo al modelo canónico, la selección de proteínas transmembrana para ser internalizadas por endocitosis mediada por vesículas cubiertas de clatrina involucra la presencia de señales en los dominios citoplásmicos de las proteínas cargo. Estas señales son reconocidas por distintos receptores o adaptadores que forman parte de la cubierta proteica y es mediante esta interacción que las proteínas cargo transmembrana son concentradas en los sitios de la MP donde inicia la endocitosis. En este trabajo de tesis hemos estudiado la influencia de la geometría del dominio transmembrana (TMD) en la endocitosis de proteínas con solo un paso transmembrana. Utilizando TMDs quiméricos mostramos que la geometría del TMD actúa como un determinante de transporte a nivel de la MP. Particularmente, demostramos que un TMD largo con una mitad exoplásmica conformada por aminoácidos voluminosos resulta en una señal de polaridad y endocitosis en Saccharomyces cerevisiae. Una búsqueda bioinformática en el proteoma de este organismo modelo identificó varias proteínas con un hemi- MD exoplásmico largo y voluminoso, lo que nos permitió validar nuestros resultados utilizando TMDs endógenos de levadura. Por otro lado, también demostramos que un hemi- TMD exoplásmico largo y voluminoso es funcional como señal de endocitosis en células de mamífero en cultivo. Además, realizamos diferentes análisis para comprender cómo proteínas con un hemi-TMD exoplásmico largo y voluminoso resultan endocitadas. Encontramos que la internalización de las mismas es afectada de forma indirecta por la ubiquitin ligasa Rsp5. Por otro lado, mostramos que la geometría de los TMD s determina la partición de los mismos a diferentes dominios lipídicos in vivo. La identificación de este novedoso determinante de endocitosis dado por la geometría del TMD muestra una nueva forma mediante la cual las proteínas cargo pueden ser reconocidas e incluidas en la vía endocítica. Esta forma se basa en un principio fundamental diferente de las señales endocíticas previamente conocidas y, por lo tanto, agrega nuevas perspectivas sobre el mecanismo de endocitosis. 2023-06-01 Bigliani, Gonzalo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina. Valdez Taubas, Javier. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Touz, María Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Alvarez, Cecilia Inés. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Ambroggio, Ernesto Esteban. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Aguilar, Pablo S. Universidad de Buenos Aires. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina.

Primary cilia are microtubule-based sensory organelles that are involved in the organization of numerous key signals during development and in differentiated tissue homeostasis. In fact, the formation and resorption of cilia highly depends on the cell cycle phase in replicative cells, and the ubiquitin proteasome pathway (UPS) proteins, such as E3 ligases and deubiquitinating enzymes, promote microtubule assembly and disassembly by regulating the degradation/availability of ciliary regulatory proteins. Also, many differentiated tissues display cilia, and mutations in genes encoding ciliary proteins are associated with several human pathologies, named ciliopathies, which are multi-organ rare diseases. The retina is one of the organs most affected by ciliary gene mutations because photoreceptors are ciliated cells. Photoreception and phototransduction occur in the outer segment, a highly specialized neurosensory cilium. In this review, we focus on the function of UPS proteins in ciliogenesis and cilia length control in replicative cells and compare it with the scanty data on the identified UPS genes that cause syndromic and non-syndromic inherited retinal disorders. Clearly, further work using animal models and gene-edited mutants of ciliary genes in cells and organoids will widen the landscape of UPS involvement in ciliogenesis and cilia homeostasis.

Protein modifications by phosphorylation or ubiquitylation have been selected throughout evolution as efficient regulatory mechanisms of cellular processes. Cell migration is a complex, highly coordinated process where these mechanisms must participate in an integrated manner to transmit signaling during migration. In this study, we show that the ubiquitin ligase HERC1 regulates the p38 signaling pathway, and that this regulation is mediated by the MAPK kinase MKK3. Moreover, we demonstrate a crosstalk between RAF and MKK3/p38 pathways where RAF acts upstream of MKK3. Mechanistically, HERC1 regulates the protein levels of C-RAF and MKK3. Thus, HERC1 ubiquitylates C-RAF, targeting it for proteasomal degradation, and RAF proteins regulate MKK3 mRNA levels. Accordingly, HERC1 knockdown induces C-RAF stabilization and activation of RAF proteins; in turn, this activation increases MKK3, which phosphorylates and activates p38. The importance of these observations is demonstrated by HERC1 regulation of cell migration through regulation of p38 signaling via a RAF-dependent mechanism. Thus, HERC1 plays an essential role as a regulator of crosstalk between RAF/MKK3/p38 signaling pathways during cell migration.

HERC1 is a ubiquitin ligase protein, which, when mutated, induces several malformations and intellectual disability in humans. The animal model of HERC1 mutation is the mouse tambaleante characterized by: (1) overproduction of the protein; (2) cerebellar Purkinje cells death by autophagy; (3) dysregulation of autophagy in spinal cord motor neurons, and CA3 and neocortical pyramidal neurons; (4) impairment of associative learning, linked to altered spinogenesis and absence of LTP in the lateral amygdala; and, (5) motor impairment due to delayed action potential transmission, decrease synaptic transmission efciency and altered myelination in the peripheral nervous system. To investigate the putative role of HERC1 in the presynaptic dynamics we have performed a series of experiments in cultured tambaleante hippocampal neurons by using transmission electron microscopy, FM1-43 destaining and immunocytochemistry. Our results show: (1) a decrease in the number of synaptic vesicles; (2) reduced active zones; (3) less clathrin immunoreactivity and less presynaptic endings over the hippocampal main dendritic trees; which contrast with (4) a greater number of endosomes and autophagosomes in the presynaptic endings of the tambaleante neurons relative to control ones. Altogether these results show an important role of HERC1 in the regulation of presynaptic membrane dynamics. Junta de Andalucía BIO-209 MINECO-DGICYT BFU2015-64536-R Junta de Andalucía BIO-122 MINECO-DGICYT BFU2015-64536-R MINECO-AEI/FEDER, UE, BFU2016-80295-R

The induction of protein degradation in a highly selective and efficient way by means of druggable molecules is known as targeted protein degradation (TPD). TPD emerged in the literature as a revolutionary idea: a heterobifunctional chimera with the capacity of creating an interaction between a protein of interest (POI) and a E3 ubiquitin ligase will induce a process of events in the POI, including ubiquitination, targeting to the proteasome, proteolysis and functional silencing, acting as a sort of degradative knockdown. With this programmed protein degradation, toxic and disease-causing proteins could be depleted from cells with potentially effective low drug doses. The proof-of-principle validation of this hypothesis in many studies has made the TPD strategy become a new attractive paradigm for the development of therapies for the treatment of multiple unmet diseases. Indeed, since the initial protacs (Proteolysis targeting chimeras) were posited in the 2000s, the TPD field has expanded extraordinarily, developing innovative chemistry and exploiting multiple degradation approaches. In this article, we review the breakthroughs and recent novel concepts in this highly active discipline., Research funded by Consejo Superior de Investigaciones Científicas (Spanish National Research Council) (project 202020E161, CSIC-COV19). Additional partial financial support from Project CTQ2017-85378-R (AEI/FEDER, UE) from the Spanish Ministry of Science, Innovation and Universities, is acknowledged., Publisher’s version

The p53 tumor suppressor protein is a transcription factor that plays a prominent role in protecting cells from malignant transformation. Protein levels of p53 and its transcriptional activity are tightly regulated by the ubiquitin E3 ligase MDM2, the gene expression of which is transcriptionally regulated by p53 in a negative feedback loop. The p53 protein is transcriptionally active as a tetramer, and this oligomerization state is modulated by a complex formed by NEURL4 and the ubiquitin E3 ligase HERC2. Here, we report that MDM2 forms a complex with oligomeric p53, HERC2, and NEURL4. HERC2 knockdown results in a decline in MDM2 protein levels without affecting its protein stability, as it reduces its mRNA expression by inhibition of its promoter activation. DNA damage induced by bleomycin dissociates MDM2 from the p53/HERC2/NEURL4 complex and increases the phosphorylation and acetylation of oligomeric p53 bound to HERC2 and NEURL4. Moreover, the MDM2 promoter, which contains p53‐response elements, competes with HERC2 for binding of oligomeric, phosphorylated and acetylated p53. We integrate these findings in a model showing the pivotal role of HERC2 in p53‐MDM2 loop regulation. Altogether, these new insights in p53 pathway regulation are of great interest in cancer and may provide new therapeutic targets., The ubiquitin ligase MDM2 regulates protein levels of the p53 tumor suppressor protein. p53 is a transcription factor that regulates MDM2 expression. This study reports the formation of a NEURL4/HERC2/oligomeric p53/MDM2 complex and how it participates in the stability of p53 and MDM2 proteins and in the transcriptional activation of p53 in response to DNA damage.

