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Sara Catarina Timóteo Dos Santos Domingues Identificação De Universidade de Aveiro Secção Autónoma de Ciências da Saúde 201 2 Sara Catarina Timóteo Identificação de Complexos Proteicos na Doença de dos Santos Domingues Alzheimer Identification of Protein Complexes in Alzheimer’s Disease Universida de de Aveiro Secção Autónoma de Ciências da Saúde 20 12 Sara Catarina Timóteo Identificação de Complexos Proteicos na Doença de dos Santos Domingues Alzheimer Identification of Protein Complexes in Alzheimer’s Disease Tese apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Doutor em Ciências Biomédicas, realizada sob a orientação científica da Prof. Doutora Odete Abreu Beirão da Cruz e Silva, Professora Auxiliar com Agregação da Secção Autónoma de Ciências da Saúde da Universidade de Aveiro. Apoio financeiro da FCT e do FSE no âmbito do III Quadro Comunitário de Apoio (Bolsa de Doutoramento SFRH/ BD/ 21559/ 2005 ). o júri presidente Doutor Artur da Rosa Pires Professor Catedrático da Universidade de Aveiro Doutor Uwe Konietzko Investigador Sénior da Universidade de Zurique Doutor José António Henriques de Conde Belo Professor Associado com Agregação da Universidade do Algarve Doutora Patrícia Espinheira de Sá Maciel Professora Associada da Escola de Ciências da Saúde da Universidade do Minho Doutora Odete Abreu Beirão da Cruz e Silva Professora Auxiliar com Agregação da Universidade de Aveiro Doutora Margarida Sâncio da Cruz Fardilha Professora Auxiliar Convidada da Universidade de Aveiro agradecimentos I would like to express my gratitude to FCT, for the financial support. To the Centre for Cell Biology and to the Biology and Health Sciences Departments from the University of Aveiro, for providing the necessary conditions. To my supervisor Prof. Odete da Cruz e Silva for giving me the opportunity to participate in the APP interactome project, for encouraging me and supporting my projects and ideas. To Prof. Edgar da Cruz e Silva (1958-2010) for the scientific thinking that challenged mainstream beliefs and also for the wonderful stories that always taught us something. I am grateful for the beneficial scientific discussions, constant positive input and for instilling me self-confidence. Prof. Edgar was a dedicated mentor that will always be remembered. The healthy synergy between the Neurosciences and Signal Transduction Groups was so beneficial for my doctoral education that I will always be very grateful to both, Prof. Odete and Prof. Edgar. To our collaborator Dr. Uwe Konietzko who welcomed me in his lab with open arms and helped me with the microscopy analyses. I thank Dr. Uwe for collaborating in the RanBP9 project, providing unique tools that made possible the analysis of nuclear structures, and for all the transfections and microscopy work. To all my colleagues, present and former members of the Neurosciences and Signal Transduction laboratories. To the graduation and master students that worked with me, contributing also to this project. Ao meu marido Nuno, pelo incansável apoio e paciência, por toda ajuda e disponibilidade que tornaram possível a realização desta tese. Ao meu querido Francisco, pela compreensão e carinho, apesar da tenra idade … Aos meus pais e toda a família pelo apoio e bons momentos de descontração. palavras-chave Doença de Alzheimer, APP, AICD, interactoma, fosforilação, Fe65, splicing alternativo, RanBP9, sinalização nuclear. resumo A Doença de Alzheimer (AD) é a maior doença neurodegenerativa a nível mundial, e a principal causa de demência na população idosa. O processamento da proteína precursora de amilóide (APP) pelas β- e g- secretases origina o peptídeo Aβ, que agrega em oligómeros neurotóxicos e em placas senis. Estes são eventos-chave na patogénese da DA que levam à rutura da neurotransmissão sináptica, morte neuronal e inflamação neuronal do hipocampo e córtex cerebral, causando perda de memória disfunção cognitiva geral. Apesar dos grandes avanços no conhecimento do papel do processamento da APP na DA, a sua função fisiológica ainda não foi totalmente elucidada. Os mapas de interações proteína-proteína (PPI) humanos têm desempenhado um papel importante na investigação biomédica, em particular no estudo de vias de sinalização e de doenças humanas. O método dois-híbrido em levedura (YTH) consiste numa plataforma para a produção rápida de redes de PPI em larga-escala. Neste trabalho foram realizados vários rastreios YTH com o objetivo de identificar proteínas específicas de cérebro humano que interagissem com a APP, ou com o seu domínio intracelular (AICD), tanto o tipo selvagem como com os mutantes Y687F, que mimetizam o estado desfosforilado do resíduo Tyr-687. De facto, a endocitose da APP e a produção de A β estão dependentes do estado de fosforilação da Tyr-687. Os rastreios YTH