Tarcísio Liberato De Souza Júnior PROTEÔMICA APLICADA AO ESTUDO DO PROCESSAMENTO N- TERMINAL PROTEICO NO SECRETOMA DE CÉLULA

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Tarcísio Liberato De Souza Júnior PROTEÔMICA APLICADA AO ESTUDO DO PROCESSAMENTO N- TERMINAL PROTEICO NO SECRETOMA DE CÉLULA Tarcísio Liberato de Souza Júnior PROTEÔMICA APLICADA AO ESTUDO DO PROCESSAMENTO N- TERMINAL PROTEICO NO SECRETOMA DE CÉLULAS TUMORAIS Tese apresentada à Universidade Federal de São Paulo – Instituto de Ciência e Tecnologia, para obtenção do título de Doutor em Ciências. São José dos Campos 2018 Tarcísio Liberato de Souza Júnior PROTEÔMICA APLICADA AO ESTUDO DO PROCESSAMENTO N- TERMINAL PROTEICO NO SECRETOMA DE CÉLULAS TUMORAIS Tese apresentada à Universidade Federal de São Paulo – Instituto de Ciência e Tecnologia, para obtenção do título de Doutor em Ciências. Orientador: Dr. André Zelanis Palitot Pereira São José dos Campos 2018 Liberato de Souza Júnior, Tarcísio Proteômica aplicada ao estudo do processamento N- terminal proteico no secretoma de células tumorais/ Tarcísio Liberato de Souza Júnior. – São José dos Campos, 2018. XX, 122f Tese (Doutorado) – Universidade Federal de São Paulo. Instituto de Ciência e Tecnologia. Programa de Pós- Graduação em Biotecnologia Proteomics appliedd to the study of N-terminal protein processing in tumoral cells 1. Proteômica. 2. N-terminômica. 3. Melanoma. 4. Câncer UNIVERSIDADE FEDERAL DE SÃO PAULO INSTITUTO DE CIÊNCIA E TECNOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM BIOTECNOLOGIA Chefe do Departamento: Prof. Dr. Eduardo Antonelli Coordenador do curso de pós-graduação: Profa. Dra. Cláudia Barbosa Ladeira de Campos iii Tarcísio Liberato de Souza Júnior PROTEÔMICA APLICADA AO ESTUDO DO PROCESSAMENTO N-TERMINAL PROTEICO NO SECRETOMA DE CÉLULAS TUMORAIS Presidente da banca: Prof. Dr. André Zelanis Palitot Pereira _________________________ Banca examinadora: Prof. Dr. _________________________________________________ Prof. Dr. _________________________________________________ Prof. Dr. _________________________________________________ Prof. Dr. _________________________________________________ iv “Aos meus pais pelo exemplo de vida e amor incondicional, sempre me encorajando frente aos obstáculos da vida. À minha queria irmã Cristiane e irmãos Felipe e Fernando, por todo amor que nos une a cada dia. Amo todos vocês!” v AGRADECIMENTOS Antes de mais nada, agradeço a meu amigo e orientador Dr. André Zelanis Palitot Pereira, por sua dedicação a este projeto, e o empenho ao meu processo de doutoramento. Não tenho como expressar a minha gratidão quanto a sua generosidade, convivção, avidez quanto à idealização desse projeto, bem como suas orientações singulares. A sua dedicação ao ensino e a pesquisa são notórias, motivadoras, que me inspira a crescer como profissional, e a continuar no caminho da ciência. Desejo a você André, que toda orientação e conhecimento a mim passado, seja revertido em sucesso na sua carreira. Muito Obrigado! Gostaria de agradecer ao programa de Pós-graduação em Biotecnologia da Universidade Federal de São Paulo e todos os seus membros constituintes. Agradeço a todos os pesquisadores que colaboraram para o desenvolvimento deste estudo, em especial à professora Dra. Dayane Batista Tada, do Laboratório de Desenvolvimento de nanopartículas da UNIFESP, ao Dr. Martin Rodrigo Alejandro Wurtele Alfonso do Laboratório de Bioquímica e Biologia Estrutural da UNIFESP, à Dra. Solange Maria de Toledo Serrano, do Laboratório Especial de Toxinologia Aplicada do Instituto Butantan, ao Dr. Alexandre Keiji Tashima, do Laboratório de Proteômica da UNIFESP-SP, ao professor Dr. Oliver Schilling, do Instituto de Medicina e Biologia Molecular da Universidade de Freiburg, Alemanha. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES, pelo apoio financeiro que tornou viável o desenvolvimento deste trabalho. À Fundação de Amparo à Pesquisa do Estado de São Paulo, pelo apoio ao projeto ... n° 2014/06579-3 Gostaria de agradecer ao Dr. Eduardo Shigueo Kitano, à M.Sc Débora Andrade e a Dra. Milene Menezes. Agradeço em especial os meus amigos e colegas de laboratório, Msc. Dayelle Pessotti, Isabella Fukushima, Francine Fontes, Maurício Camacho, Rafael Berton, vi Msc. Daniela Formaggio, Msc. Thaís Dolzany, por toda parceria, companheirismo. Esse trabalho não teria sido concretizado sem a presença de cada um de vocês, muito obrigado! Aos meus pais, à minha irmã Cristiane, Felipe, Nando, Samantha, Mimim e Heleninha. Obrigado a todos que participaram direta ou indiretamente desse trabalho ao longo desses anos. Obrigado a todos! vii SUMÁRIO 1 INTRODUÇÃO ........................................................................................... 21 1.1 MELANOMA ........................................................................................... 21 1.2 PROTEÍNAS SECRETADAS (SECRETOMA) ................................................. 