Homologous to the E6AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing proteins (HERCs) belong to the superfamily of ubiquitin ligases. HERC proteins are divided into two subfamilies, Large and Small HERCs. Despite their similarities in terms of both structure and domains, these subfamilies are evolutionarily very distant and result from a convergence phenomenon rather than from a common origin. Large HERC genes, HERC1 and HERC2, are present in most metazoan taxa. They encode very large proteins (approximately 5,000 amino acid residues in a single polypeptide chain) that contain more than one RCC1-like domain as a structural characteristic. Accumulating evidences show that these unusually large proteins play key roles in a wide range of cellular functions which include neurodevelopment, DNA damage repair, and cell proliferation. To better understand the origin, evolution, and function of the Large HERC family, this minireview provides with an integrated overview of their structure and function and details their physiological implications. This study also highlights and discusses how dysregulation of these proteins is associated with severe human diseases such as neurological disorders and cancer.

111 p.-31 fig.-8 tab., [EN]Leishmania infantum is a protozoan parasite that causes leishmaniasis in the northern Mediterranean basin. It presents a complex biological cycle with an extracellular phase in the digestive tract of the insect vector and an intracellular phase within the mammalian host. Gene expression throughout the extracellular phase presents variations that seem to be aimed at preparing the parasite to resist the activity of the host immune system in what has been called pre-adaptation theory. In this pre-infection phase, the gene corresponding to the ubiquitin conjugate protein E2 (LinJ.33.2910) appears over-expressed. Given the importance of the functionality of this protein (LiUbc1) along the evolutionary scale, the characterization of this protein in a primitive eukaryotic such as L. infantum is of interest. We have compared this protein with its homologous proteins in other protozoa of the same family and of other lower and higher eukaryotes. It has been seen that homology remains within the genus, decreases in the same family and falls with respect to those of other higher eukaryotes. Molecular modeling of the protein suggests that it is a homodimer similar to that of the structure of Saccharomyces cerevisiae and shows partial homology with that of Homo sapiens. We have proceeded to the cloning of the gene and its expression, purifying the recombinant protein. Its expression has been studied in two different isolates of the parasite and differences between them have been detected. The existence of a dimeric and a monomeric form has been confirmed as predicted by molecular models. Knock-in promastigotes have been obtained that over-express the LiUbc1 protein and its infective capacity has been studied in vitro. It has been detected that over-expression favors the survival of the amastigote phase in the first 24h of the infection in vitro. This suggests that the LiUbc1 protein may be related to the infective capacity of the parasite. The location of the LiUbc1 protein has been studied in wild type and knock-in promastigotes. It has been found to have a location in subcellular structures (nuclear membrane, kinetoplast and flagellar pocket). These structures match the regulatory function assigned to the protein. A challenge experiment against infection in the Mesocricetus auratus model has shown that inoculation of the LiUbc1 protein induces protection in animals against unvaccinated controls. These experiments confirm that the LiUbc1 protein could be a vaccine candidate., [ES]Leishmania infantum es protozoo parásito causante de leishmaniasis en la cuenca norte Mediterránea. Presenta un ciclo biológico complejo con una fase extracelular en el aparato digestivo del insecto vector y una fase intracelular dentro del hospedador mamífero. La expresión génica a lo largo de la fase extracelular presenta variaciones que parece que van dirigidas a preparar al parásito para resistir la actividad del sistema inmune del huésped en lo que se ha llamado teoría de la pre-adaptación. En esta fase previa a la infección, el gen correspondiente a la proteína conjugadora de ubiquitina E2 (LinJ.33.2910), aparece sobre expresado. Dada la importancia de la funcionalidad de esta proteína (LiUbc1) a lo largo de la escala evolutiva, resulta de interés la caracterización de esta proteína en un eucariota primitivo como es L. infantum. Se ha procedido a comparar dicha proteína con sus proteínas homólogas en otros protozoos de la misma familia y de otros eucariotas inferiores y superiores. Se ha visto que la homología se mantiene dentro del género, disminuye en la misma familia y desciende respecto a las de otros eucariotas superiores. El modelado de la proteína sugiere que se trata de un homodímero similar al de la estructura de Saccharomyces cerevisiae y muestra una homología parcial con la de Homo sapiens. Se ha procedido al clonaje del gen y a la expresión del mismo, purificando la proteína recombinante. Se ha estudiado su expresión en dos aislados distintos del parásito y se han detectado diferencias entre los mismos. Se ha confirmado la existencia de una forma dimérica y otra monomérica como predecían los modelos moleculares. Se han obtenido promastigotes knock-in que la sobre-expresan la proteína LiUbc1 y se ha estudiado su capacidad infectiva in vitro. Se ha detectado que la sobre-expresión favorece la supervivencia de la fase amastigote en las primeras 24h de la infección. Esto sugiere que la proteína LiUbc1 puede estar relacionada con la capacidad infectiva del parásito. Se ha estudiado la localización de la proteína LiUbc1 en ambos aislados y además en los promastigotes knock-in. Se ha visto que tiene una localización en estructuras subcelulares (membrana nuclear, kinetoplasto y bolsillo flagelar). Estas estructuras concuerdan con la función de regulación asignada a la proteína. En un experimento de reto frente a la infección en el modelo de Mesocricetus auratus se ha mostrado que la inoculación de la proteína LiUbc1 induce protección en los animales frente a los controles no vacunados. Estos experimentos confirman que la proteína LiUbc1 puede ser un candidato vacunal.

Orientador: Prof. Dr. Silvio Marques Zanata Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia. Defesa : Curitiba, 30/09/2019 Inclui referências: p. 54-60 Resumo: A degradação seletiva de muitas proteínas intracelulares é realizada pela via ubiquitina, responsável por mecanismos homeostáticos celulares como a regulação de vias de sinalização, endocitose e apoptose. Este processo é reversível e regulado negativamente pelas enzimas deubiquitinases (DUB), dentre as quais a família USP (ubiquitin specific protease) possui grande importância no papel funcional da proliferação e sobrevivência celular. Alterações na via ubiquitina são descritas em condições patológicas, devido a ocorrência de danos em proteínas celulares, acúmulo intracelular proteico e redução da atividade proteossômica, podendo resultar em supressão da apoptose e progressão descontrolada do ciclo celular e suas diversas sinalizações. Dentre as isoformas de USP2, a USP2a tem sido amplamente associada à patogenia de tumores, seja por sua interferência em proteínas do ciclo celular, oncoproteínas, e na via de degradação de receptores tirosina cinase, como o EGFR (receptor de fator de crescimento epidermal). Por outro lado, existem poucos estudos relacionados à caracterização de USP2b, que poderiam identificar sua possível inferência em processos patológicos. Deste modo, o objetivo foi construir ferramentas e estabelecer condições experimentais para a investigação bioquímica das deubiquitinases USP2a e USP2b. USP2b recombinante com cauda de histidina foi produzida em bactérias E. coli Rosetta2(DE3) pLysS e submetida a protocolos de purificação. Embora sem desfecho positivo na expressão da USP2b nas condições citadas, obteve-se com sucesso a expressão de USP2a e USP2b em células de mamíferos (HeLa/HEK293T) utilizando-se diferentes métodos de transfecção (lipofectamina e co-precipitação com cálcio), possibilitando a identificação de picos de expressão conforme a metodologia aplicada. Experimentos permitiram a identificação de USP2a endógena enriquecida na fração 30% de saturação de sulfato de amônio. Além disso, o anticorpo monoclonal anti-USP2a secretado pelo hibridoma 4GD2, previamente elaborado pelo grupo, permitiu a imunoprecipitação de USP2a tanto endogenamente expressa por células HeLa como superexpressa na linhagem HEK293T. Evidências experimentais obtidas neste trabalho sugerem que a USP2b tem a capacidade de estabilizar EGFR, de modo semelhante ao já descrito para a isoforma USP2a. Espera-se que os resultados gerados possam contribuir para o desenvolvimento de novas ferramentas terapêuticas e diagnósticas bem como no mecanismo de funcionamento destas deubiquitinases. Palavras-chave: Enzimas deubiquitinases. USP2. USP2a. USP2b. EGFR. EGF. Abstract: Selective degradation of many intracellular proteins is performed by ubiquitin pathway, which is responsible for cellular homeostatic mechanisms, such as regulation of cellular signaling, endocytosis and apoptosis. Ubiquitylating modification is reversible, negatively regulated by deubiquitinases enzymes (DUB), among which the USP family (ubiquitin specific protease) has great importance in the functional role of cell proliferation and survival. Disturbance on ubiquitin pathway are described under pathological conditions, due to cellular proteins damage, intracellular protein accumulation and reduction of proteasomic activity, may causing apoptosis suppression, and uncontrolled cell cycle and its several signals. Amog USP2 isoforms, USP2a has been widely associated in tumor pathogenesis, whether by their interference with cell cycle proteins, oncoproteins, and on tyrosine kinase receptor degradation pathway, such as EGFR (epidermal growth factor receptor). Meanwhile, there are few studies on USP2b characterization, that could recognize its possible inference in pathological processes. Therefore, the aim of this study was to create tools and establish experimental conditions for deubiquitinases USP2a and USP2b biochemical investigation. Recombinant USP2b with histidine tail was produced in E. coli Rosetta2(DE3) pLysS bacteria for purification protocols, however, not resulting on the expected expression. USP2a and USP2b expression in mammalian cells (HeLa/HEK293T) was successfully obtained by different transfection methods (lipofectamine and calcium co-precipitation), enabling identification of expression peaks according to the methodology applied. Experiments allowed identification of endogenous USP2a enriched in the fraction of 30% ammonium sulfate saturation. Also, anti-USP2a monoclonal antibody secreted by 4GD2 hybridoma, previously obtained by the group, allowed USP2a immunoprecipitation at HeLa cells endogenously expression, and on HEK293T at overexpression conditions. Experimental evidences of this study suggest that UPS2b has the ability to stabilize EGFR, in a similiar manner already described in USP2a isoform. The obtained results are expected to contribute to new therapeutic and diagnostic tools development, as well as the functioning mechanism of these deubiquitinases. Keywords: Deubiquitinases enzymes. USP2. USP2a. USP2b. EGFR. EGF.

