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New Retroviral-Like Membrane-Associated Aspartic Proteases From Novas proteases aspárticas membranares do tipo retroviral de rickettsiae: caracterização bioquímica e determinação de especificidade New retroviral-Iike membrane-associated aspartic proteases from rickettsiae: biochemical characterization and specificity profiling Tese de Doutoramento apresentada à Universidade de Coimbra para obtenção do grau de Doutor em Bioquímica (especialidade de Tecnologia Bioquímica). Doctoral thesis in the scientific area of Biochemistry (specialty Biochemistry Technology) presented to the University of Coimbra. Rui Gonçalo Batista Mamede da Cruz Coimbra - 2014 Department of Life Sciences University of Coimbra UNIVERSIDADE DE COIMBRA Front cover image illustrates Rickettsia cells (in green) invading mammalian host cells. Publishing rights were granted by Michael Taylor, the original author of the image. Agradecimentos / Acknowledgments A conclusão desta etapa não teria sido possível sem o apoio incondicional da família, amigos, orientadores, colegas e de todos aqueles que foram cruzando o meu caminho e deixando o seu contributo, em particular nos últimos quatro anos que conduziram a esta dissertação. Gostaria por isso de lhes deixar algumas palavras de profundo e sincero agradecimento. Em primeiro lugar, gostaria de agradecer à Doutora Isaura Simões e ao Professor Carlos Faro, por me terem acolhido e proporcionado as excepcionais condições para a realização deste trabalho. Em especial à Doutora Isaura Simões, expresso a minha profunda gratidão pela orientação, rigor, sentido crítico e paixão com que encara a ciência. Agradeço-lhe por acreditar em mim e por me fazer ir sempre mais além. Um agradecimento muito especial a todos aqueles que fazem e fizeram parte do laboratório de Biotecnologia de Molecular do Biocant, pelo seu contributo no meu crescimento científico, profissional e pessoal. Um agradecimento ao Pedro Castanheira pelo conhecimento que me transmitiu e pelas palavras de incentivo e encorajamento. À Carla Almeida um enorme obrigado pela disponibilidade e partilha de conhecimento e, acima de tudo, pelos sempre bons conselhos. Aos meus colegas de bancada Ana Sofia Lourenço, André Soares, Marisa Simões, Joana Furtado, Pedro Curto, Ana Rita Leal, Liliana Antunes e a todos os outros, agradeço pelos bons momentos de amizade, pela boa disposição, pelas estimulantes conversas e desabafos mútuos. Muito obrigado por tornarem este percurso mais agradável. Ao Laboratório de Proteómica, de uma forma especial ao Bruno Manadas, à Vera Mendes e à Sandra Anjos, agradeço pela disponibilidade e colaboração nas análises de espectrometria de massa. Aos restantes colegas e amigos que integram a família Biocant, o meu muito obrigado pelos bons momentos de convívio que em muito contribuíram para que que cada dia fosse encarado com particular motivação. Uma palavra de carinho a todos os colegas e amigos do CNC (dos tempos do IBILI), pelas boas recordações dos bons momentos aí vividos e pelas amizades que ficaram para a vida. Um muito obrigado. To Doctor Juan Martinez, who kindly welcomed me in his lab, and to Sean Riley, I deeply thank the knowledge and advice they shared with me throughout my stay, as well as the great opportunity to know a different laboratorial reality, and the meaningful personal experience it all meant. I extend my thanks to Abby and all members of Doctor Macaluso’s Lab for making my life in Baton Rouge so enjoyable. I would like also to thank Doctor Christopher Overall and his lab team, especially to Pitter Huesgen, for the guidance and all the support during my stay in Vancouver, for all the collaboration, shared knowledge and friendship that made that time in Vancouver an exceptional experience. A todos os meus amigos, em particular ao Rui, um enorme obrigado por estarem sempre presentes e por me permitirem aproveitar o que de melhor a vida tem para oferecer. Muito obrigado pela amizade, companhia e afecto. Agradeço à minha família todo o apoio incondicional. De uma forma muito especial, agradeço aos meus pais por me terem permitido ser o que sou hoje, pois tudo o que consegui a eles o devo. Agradeço-lhes por estarem ao meu lado em todos os momentos, pela preocupação constante e por serem um exemplo de integridade, humildade e trabalho. Ao meu irmão por ser um amigo e por tudo o que me ensinou ao longo da vida. Tenho um grande orgulho em ser vosso filho e irmão. Por fim, um desmedido obrigado à Dulce por ser o meu porto seguro. As palavras serão sempre poucas para expressar tudo aquilo que representas para mim. Agradeço-te acima de tudo por seres parte daquilo que eu sou. As minhas conquistas serão sempre igualmente tuas… The work herein presented is part of the manuscript entitled, RC1339/APRc from Rickettsia conorii is a Novel Aspartic Protease with Properties of Retropepsin-Like Enzymes, accepted for publication at PLOS Pathogens (manuscript reference: PPATHOGENS-D-14-00592). Rui Cruz, Pitter Huesgen, Sean P. Riley, Alexander Wlodawer, Carlos Faro, Christopher M. Overall, Juan J. Martinez and Isaura Simões. 2014. RC1339/APRc from Rickettsia conorii is a Novel Aspartic Protease with Properties of Retropepsin-Like Enzymes. PLOS Pathogens (DOI: 10.1371/journal.ppat.1004324). Este trabalho foi financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade – COMPETE e por Fundos Nacionais através da FCT – Fundação para a Ciência e a Tecnologia no âmbito do projeto PTDC/SAU-MII/107942/2008; Rui Cruz usufruiu de uma bolsa de Doutoramento com a referência SFRH/BD/47638/2008. Table of contents Resumo .................................................................................................................................. xi Abstract ................................................................................................................................ xv Abbreviations ...................................................................................................................... xix Chapter I. Introduction ........................................................................................................... 1 1.1. Proteolytic enzymes ................................................................................................. 3 General description ......................................................................................................... 3 Aspartic proteases ........................................................................................................... 4 Distribution and relevance .......................................................................................... 5 Structure and processing activity ................................................................................ 7 Catalytic mechanism, specificity and inhibition......................................................... 13 Evolutionary relationships of aspartic proteases ...................................................... 19 1.2. Rickettsiae and Rickettsioses .................................................................................. 24 Bacteriology and Epidemiology ..................................................................................... 24 Bacteriology .............................................................................................................. 24 Epidemiology ............................................................................................................. 25 Phylogenomics .............................................................................................................. 27 Taxonomy and Phylogeny ......................................................................................... 27 Genomics .................................................................................................................. 30 Pathogenesis ................................................................................................................. 32 Life cycle .................................................................................................................... 32 Virulence ................................................................................................................... 35 Diagnostics and Therapeutics ........................................................................................ 42 1.3. Research objectives ................................................................................................ 45 Chapter II. Biochemical and enzymatic characterization of APRc ....................................... 47 2.1. Introduction............................................................................................................ 49 2.2. Materials and Methods .......................................................................................... 51 Materials ....................................................................................................................... 51 Bioinformatics analysis .................................................................................................. 51 DNA constructs ............................................................................................................. 51 Expression and purification of the soluble forms of APRc ............................................. 53 Autoprocessing activity of APRc in soluble extracts of E. coli ........................................ 54 In vitro auto-processing studies .................................................................................... 54 Analytical size-exclusion chromatography ...................................................................
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