24 1.3 ANÁLISE SISTÊMICA DA EXTREMIDADE N-TERMINAL DE PROTEÍNAS: .......... 27 1.3.1 N-terminômica .................................................................................. 27 1.3.2 Acetilação do N-Terminal ................................................................. 29 1.3.3 Formação de Piroglutamato (glutamato e glutamina N-terminal) .... 30 1.3.4 Formilação ........................................................................................ 31 1.3.5 Propionilação .................................................................................... 31 1.3.6 Miristoilação e Palmitoilação ............................................................ 32 1.3.7 Mono-metilação, Di-metilação e Tri-metilação ................................. 32 1.3.8 N-degrons ......................................................................................... 33 1.4 PROTEASES, PROCESSAMENTO PROTEOLÍTICO E CÂNCER ....................... 34 2 OBJETIVOS ............................................................................................... 39 2.1 OBJETIVO GERAL ................................................................................... 39 2.2 OBJETIVOS ESPECÍFICOS ....................................................................... 39 3 MATERIAL E MÉTODOS .......................................................................... 40 3.1 LINHAGENS CELULARES ......................................................................... 40 3.2 AMOSTRAS DE PACIENTES ...................................................................... 42 3.3 CULTIVO CELULAR ................................................................................. 46 3.4 CARACTERIZAÇÃO DAS LINHAGENS CELULARES ...................................... 46 3.4.1 Curva de Crescimento ...................................................................... 46 3.4.2 Efeito do carenciamento na proliferação celular .............................. 47 3.4.3 Tempo de aderência em cultura ....................................................... 47 3.4.4 Viabilidade (exclusão do corante azul de tripan) .............................. 48 3.5 COLETA DO SECRETOMA ........................................................................ 48 3.6 QUANTIFICAÇÃO DE PROTEÍNAS ............................................................. 49 3.7 ELETROFORESE DE PROTEÍNAS EM GEL DE SDS-POLIACRILAMIDA ........... 49 3.8 ZIMOGRAFIA .......................................................................................... 49 3.9 PREPARO DAS AMOSTRAS DE PACIENTES COM MELANOMA ....................... 50 3.9.1 Desparafinização dos tecidos tumorais ............................................ 51 viii 3.9.2 Microdissecação ............................................................................... 51 3.10 ANÁLISE PROTEÔMICA ............................................................................. 51 3.10.1 Extração de proteínas das amostras de pacientes com melanoma e análise proteômica (shotgun) ................................................................. 51 3.10.2 Análise proteômica (shotgun) dos secretomas celulares ............... 52 3.10.3 Seleção negativa e enriquecimento de N-terminais proteicos por sulfonação dos peptídeos internos ............................................................ 54 3.10.4 TAILS - Seleção negativa e enriquecimento de N-terminais proteicos utilizando dendrímero de aldeídos ............................................. 56 3.11 PROCESSAMENTO DOS DADOS ............................................................... 60 3.11.1 Análise proteômica quantitativa ...................................................... 60 3.11.2 Análise N-terminômica e anotação funcional ................................. 61 3.11.2.1 Identificação e quantificação .................................................... 61 3.11.2.2. Anotação funcional/posicional ................................................. 62 3.12 ANÁLISE BIOINFORMÁTICA ...................................................................... 63 3.12.1 Normalização dos dados e análise pareada (teste-t) ..................... 63 3.12.2 Soft clustering, enriquecimento de categorias do Gene Ontology (GO) e heatmaps ....................................................................................... 64 4 RESULTADOS .......................................................................................... 65 4.1 CARACTERIZAÇÃO DAS LINHAGENS CELULARES ....................................... 65 4.2 QUANTIFICAÇÃO PROTEICA NOS SECRETOMAS CELULARES ...................... 66 4.3 PERFIL ELETROFORÉTICO DOS SECRETOMAS EM GEL DE SDS- POLIACRILAMIDA ............................................................................................. 66 4.4 ZIMOGRAFIA .......................................................................................... 67 4.5 ANÁLISE PROTEÔMICA (SHOTGUN) DOS SECRETOMAS CELULARES ............... 68 4.5.1 Identificações proteicas
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