© 2021 by the authors., We study self-association of ubiquitin and the disordered protein ACTR using the most commonly used water models. We find that dissociation events are found only with TIP4P-EW and TIP4P/2005, while the widely used TIP3P water model produces straightforward aggregation of the molecules due to the absence of dissociation events. We also find that TIP4P/2005 is the only water model that reproduces the fast association/dissociation dynamics of ubiquitin and best identifies its binding surface. Our results show the critical role of the water model in the description of protein–protein interactions and binding., This research was funded by the Spanish government, grant numbers CTQ2016-78636-P and PGC2018-099277-B-C21 (MCIU/AEI/ERDF) and the Catalan government, grant number 2017 SGR 324., Publisher’s version

The E3 ubiquitin ligase RNF8 plays critical roles in maintaining genomic stability by promoting the repair of DNA double-strand breaks (DSBs) through ubiquitin signaling. Abnormal activation of Notch signaling and defective repair of DSBs promote breast cancer risk. Here, we found that low expression of the full-length RNF8 correlated with poor prognosis for breast cancer patients. Our data revealed that in addition to its role in the repair of DSBs, RNF8 regulated Notch1 signaling and cell-fate determination of mammary luminal progenitors. Mechanistically, RNF8 acted as a negative regulator of Notch signaling by ubiquitylating the active NOTCH1 protein (N1ICD), leading to its degradation. Consistent with abnormal activation of Notch signaling and impaired repair of DSBs in Rnf8-mutant mammary epithelial cells, we observed increased risk of mammary tumorigenesis in mouse models for RNF8 deficiency. Notably, deficiency of RNF8 sensitized breast cancer cells to combination of pharmacological inhibitors of Notch signaling and poly(ADP-ribose) polymerase (PARP), suggesting implications for treatment of breast cancer associated with impaired RNF8 expression or function.

La formación y progresión de tumores está regulada en última instancia por la abundancia y la actividad de factores proteicos oncogénicos o supresores tumorales. En línea con esto se ha demostrado que la proteólisis irregular de factores proteicos implicados en la regulación del ciclo celular, la transcripción génica y la apoptosis contribuye a la transformación oncogénica y a la carcinogénesis. En células eucariotas, la homeostasis proteica está finamente regulada por el Sistema Ubiquitina/Proteosoma (Ubiquitin Proteosome System, UPS), que consiste en una compleja red enzimática encargada de transferir moléculas de ubiquitina a un sustrato y de este modo promover su degradación a través del proteosoma o modular su ubicación espacio temporal en una vía celular determinada. Por lo tanto, el estudio a nivel molecular de la cascada de ubiquitinación, junto con el desarrollo de compuestos que modulan componentes específicos del UPS, podrían facilitar el desarrollo y mejoramiento de nuevos y mejores tratamientos de distintas patologías. En este sentido, las ligasas de ubiquitina (o E3s) son excelentes candidatas debido a que estas enzimas son los efectores finales de la cascada de ubiquitinación y dictan la especificidad de la maquinaria del UPS. La E3 ligasa CRL4^Cdt2 desempeña un rol fundamental en la regulación de la replicación y reparación del ADN, en condiciones normales como así también en respuesta al daño al ADN. Diferentes evidencias indican que las alteraciones en los niveles de CRL4^Cdt2 pueden contribuir a la aparición y progresión del cáncer. Todos los sustratos conocidos de CRL4^Cdt2 tienen un dominio PIP (PCNA Interacting Protein) que interactúa con PCNA (Proliferating Cell Nuclear Antigen) asociado a la cromatina, y es precisamente la unión de los sustratos a PCNA lo que asegura su degradación. Por este motivo, con el objeto de identificar nuevos sustratos de CRL4^Cdt2 que permitan ahondar en el mecanismo molecular de acción de este complejo, se realizó una purificación por afinidad en tándem de Cdt2 sobre-expresado y posterior espectrometría de masa en tándem en la fracción cromatínica. A partir de estos resultados se escogieron distintos candidatos teniendo en cuenta la frecuencia de aparición de los mismos en las distintas purificaciones y se logró validar la interacción de Cdt2 sobre-expresado en la cromatina con G9a y GLP. Estas proteínas son metiltransferasas de histonas que catalizan la mono y la dimetilación de la Histona 3 en la Lisina 9, esta marca epigenética se encuentra relacionada con silenciamiento transcripcional y es indispensable para la constitución de la heterocromatina. Debido a que G9a tiene la capacidad de metilar también proteínas no histónicas realizamos un análisis de modificaciones postraduccionales de Cdt2 en una línea control y en líneas knockout que no expresan G9a y GLP (generadas por el sistema CRISPR/Cas9), donde no se encontraron péptidos de Cdt2 metilados diferencialmente en la línea control, aunque se requieren estudios adicionales y más detallados para obtener una respuesta más sólida y definitiva. Logramos identificar y validar la interacción de Cdt2 con otras dos proteínas que interactúan con el complejo G9a/GLP y median su interacción con el ADN. Utilizando ARN de interferencia de Cdt2 comprobamos que la vida media de las proteínas que forman parte de este complejo no se encuentra regulada por esta E3 ligasa, así como tampoco la localización subcelular. Debido a que el silenciamiento de Cdt2 ocasiona re-replicación e inestabilidad genómica, desarrollamos también una línea celular que no expresa Cdt2 endógeno y expresa una construcción Cdt2-Flag que es inducible con doxiciclina. Los experimentos de vida media en diferentes clones de esta línea celular confirmaron los resultados obtenidos con ARN de interferencia. Por último, es importante destacar que CRL4^Cdt2 también cumple un rol importante en la regulación de la respuesta celular a estrés genotóxico. Asimismo, recientemente se ha reportado que G9a interviene en la regulación de la respuesta al daño al ADN. Por lo tanto, estamos actualmente estudiando si la interacción funcional entre CRL4^Cdt2 y G9a/GLP posee una función importante en la modulación de la respuesta celular a distintos daños. En este sentido hemos obtenidos resultados preliminares muy alentadores. Esperamos que en estudios posteriores podamos ahondar en la caracterización molecular del eje CRL4Cdt2/G9a y la función que cumple en el control de la integridad genómica. The formation and progression of tumors is ultimately regulated by the abundance and activity of oncogenic protein factors or tumor suppressors. Accordingly, aberrant proteolysis of substrates involved in the regulation of cell cycle, gene transcription, and apoptosis has been shown to contribute to carcinogenesis. In eukaryotic cells, protein homeostasis is tightly regulated by the Ubiquitin Proteasome System (UPS), which consists of a complex enzymatic network responsible for transferring molecules of ubiquitin to a substrate and thereby promote its degradation through proteasome or modulate its spatio-temporal localization in a specific cell pathway. Therefore, the molecular study of ubiquitination cascades, together with the development of compounds that modulate specific components of the UPS, could facilitate the development and improvement of new and better strategies for the treatment of different pathologies. Accordingly, the ubiquitin ligase protein group (or E3s) represents an excellent candidate, because these enzymes are the final effectors of the ubiquitination cascade and thus dictate the specificity of UPS machinery. E3 ligase CRL4^Cdt2 plays a key role in the regulation of DNA replication and repair, in normal conditions as well as in response to DNA damage. Different lines of evidence indicate that alterations in the levels of CRL4Cdt2 may contribute to the onset and progression of cancer. All known substrates of CRL4^Cdt2 have a PIP domain (PCNA Interacting protein) that interacts with PCNA (Proliferating Cell Nuclear Antigen) associated with chromatin, and it is precisely the binding of the substrates to PCNA which ensures its timely degradation. Hence, with the aim to gain further insight into the molecular mechanisms of action of CRL4^Cdt2, we performed a tandem affinity purification of overexpressed Cdt2 and subsequent tandem mass spectrometry using only the chromatin cellular fraction. Using this strategy, among the most abundant putative Cdt2 protein interacting factors, we identified and validated the interaction between overexpressed Cdt2 and G9a/GLP. These two proteins are histone methyltransferases that catalyze the mono and dimethylation of Histone 3 in Lysine 9, which plays an important role in the transcriptional silencing process and is necessary for heterochromatin domains formation. G9a has also the ability to methylate non-histone proteins, and for this reason we performed post-translational modifications analysis of Cdt2 on knockout cell lines that do not express G9a and GLP, that were generated by the CRISPR/Cas9 system. So far, we do not find significant differences between normal and knockout cell lines although further and more detailed studies are required to obtain a more robust and definitive answer. We were able to identify and validate the interaction of Cdt2 with two other proteins that interact with the G9a/GLP and mediate their interaction with the DNA. Using interference RNA against Cdt2, we found that CRL4^Cdt2 does not regulate the half-life of the G9a/GLP protein complex, neither its subcellular localization. Because Cdt2 silencing causes re-replication and genomic instability, we also developed a cell line that does not express endogenous Cdt2 but harbors a doxycycline inducible Cdt2-Flag construct that allowed us to perform half-life experiments without affecting overall cellular homeostasis. Using different clones, we confirmed the results obtained with small interfering RNA. Finally, as mentioned before, it is well established that CRL4Cdt2 plays an important role in the regulation of the genotoxic stress response. Accordingly, recent reports indicate that G9a also intervenes in the regulation of the response to DNA damage. Therefore, we are currently studying whether the functional interaction between CRL4^Cdt2 and the G9a/GLP complex plays an important role in the modulation of the cellular response to different DNA damage agents. In this regard, we have obtained very encouraging preliminary results. Further studies are required to unveil the specific and intricate molecular mechanism of action of the CRL4^Cdt2/G9a axis and its role in the control of genomic stability. Fil: Enriqué Steinberg, Juliana Haydeé. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Tesi di Laurea Magistrale in Scienze Chimiche di Francesco Mura