permitiram assim obter de redes proteínas que interagem com a APP, utilizando como “isco” a APP, APP Y687F e AICD Y687F . Os clones positivos foram isolados e identificados através de sequenciação do cDNA. A maior parte dos clones identificados, 118, correspondia a sequências que codificam para proteínas conhecidas, resultando em 31 proteínas distintas. A análise de proteómica funcional das proteínas identificadas neste estudo e em dois projetos anteriores (AICD Y687E , que mimetiza a fosforilação, e AICD tipo selvagem), permitiram avaliar a relevância da fosforilação da Tyr-687. Três clones provenientes do rastreio YTH com a APP Y687F foram identificados como um novo transcrito da proteína Fe65, resultante de splicing alternativo, a Fe65E3a ( GenBank Accession : EF103274), que codifica para a isoforma p60Fe65. A p60Fe65 está enriquecida no cérebro e os seus níveis aumentam durante a diferenciação neuronal de células PC12, evidenciando o potencial papel que poderá desempenhar na patologia da DA. A RanBP9 é uma proteína nuclear e citoplasmática envolvida em diversas vias de sinalização celulares. Neste trabalho caracterizou-se a nova interação entre a RanBP9 e o AICD, que pode ser regulada pela fosforilação da Tyr-687. Adicionalmente, foi identificada uma nova interação entre a RanBP9 e a acetiltransferase de histonas Tip60. Demonstrou-se ainda que a RanBP9 tem um efeito de regulação inibitório na transcrição mediada por AICD, através da interação com a Tip60, afastando o AICD dos locais de transcrição ativos. O estudo do interactoma da APP/AICD, modelado pela fosforilação da Tyr-687, revela que a APP poderá estar envolvida em novas vias celulares, contribuindo não só para o conhecimento do papel fisiológico da APP, como também auxilia a revelar as vias que levam à agregação de A β e neurodegeneração. A potencial relevância deste trabalho relaciona-se com a descoberta de algumas interações proteicas/vias de sinalização que podem que podem ser relevantes para o desenvolvimento de novas estratégias terapêuticas na DA. keywords Alzheimer’s disease, APP, AICD, interactome, phosphorylation, Fe65, alternative splicing, RanBP9, nuclear signaling. abstract Alzheimer’s disease (AD ) is the most prevalent neurodegenerative disorder worldwide and the leading cause of dementia in the elderly. Processing of amyloid-β precursor protein (APP) by β - and g-secretases produces Aβ, which aggregates into neurotoxic oligomers and senile plaques. These are key events in the pathogenesis of AD that lead to the disruption of synaptic neurotransmission, neuronal cell death, and inflammation in the hippocampus and cerebral cortex, thus causing memory loss and global cognitive dysfunction. Despite advances in understanding the role of APP processing in AD, the normal physiological function of this protein has proven more difficult to elucidate. Human protein-protein interaction (PPI) maps play an increasingly important role in biomedical research and have been shown to be highly valuable in the study of a variety of human diseases and signaling pathways. The yeast two- hybrid (YTH) system provides a platform for the rapid generation of large scale PPI networks. Several YTH screens were performed to identify human brain- specific proteins interacting with APP, or with its intracellular domain (AICD), either the wild-type or the Y687F mutant, which mimics the dephosphorylated residue. In fact, APP endocytosis and Aβ generation are dependent upon Tyr - 687 phosphorylation. A human APP network comprised of the protein interactions was assembled through YTH screening, using as baits APP, APP Y687F and AICD Y687F . Positive clones were isolated and identified by DNA sequencing and database searching. The majority of these clones, 118, matched to a protein coding sequence, yielding 31 different proteins. Functional proteomics analysis of the proteins identified in this study, and two additional screens from previous projects (phospho-mutant AICD Y687E and wild-type AICD), allowed to infer the relevance of Tyr-687 phosphorylation. Three clones from YTH with APP Y687F were identified as a new splice variant of the APP binding protein Fe65, Fe65E3a (GenBank Accession EF103274), encoding the p60Fe65 isoform. Fe65E3a is expressed preferentially in the brain and the p60Fe65 protein levels increased during PC12 cell differentiation. This novel Fe65 isoform and the regulation of the splicing events leading to its production, may contribute to elucidating neuronal specific roles of Fe65 and its contribution to AD pathology. RanBP9 is an evolutionarily conserved nucleocytoplasmic protein implicated as a scaffolding protein in several signaling pathways. In this work a novel interaction between RanBP9 and AICD, which can be regulated by Tyr-687 phosphorylation,
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