Sumoylation is a reversible post-translational modification that regulates different cellular processes by conjugation/deconjugation of SUMO moieties to target proteins. Most work on the functional relevance of SUMO has focused on cell cycle, DNA repair and cancer in cultured cells, but data on the inter-dependence of separate components of the SUMO pathway in highly specialized tissues, such as the retina, is still scanty. Nonetheless, several retinal transcription factors (TFs) relevant for cone and rod fate, as well as some circadian rhythm regulators, are regulated by sumoylation. Here we present a comprehensive survey of SUMO pathway gene expression in the murine retina by quantitative RT-PCR and in situ hybridization (ISH). The mRNA expression levels were quantified in retinas obtained under four different light/dark conditions, revealing distinct levels of gene expression. In addition, a SUMO pathway retinal gene atlas based on the mRNA expression pattern was drawn. Although most genes are ubiquitously expressed, some patterns could be defined in a first step to determine its biological significance and interdependence. The wide expression of the SUMO pathway genes, the transcriptional response under several light/dark conditions, and the diversity of expression patterns in different cell layers clearly support sumoylation as a relevant post-translational modification in the retina. This expression atlas intends to be a reference framework for retinal researchers and to depict a more comprehensive view of the SUMO-regulated processes in the retina.

The 26S proteasome is the central proteolytic machinery of the ubiquitin proteasome system (UPS), which is involved in the degradation of ubiquitinated protein substrates. Recently, UPS inhibition has been shown to be a key factor in fatty liver graft preservation during organ cold storage using University of Wisconsin solution (UW) and Institute Georges Lopez (IGL-1) solutions. However, the merits of IGL-1 and histidine-tryptophan-ketoglutarate (HTK) solutions for fatty liver preservation have not been compared. Fatty liver grafts from obese Zücker rats were preserved for 24 h at 4 ◦C. Aspartate aminotransferase and alanine aminotransferase (AST/ALT), glutamate dehydrogenase (GLDH), ATP, adenosine monophosphate protein kinase (AMPK), e-NOS, proteasome activity and liver polyubiquitinated proteins were determined. IGL-1 solution prevented ATP breakdown during cold-storage preservation of steatotic livers to a greater extent than HTK solution. There were concomitant increases in AMPK activation, e-NOS (endothelial NOS (NO synthase)) expression and UPS inhibition. UPS activity is closely related to the composition of the solution used to preserve the organ. IGL-1 solution provided significantly better protection against ischemia-reperfusion for cold-stored fatty liver grafts than HTK solution. The effect is exerted through the activation of the protective AMPK signaling pathway, an increase in e-NOS expression and a dysregulation of the UPS., This work was supported by Instituto de Salud Carlos III (ISCIII) through the FIS project PI12/0056 co-funded by FEDER from Regional Development European Funds (European Union).

p53 is a transcription factor that regulates important cellular processes related to tumor suppression, including induction of senescence, apoptosis, and DNA repair as well as the inhibition of angiogenesis and cell migration. Therefore, it is critical to understand the molecular mechanism that regulates it. p53 tetramerization is a key step in its activation process and the regulation of this oligomerization, an important control point. The E3 ubiquitin ligase HERC2 controls the p53 transcriptional activity by regulation of its oligomerization state. HERC2-interacting proteins such as the adaptor-like protein with six neuralized domains NEURL4 are also candidates to regulate p53 activity. Here, we demonstrate the existence of an interaction network between NEURL4, HERC2 and p53 proteins. We report a functional interaction between NEURL4 and p53, involving the C-terminal region of p53 and the neuralized domains 3 and 4 of NEURL4. Through this interaction, NEURL4 regulates the transcriptional activity of p53. Thus, NEURL4 depletion reduced the transcriptional activity whereas NEURL4 overexpression increased it. In both cases, p53 stability was not affected. Although NEURL4 may interact with p53 independently of the E3 ubiquitin ligase HERC2, we observed that both proteins are needed to regulate the transcriptional activity of p53. Clonogenic assays confirmed the functional relevance of this interaction observing a decrease in cell growth by NEURL4 overexpression correlated to the increase of cellular cycle inhibitor p21 by p53 activation. Under these conditions, NEURL4 activated p53 oligomerization. All these findings identify NEURL4 as a novel regulator of the p53's signaling.

La sumoïlació és una modificació posttraduccional (PTM) que consisteix en la conjugació covalent i reversible d’una proteïna SUMO (Small ubiquitin-like modifier) al residu lisina d’una proteïna diana. Igual que la ubiquitina, el cicle metabòlic de SUMO consta d’una conjugació, on hi participen tres tipus diferents d’enzims (E1 activadors, E2 conjugadors i E3 lligases), i d’una deconjugació, que es dóna a través de l’acció de proteases. A nivell funcional, la sumoïlació regula processos cel·lulars tan diversos com la replicació, la reparació del DNA, la localització, activitat i degradació de proteïnes, o la transcripció, entre d’altres. Els estudis existents sobre la sumoïlació se centraven en la modificació de proteïnes diana concretes, pel que mancaven estudis de la interdependència dels elements de la maquinària de SUMO en la regulació i el manteniments de teixits especialitzats. Un d’aquests teixits és la retina, que recobreix l’ull per la seva cara interna i s’encarrega de la captació i el processat de l’estímul lumínic. Forma part del sistema nerviós central, pel que es tracta de teixit neuronal, format per tres capes nuclears de neurones i les seves connexions. Les cèl·lules encarregades d’iniciar la cadena de la fototransducció són els fotoreceptors: cons i bastons; que comparteixen una mateixa estructura, i que difereixen en els fotopigments que hi contenen, fet que els dóna diferents funcionalitats lumíniques. Per aquest motiu, un dels objectius d’aquesta tesi va ser l’estudi de l’expressió dels gens de la via de SUMO a la retina de ratolí adult, per mitjà de la seva quantificació per RT-qPCR i de la seva localització en talls de retina per hibridació in situ. Es van detectar l’expressió de tots els gens de la maquinària de SUMO, amb nivells variables en concordança amb la seva importància dins del cicle. . A més, atès que la llum és un dels marcadors externs que controlen els ritmes circadiaris de l’organisme, es va estudiar l’expressió dels gens de la via de SUMO en diferents condicions del cicle de llum/foscor, detectant alguns patrons diferencials d’entre el conjunt de gens estudiats. Pel que fa a la localització de l’expressió, els gens de la via de SUMO van donar diferents patrons de localització a les diverses capes de la retina, coincidint tots a la capa de fotoreceptors. A continuació, es va estudiar la sumoïlació del factor de transcripció (TF) de fotoreceptors NR2E3, que juga un paper central en el desenvolupament i manteniment dels bastons, activant els gens específics de bastons i silenciant els de cons. Aquesta repressió està regulada, en part, per la seva sumoïlació a través de la E3 lligasa PIAS3, tot i que aquesta no és la única que modificava el TF. En aquest context, es va plantejar l’estudi de noves interaccions del TF amb altres E3 lligases de SUMO. Per mitjà de la tècnica de BRET es va poder trobar una nova interacció amb HDAC4, posteriorment confirmada per CoIP. A més, l’estudi comparatiu de la forma humana d’NR2E3 WT amb el seu mutant no sumoïlable (123) va permetre detectar diferències d’interacció amb PIAS3, més baixa amb el TF mutant. D’altra banda, la tècnica del BioID va permetre el descobriment d’una segona interacció amb una E3 lligasa de SUMO: CBX4. Per últim, amb la comparació de les formes WT i no sumoïlable d’NR2E3 es va detectar una nova conjugació de SUMO amb el TF, que no es veia alterada per cap de les E3 lligases descrites com a interactores. En conjunt, aquest treball demostra la importància de la sumoïlació en la regulació i el manteniment de teixits i tipus cel·lulars, com ho són la retina i els fotoreceptors., Sumoylation is a reversible post-translational modification that regulates different cellular processes by conjugation/deconjugation of SUMO moieties to target proteins. Most work on the functional relevance of SUMO has focused on cell cycle, DNA repair and cancer in cultured cells, but data on the inter-dependence of separate components of the SUMO pathway in highly specialized tissues, such as the retina, is still scanty. Nonetheless, several retinal transcription factors (TFs) relevant for cone and rod fate, as well as some circadian rhythm regulators, are regulated by sumoylation. Here we present a comprehensive survey of SUMO pathway gene expression in the murine retina by quantitative RT-qPCR and in situ hybridization (ISH). The mRNA expression levels were quantified in retinas obtained under four different light/dark conditions, revealing distinct levels of gene expression. In addition, a SUMO pathway retinal gene atlas based on the mRNA expression pattern was drawn. Although most genes are ubiquitously expressed, some patterns could be defined in a first step to determine its biological significance and interdependence. On the other hand, the orphan nuclear receptor NR2E3 is a retinal transcription factor (TF) that plays an essential role in the development and maintenance of rod photoreceptors by both activating rod-specific and repressing cone-specific genes. This repression is partially regulated by the E3 SUMO ligase PIAS3, which sumoylates NR2E3 in three specific –and evolutionarily conserved– lysine residues, turning it into a transcriptional repressor. However, PIAS3 is not the unique E3 SUMO ligase acting upon this TF. In this context, we aimed to find new interactions with other E3 SUMO ligases in order to elucidate the relevance of this post-translational modification in regulating the function of NR2E3. Two new interactions with the E3 SUMO ligase HDAC4 and CBX4 were discovered. In addition, the nonsumoylable mutant NR2E3 123 showed a diminished interaction with PIAS3 in comparison to the wild-type (and sumoylable) NR2E3 (WT). Moreover, we detected a new SUMO-conjugated NR2E3 form not detectable in NR2E3 123, although neither PIAS3, CBX4 nor HDAC4 seem to participate in this SUMO conjugation. Although the physiological role of these newly found interactions is still unknown, they provide new insights in the regulation of the transcriptional activity of this retinal TF by SUMO modification.

[cat] La sumoïlació és una modificació posttraduccional (PTM) que consisteix en la conjugació covalent i reversible d’una proteïna SUMO (Small ubiquitin-like modifier) al residu lisina d’una proteïna diana. Igual que la ubiquitina, el cicle metabòlic de SUMO consta d’una conjugació, on hi participen tres tipus diferents d’enzims (E1 activadors, E2 conjugadors i E3 lligases), i d’una deconjugació, que es dóna a través de l’acció de proteases. A nivell funcional, la sumoïlació regula processos cel·lulars tan diversos com la replicació, la reparació del DNA, la localització, activitat i degradació de proteïnes, o la transcripció, entre d’altres. Els estudis existents sobre la sumoïlació se centraven en la modificació de proteïnes diana concretes, pel que mancaven estudis de la interdependència dels elements de la maquinària de SUMO en la regulació i el manteniments de teixits especialitzats. Un d’aquests teixits és la retina, que recobreix l’ull per la seva cara interna i s’encarrega de la captació i el processat de l’estímul lumínic. Forma part del sistema nerviós central, pel que es tracta de teixit neuronal, format per tres capes nuclears de neurones i les seves connexions. Les cèl·lules encarregades d’iniciar la cadena de la fototransducció són els fotoreceptors: cons i bastons; que comparteixen una mateixa estructura, i que difereixen en els fotopigments que hi contenen, fet que els dóna diferents funcionalitats lumíniques. Per aquest motiu, un dels objectius d’aquesta tesi va ser l’estudi de l’expressió dels gens de la via de SUMO a la retina de ratolí adult, per mitjà de la seva quantificació per RT-qPCR i de la seva localització en talls de retina per hibridació in situ. Es van detectar l’expressió de tots els gens de la maquinària de SUMO, amb nivells variables en concordança amb la seva importància dins del cicle. . A més, atès que la llum és un dels marcadors externs que controlen els ritmes circadiaris de l’organisme, es va estudiar l’expressió dels gens de la via de SUMO en diferents condicions del cicle de llum/foscor, detectant alguns patrons diferencials d’entre el conjunt de gens estudiats. Pel que fa a la localització de l’expressió, els gens de la via de SUMO van donar diferents patrons de localització a les diverses capes de la retina, coincidint tots a la capa de fotoreceptors. A continuació, es va estudiar la sumoïlació del factor de transcripció (TF) de fotoreceptors NR2E3, que juga un paper central en el desenvolupament i manteniment dels bastons, activant els gens específics de bastons i silenciant els de cons. Aquesta repressió està regulada, en part, per la seva sumoïlació a través de la E3 lligasa PIAS3, tot i que aquesta no és la única que modificava el TF. En aquest context, es va plantejar l’estudi de noves interaccions del TF amb altres E3 lligases de SUMO. Per mitjà de la tècnica de BRET es va poder trobar una nova interacció amb HDAC4, posteriorment confirmada per CoIP. A més, l’estudi comparatiu de la forma humana d’NR2E3 WT amb el seu mutant no sumoïlable (123) va permetre detectar diferències d’interacció amb PIAS3, més baixa amb el TF mutant. D’altra banda, la tècnica del BioID va permetre el descobriment d’una segona interacció amb una E3 lligasa de SUMO: CBX4. Per últim, amb la comparació de les formes WT i no sumoïlable d’NR2E3 es va detectar una nova conjugació de SUMO amb el TF, que no es veia alterada per cap de les E3 lligases descrites com a interactores. En conjunt, aquest treball demostra la importància de la sumoïlació en la regulació i el manteniment de teixits i tipus cel·lulars, com ho són la retina i els fotoreceptors., [eng] Sumoylation is a reversible post-translational modification that regulates different cellular processes by conjugation/deconjugation of SUMO moieties to target proteins. Most work on the functional relevance of SUMO has focused on cell cycle, DNA repair and cancer in cultured cells, but data on the inter-dependence of separate components of the SUMO pathway in highly specialized tissues, such as the retina, is still scanty. Nonetheless, several retinal transcription factors (TFs) relevant for cone and rod fate, as well as some circadian rhythm regulators, are regulated by sumoylation. Here we present a comprehensive survey of SUMO pathway gene expression in the murine retina by quantitative RT-qPCR and in situ hybridization (ISH). The mRNA expression levels were quantified in retinas obtained under four different light/dark conditions, revealing distinct levels of gene expression. In addition, a SUMO pathway retinal gene atlas based on the mRNA expression pattern was drawn. Although most genes are ubiquitously expressed, some patterns could be defined in a first step to determine its biological significance and interdependence. On the other hand, the orphan nuclear receptor NR2E3 is a retinal transcription factor (TF) that plays an essential role in the development and maintenance of rod photoreceptors by both activating rod-specific and repressing cone-specific genes. This repression is partially regulated by the E3 SUMO ligase PIAS3, which sumoylates NR2E3 in three specific –and evolutionarily conserved– lysine residues, turning it into a transcriptional repressor. However, PIAS3 is not the unique E3 SUMO ligase acting upon this TF. In this context, we aimed to find new interactions with other E3 SUMO ligases in order to elucidate the relevance of this post-translational modification in regulating the function of NR2E3. Two new interactions with the E3 SUMO ligase HDAC4 and CBX4 were discovered. In addition, the nonsumoylable mutant NR2E3 123 showed a diminished interaction with PIAS3 in comparison to the wild-type (and sumoylable) NR2E3 (WT). Moreover, we detected a new SUMO-conjugated NR2E3 form not detectable in NR2E3 123, although neither PIAS3, CBX4 nor HDAC4 seem to participate in this SUMO conjugation. Although the physiological role of these newly found interactions is still unknown, they provide new insights in the regulation of the transcriptional activity of this retinal TF by SUMO modification.

Un gran número de enfermedades neurodegenerativas se caracterizan por la acumulación de agregados de proteínas amiloidogénicas como la Tau, Alfa-sinucleína y Beta-amiloide. Previa a la formación de agregados estables, aparecen especies intermedias de estas proteínas: los oligómeros. Los oligómeros pueden ser más tóxicos y patológicamente más significativos en enfermedades neurodegenerativas como el Alzheimer (EA) y el Parkinson (EP). Las neuronas son células que presentan una fuerte actividad metabólica asociadaauna elevada actividad transcripcional. Tratandose de células de vida larga las hacen vulnerables al daño oxidativo del genoma, incluyendo roturas de la cadena de DNA y mutaciones de sus bases. Existe una creciente evidencia de una acumulación de lesiones en elDNAque no se reparan en el sistema nervioso central (SNC) durante el envejecimiento acelerado y la neurodegeneración progresiva. Los metales pro-oxidantes, las especies reactivas de oxígeno (ROS) y las proteínas amiloidogénicas mal plegadas contribuyen a las alteraciones en el genoma del SNC durante el envejecimiento acelerado y la neurodegeneración progresiva. Multiples daños en el SNC impiden la reparacióndel DNA, señalando una respuesta al daño en el DNA (DDR) que conduce a la acumulación de daños persistentes, una característica común en las enfermedades neurodegenerativas esporádicas. La ubiquitina y los pequeños modificadores similares a la ubiquitina como las proteínas SUMO son los marcadores comunes que señalan la degradación. Aunque la modificación de grandes agregados amiloides por ubiquitinación está bien establecida, se sabe muy poco sobre el papel que la ubiquitina puede jugar en el procesamiento de los oligómeros y la importancia de la sumoilación. Muchas proteínas implicadas en la neurodegeneración están sumoiladas, notablemente por ejemplo la proteína Tau en los cerebros con EA. Una mejor comprensión de las modificaciones covalentes de los oligómeros podría tener un enorme impacto en el desarrollo de agentes terapéuticos para las enfermedades neurodegenerativas. Hemos revisado los últimos avances de las implicaciones etiológicas del daño del DNA versus el desequilibrio en la reparación, con la anormal señalización DDR en el desencadenamiento de la neurodegeneración y el potencial del DDR como objetivo para la mejora de las enfermedades neurodegenerativas. También nos hemos centrado en la proteólisis de Tau y otras proteínas amiloidogénicas inducidas por modificacionescovalentes,así como la relación entre los oligómeros de Tau y la sumoilación. A large number of neurodegenerative diseases are characterized by the accumulation of aggregates of amyloidogenic proteins such as Tau, Alpha-synuclein and Beta-amyloid. Prior to the formation of stable aggregates, intermediate species of these proteins appear Oligomers. Oligomers may be more toxic and pathologically more significant in neurodegenerative diseases such as Alzheimer's (EA) and Parkinson's (EP). Neurons are cells with a strong metabolic activity associated with a high transcriptional activity. When dealing with long-lived cells, they make them vulnerable to oxidative damage in the genome, including Double-strand DNA breaks and mutations of their bases. There are increased evidences of an accumulation of DNA damage that does not repair in the central nervous system (SNC) during accelerated aging and progressive neurodegeneration. Pro-oxidant metals, reactive oxygen species (ROS), and poorly folded amyloidogenic proteins contribute to alterations in the SNC genome during accelerated aging and progressive neurodegeneration. Multiple CNS damages prevent DNA repair by pointing to a DNA damage (DDR) response that leads to persistent damage accumulation, a common feature in sporadic neurodegenerative diseases. Ubiquitin and ubiquitin-like modifiers such as SUMO proteins are common markers to signal degradation. Although the modification of large amyloid aggregates by ubiquitination is well established, very little is known about the role that ubiquitin can play in the processing of oligomers and the importance of sumoylation. Many proteins involved in neurodegeneration are sumoylated, notably for example the Tau protein in brains with EA. A better understanding of the covalent modifications of the oligomers could have a huge impact on the development of therapeutic agents for neurodegenerative diseases. We have reviewed the latest advances in the etiological implications of DNA damage versus imbalance in repair, with abnormal DDR signaling in the onset of neurodegeneration and the potential for DDR as a target for the improvement of neurodegenerative diseases. We have also focused the work on the proteolysis of Tau and other amyloidogenic proteins induced by covalent modifications, as well as the relationship between Tau oligomers and sumoylation. Grado en Medicina

[eng] Protein post-translational modifications are regulatory mechanisms that cells use in response to intra- and extracellular signals. These signals modulate a panoply of conjugating enzymes that modify proteins post-translationally by the conjugation of a small functional group or peptide. The consequences of these modifications are very diverse, but they all present a common feature: they shift protein fate, localization or function. Besides, in the case of covalent protein modifications, such as ubiquitination, these regulatory mechanisms are reversible and dynamic. In mammals, only two E1 and around thirty E2 ligases have been described to participate in the ubiquitin cycle, in contrast to the approximately 600 E3 ligases identified. Note that this is a highly dynamic process and thus, cells also contain a group of deubiquitinating enzymes (DUBs), responsible for detaching ubiquitin from its substrates. DUBs process the ubiquitin precursor that has been transcribed from several genes as fusion proteins; deubiquitinate substrates and rescue them from protein degradation; and finally, they recycle Ub molecules from proteins committed to be degraded20. DUBs play important roles in disease and cellular processes; however and despite its evident importance in the organism, data on their mode of regulation and substrate specificity is still scarce21 The retina is the part of the eye responsible for capturing light stimuli from our surroundings. It is a neuronal tissue located at the posterior part of the eye, which captures light photons, converts the luminic energy into electrochemical stimuli and finally sends visual information to the brain where it is integrated. Vertebrate retina is formed by seven neuronal cell types organized in six precise functional and structural layers. The main function of the retina, the phototransduction, is carried out by two types of photoreceptor cells (PhR), cones and rods (see below). Photoreceptors are highly specialized cellular types, which differ both structurally and functionally, but that develop from the same photoreceptor progenitor precursor. Therefore, there must be a fine and tight genetic control to successfully achieve a properly structured and functional tissue. The regulation by photoreceptor-specific transcription factors has been amply described1,35,36; however, it is not their bare action that determines photoreceptor fate, since SUMO conjugation –and possibly other post-tranlational modifications, such ubiquitination– of TFs play a key role in this process37 In mammals, several comprehensive surveys of DUBs have been reported resulting in: in silico inventories of the DUBs in the human genome20,21; identification of protein interactors by cell-based proteomics analysis45; studies of subcellular localization46; functional involvement in maintaining genome integrity47. However, detailed expression and functional analysis for most DUBs on particular tissues or organs, such as the retina, is still missing. For these reasons, this work is intended as a study of deubiquitinating enzymes in the retina and their possible role in photoreceptor development and homeostasis. To that end, several objectives have been set and reached: 1. Analysis of the involvement of deubiquitinating enzymes (DUBs) in the mouse retina: I. Analysis of mRNA expression and pattern of expression via Real Time qPCR and in situ hybridization. II. Determination of the protein localization via fluorescent immunohistochemistry. III. Sequence and functional conservation analysis through phylogenetic and phenotypic studies. IV. Transcriptomic analysis of DUBs’ mRNA expression in the retina during developmentPreliminary analysis of the functional role of selected DUBs in mouse retinal development, via in vivo gene knockdown. 2. Devising an in-vivo cell system to study the role of DUB enzymes on the regulation of retinal promoters: I. Establishing a cell culture system of study II. Knockdown of DUB genes via shRNA and siRNA silencing techniques. 3. Identification of CRX post-translational modifications, particularly ubiquitynation., [spa] Las modificaciones post-traduccionales de proteínas son mecanismos de regulación que las células utilizan en respuestas a intra y extracelulares. En el caso de las modificaciones covalentes con proteínas, como lo es la ubiquitinación, estos mecanismos de regulación son reversibles y dinámicos. La ubiquitinación se hace reversible por la acción de las enzimas deubiquitinantes (DUBs), encargados de hidrolizar el enlace entre la ubiquitina y sus proteínas sustrato. Las DUBs juegan importantes roles en enfermedades y procesos celulares, no obstante, todavía hay poca información sobre su implicación y regulación de tejidos específicos, como la retina. La retina órgano de la visión responsable de capturar estímulos lumínicos, convertirlos en estímulos electroquímicos y finalmente mandar esta información hacia el córtex visual. Existen dos tipos de fotoreceptores, los conos y los bastones, que difieren funcional y estructuralmente, pero que provienen de una misma célula precursora. Se ha descrito que esta estricta diferenciación se lleva a cabo gracias a la acción combinada de factores de transcripción específicos junto a las modificaiones post-traduccionales, como lo és la molecula ubiquitin-like, SUMO. El presente trabajo pretende analizar la acción de los enzimas deubiquitinantes en el desarrollo de la retina y los fotoreceptores. Para ello, se ha llevado a cabo una primera descripción de los niveles y el patrón de expresión de estos enzimas en la retina; así como un anàlisis transcriptómico en diferentes estadíos de desarrollo retinal murino y humano. Además, se ha realizado un estudio de su conservación evolutiva y su implicación en fenotipos neuronales y de retina. Asimismo, se ha puesto a punto un sistema celular que replica condiciones fisiológicas similares a las retinales y se ha realizado un análisis de silenciamiento de los genes que codifican para las DUBs utilizando shRNA, siRNA y Gapmers. Finalmente, se ha realizado un análisis de las posibles modificaciones post-traduccionales en el factor de transcripción retinal CRX.

The voltage-dependent potassium channel Kv1.3 plays essential physiological functions in the immune system. Kv1.3, regulating the membrane potential, facilitates downstream Ca2+ -dependent pathways and becomes concentrated in specific membrane microdomains that serve as signaling platforms. Increased and/or delocalized expression of the channel is observed at the onset of several autoimmune diseases. In this work, we show that adenosine (ADO), which is a potent endogenous modulator, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytic event that targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. Postsynaptic density protein 95 (PSD-95), a member of the MAGUK family, recruits Kv1.3 into lipid-raft microdomains and protects the channel against ubiquitination and endocytosis. Therefore, the Kv1.3/PSD-95 association fine-tunes the anti-inflammatory response in leukocytes. Because Kv1.3 is a promising multi-therapeutic target against human pathologies, our results have physiological relevance. In addition, this work elucidates the ADO-dependent PKC-mediated molecular mechanism that triggers immunomodulation by targeting Kv1.3 in leukocytes.

The reduction of oxidative stress is suggested to be one of the main mechanisms to explain the benefits of subnormothermic perfusion against ischemic liver damage. In this study we investigated the early cellular mechanisms induced in isolated ratliversafter15minperfusionattemperaturesrangingfromnormothermia(37 ∘ C) to subnormothermia (26 ∘ Cand22 ∘ C). Subnormothermic perfusion was found to maintain hepatic viability. Perfusion at 22 ∘ C raised reduced glutathione levels and the activity of glutathione reductase; however, lipid and protein oxidation still occurred as determined by malondialdehyde, 4-hydroxynonenal-protein adducts, and advanced oxidation protein products. In livers perfused at 22 ∘ C the lysosomal and ubiquitin proteasome system (UPS) were both activated. The 26S chymotrypsin-like ( 훽 5) proteasome activity was significantly increased in the 26 ∘ C (46%) and 22 ∘ C (42%) groups. The increased proteasome activity may be due to increased Rpt6 Ser120 phosphorylation, which is known to enhance 26S proteasome activity. Together, our results indicate that the early events produced by subnormothermic perfusion in the liver can induce oxidative stress concomitantly with antioxidant glutathione preservation and enhanced function of the lysosomal and UPS systems. Thus, a brief hypothermia could trigger antioxidant mechanisms and may be functioning as a preconditioning stimulus.

The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2120-128) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2120-128 region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2., This work was supported by the Spanish Ministry of Economy, and Competitivity (MINECO) grant BIO2013-45993R and Almirall S.A to MB, PP and BC, and by MINECO grant BIO2013– 45793R, cofinanced with structural funds (FEDER) from the European Union, to JT and MP. MP, JT, MM and GK were also supported by the European Commission’s Framework Program 7 (BioNMR, contract 261863). RB and MC were supported by MINECO grants BFU2011-22588, BFU2014-53550-P, MDM-2014-0435.

Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders.

Orientador : Prof. Dr. Silvio Marques Zanata Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 12/12/2016 Inclui referências : f. 65-70 Resumo: A ubiquitinação está envolvida em vários processos no organismo, desde ciclo celular até vias de sinalização de apoptose. Por esse amplo envolvimento, é de extrema importância uma fina regulação que é realizada por enzimas chamadas deubiquitinases (DUBs) que hidrolisam ligações isopeptídicas. Dentre todas as DUBs, USP é a família mais extensa tendo como membro USP2 que possui duas isoformas fruto de processamento alternativo: USP2a (de 69kDa) e USP2b (de 45kDa) que se diferenciam apenas na região N-terminal. Alguns estudos relacionam essas isoformas com a via da p53, miogênese, tumores de próstata, agressividade de tumores de mama e manutenção dos níveis de Na+ e pressão sanguínea. Apesar desses estudos, a distribuição tecidual da USP2 ainda não está completamente estabelecida. A presença de duas isoformas distintas de processamento e a inexistência de anticorpos comerciais que possam distinguir entre as duas isoformas justifica a produção de reagentes confiáveis para o estudo de USP2. Desde modo, o objetivo foi produzir anticorpo monoclonal, isoforma específico, para USP2a e clonar, produzir proteína recombinante em bactérias e superexpressar USP2b em células eucariontes. USP2a foi produzida em E. coli BL21(DE3)STAR fusionada com etiqueta de histidina e purificada por coluna de Ni-NTA. A proteína purificada foi usada para imunizar camundongos balb/c. O soro policlonal desses animais foi testado por western blot para a presença de anticorpo anti-USP2a quanto ao reconhecimento da proteína endógena e superexpressa em linhagens de próstata LNCaP e RWPE-1 tendo dois animais positivos usados para a fusão com mieloma. Os hibridomas após a diluição limitante foram varridos por ELISA chegando-se a um clone 4GD2 que reconheceu USP2a superexpressa em HEK293T e não reconheceu USP2b superexpressa em HEK293T, indicando que obteve-se sucesso na produção de um anticorpo monoclonal isoforma específico para USP2a. Além disso, a isoforma USP2b foi clonada com sucesso nos vetores pET28a(+) e pcDNA3.1(-)6his. Esta isoforma não foi expressa em E. coli mesmo com várias mudanças no sistema, desde cepa bacteriana até temperatura, meio e tempo de expressão, mas foi superexpressa em HEK293T. As ferramentas produzidas serão de extrema importância para estudos de mapeamento tecidual, análise de cinética e busca de novos parceiros moleculares para essas isoformas. Palavras-chave: USP2, USP2a, USP2b, anticorpo monoclonal, proteína recombinante. Abstract: Several physiological processes involve ubiquitylating modification, from cell cycle to apoptosis pathways. Because this involvement has a large range, it is extremely important that this post-translational modification be tightly regulated. Enzymes called deubiquitinases (DUBs) hydrolyze isopeptide bond and have a pivotal role in this regulation. Among all DUBs, the USP is the largest family. USP2, a member of this family, has two isoforms from alternative splicing: USP2a (69kDa) and USP2b (45kDa) and differences rely within their N-terminal regions. These isoforms have related to p53 pathway, myogenesis, prostate tumors, breast tumors aggressiveness and maintenance of Na+ levels and blood pressure. Despite these studies, the tissue distribution of USP2 is not completely established. Absences of commercial antibody that recognize specific isoforms justify the production of reliable reagents to study USP2. Thus, the aims of this study were (1) to express both USP2a and USP2b in heterologous bacterial system; (2) to overexpress USP2b in mammalian cells and (3) to produce monoclonal antibodies specific to USP2a isoform. USP2a was produced in E. coli BL21(DE3)STAR fused with polyhistidine tag and purified by Ni-NTA column. The purified protein was used to immunize Balb/c mice strain. We have tested the polyclonal serum of these animals by western blot to the presence of antibody anti-USP2a that recognize endogenous and overexpressed protein in prostate line LNCaP and RWPE-1. Two animals were positive and employed in fusion procedures. After hybridoma supernatant screenings for anti-USP2a presence by ELISA, we isolated the clone 4GD2, which was stable and still secreting antibodies after several freeze-thaw cycles. Monoclonal antibody 4GD2 recognizes USP2a overexpressed in HEK293T cells and does not cross-react with USP2b, suggesting that MoAb 4GD2 is highly specific. In addition, USP2b was successfully cloned in pET28a(+) and pcDNA3.1(-)6his vectors, both expression vectors for prokaryotic and eukaryotic systems, respectively. We were not able to express successfully USP2b in E. coli under different protocols (e.g. temperature, medium and time parameters changes), however we expressed His-tagged USP2b in HEK293T cells. The produced tools will be extremely important in further studies, such as tissue and temporal USP2 expression mapping, USP2 expression and disappearing kinetics in cell lines and characterization of new molecular partners for this DUB. Key words: USP2, USP2a, USP2b, monoclonal antibody and recombinant protein.

ULK1 is a key kinase in autophagy initiation. Nazio et al. demonstrate that the E3 ubiquitin ligase NEDD4L targets ULK1 for degradation soon after autophagy induction, whereas a simultaneous ULK1 mRNA transcription is needed for priming subsequent rounds of autophagy., Autophagy is an intracellular degradation pathway whose levels are tightly controlled to secure cell homeostasis. Unc-51–like kinase 1 (ULK1) is a conserved serine–threonine kinase that plays a central role in the initiation of autophagy. Here, we report that upon autophagy progression, ULK1 protein levels are specifically down-regulated by the E3 ligase NEDD4L, which ubiquitylates ULK1 for degradation by the proteasome. However, whereas ULK1 protein is degraded, ULK1 mRNA is actively transcribed. Upon reactivation of mTOR-dependent protein synthesis, basal levels of ULK1 are promptly restored, but the activity of newly synthesized ULK1 is inhibited by mTOR. This prepares the cell for a new possible round of autophagy stimulation. Our results thus place NEDD4L and ULK1 in a key position to control oscillatory activation of autophagy during prolonged stress to keep the levels of this process under a safe and physiological threshold.

Objetivo: Avaliar o complexo de destruição da betacatenina no carcinoma colorretal e no adenoma do colo pela expressão das proteínas betacatenina, adenomatous polyposis coli, GSK3β, axina e ubiquitina. Métodos: Amostras de tecidos de 64 doentes com carcinoma colorretal e de 53 pacientes com adenoma do colo foram analisadas. Blocos de tecidos foram submetidos ao estudo imuno-histoquímico com anticorpos policlonais nos tecidos do carcinoma, mucosa não neoplásica adjacente e adenoma. A imunorreatividade foi avaliada pela porcentagem de positividade de células coradas e pela intensidade do grau de coloração das proteínas no citoplasma e no núcleo das células. Na análise estatística, foram utilizados o coeficiente de correlação de Spearman, os testes t de Student, χ2, Mann-Whitney e de McNemar, e a análise de regressão logística univariada. Resultados: No carcinoma colorretal, as expressões da betacatenina e da adenomatous polyposis coli foram significativamente maiores do que em adenomas do colo (p

[eng] Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons of the Substantia Nigra pars compacta (SNpc) and the presence of cytoplasmic protein inclusions named Lewy Bodies. Current treatments are directed principally to ameliorate the clinical manifestations of the disease rather than suppressing the underlying neuron degeneration and death. This is due, in part, to an incomplete understanding of the pathways that lead to neurodegeneration in PD. RTP801 is induced in cellular and animal models of Parkinson's disease (PD) and is elevated in neuromelanin positive neurons in the Substantia Nigra (SN) of PD patients. In a variety of neuronal systems, RTP801 overexpression or upregulation is sufficient to trigger cell death. This involves a sequential mechanism in which it sequentially inactivates mTOR and survival kinase Akt. RTP801 is a protein with a very short half-life (2-7 minutes), so it should be strictly and dynamically controlled at post-translational level to precisely modulate mTOR pathway. Hence, elucidating which proteins mediate RTP801 degradation would be a stepping-stone to design new therapies to block neurodegeneration. NEDD4, is an E3 ubiquitin ligase highly expressed in mammalian neurons that has been directly linked to PD pathogenesis since it has been reported to promote α-synuclein degradation and to be protective against its toxicity. In this work, we report that NEDD4 contributes to RTP801 protein degradation. We show in neuronal cells that there is a pool of RTP801 degraded via lysosomal pathway. We also demonstrate that both proteins interact and that NEDD4 enhances RTP801 polyubiquitination by preferentially conjugating K63 ubiquitin chains. Importantly, NEDD4 regulates RTP801 protein levels in cultured cells and in a conditional knockout mouse model. We also provide evidence that NEDD4 protects against RTP801 toxicity by mediating its polyubiquitination. Furthermore, NEDD4 levels are decreased in the 6-OHDA PD cellular model and its restoration protects against 6-OHDA-induced cell death by reducing RTP801 protein levels. Moreover, NEDD4 loss of function is toxic in neurons due to RTP801 elevation and the subsequent mTOR/Akt inactivation. A role for NEDD4 in PD is supported by observation of diminished NEDD4 in nigral neurons from postmortem sporadic PD brains. In line with these findings, the NEDD4 signaling inducer NAB2 reduces RTP801 protein levels in control and in 6-OHDA-treated cortical neurons. However, NAB2 is not sufficient to confer protection against 6-OHDA toxicity, because the compound itself represses mTOR signaling pathway compromising cell survival., [spa] RTP801/REDD1 es una proteína pro-apoptótica que se encuentra aumentada en modelos celulares y animales y en muestras humanas de la enfermedad de Parkinson. RTP801 es suficiente y necesaria para promover muerte celular mediante un mecanismo de inhibición de la quinasas de supervivencia mTOR y Akt. NEDD4 es una E3 ubiquitina ligasa que ubiquitiniza proteínas señalizándolas para su posterior degradación. Esta proteína se ha relacionado recientemente con la enfermedad de Parkinson, ya que se ha visto que ubiquitiniza la proteína alfa-sinucleína. En este trabajo, se ha descrito que la RTP801 es un sustrato de la ubiquitina ligasa NEDD4. Se ha demostrado que ambas proteínas interaccionan y que NEDD4 ubiquitiniza RTP801 con cadenas de ubiquitina del tipo K63, señalizándola para su posterior degradación lisosomal. También, se ha observado que NEDD4 regula los niveles de RTP801 en sistemas neuronales, y que la pérdida de función de NEDD4 es tóxica debido al incremento de RTP801 y la consecuente inhibición de las quinasas mTOR/Akt. De forma interesante, la NEDD4 ectópica es capaz de proteger ante la toxicidad provocada por la sobreexpresión de RTP801 y ante la toxina parkinsoniana 6-OHDA. Además, la proteína NEDD4 se encuentra disminuida en neuronas nigrales de la SNpc de cerebros post mortem con la enfermedad de Parkinson. De forma consistente, el compuesto NAB2, un activador de NEDD4, disminuye los niveles de la RTP801 en neuronas. No obstante, la acción de este compuesto no es suficiente para proteger ante la toxicidad de la 6-OHDA. También, se ha identificado una posible regulación entre las E3 ligasas parkina y NEDD4, que conferiría un grado de complejidad mayor a la regulación de la RTP801. Diferentes mutantes de la proteína RTP801 han sido generados para estudiar cómo afecta la ubiquitinización a su estabilidad y función. De forma interesante, el mutante RTP801-K185R pierde la función pro-apoptótica de la RTP801, poniendo en relieve que la ubiquitinización en este residuo es crítica para su función. Finalmente, mediante estudios proteómicos se han identificado numerosos interactores de RTP801 putativos, entre los cuáles se encuentra la clatrina y la proteína adaptadora AP-2, sugiriendo un posible papel de la RTP801 en procesos relacionados con la endocitosis mediada por clatrina.