This research programme investigated the expression of prolyl hydroxylase (PHD) proteins in rodent tissues. The importance of PHD enzymes lies in their ability to render oxygen sensitivity to Hypoxia inducible factor (HIF), the principal mediator of intracellular oxygen homeostasis. The first part of this study focused on developing and validating anti-sera capable of detecting PHD proteins in rodent tissues. With these reagents, it was possible to assess the relative expression of each PHD protein in a number of different rat tissues. PHD2 was the most abundant isoform in all tissues studied. In contrast, an abundance of PHD1 was observed only in testis and skeletal muscle. A number of different tissue species of PHD3 were identified and their abundance was found to vary between different tissues. These observations provide further evidence of the principal role of PHD2 in regulating HIF in vivo, but also point towards additional roles for PHD1 and PHD3 in selected tissues. They highlight the potential for there being a complex interplay between different PHD enzymes which could, in the future, prove potential targets for therapeutic manipulation. This study also provides additional insights into the mechanisms underlying the phenotypes observed in PHD deletional mouse models which appear, in many cases, to be directly related to the abundance of a given PHD isoform. The emerging role of PHD3 as a promoter of sympathetic lineage apoptosis prompted further study of PHD3 expression in rat neuronal tissues. An abundance of PHD3 was demonstrated throughout the rat sympathetic nervous system, a finding which appeared at odds with its known role as a promoter of neuronal apoptosis and resulted in a series of collaborative studies which demonstrated a sympatho-adrenal phenotype in wild type compared to PHD3-/- mice. Further collaborative studies utilising wild type mice and those deleted of specific PHD isoforms, were carried out to assess the significance of the abundance of PHD3 and PHD1 noted here in rat hippocampus and testis respectively. While neither study demonstrated statistically significant phenotypes, these observations remain of interest and areas for future research.

Orientador: Andre Luís Berteli Ambrósio / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T16:57:05Z (GMT). No. of bitstreams: 1 Fala_AngelaMaria_M.pdf: 4666864 bytes, checksum: 3bcb145c1dc353b15b43b75e5723a310 (MD5) Previous issue date: 2013 / Resumo: A adaptação das células cancerosas ao microambiente é o ponto central que leva ao fenótipo invasivo e metastático, e é garantida principalmente através do controle preciso da expressão gênica. A resposta às necessidades energéticas e biossintéticas e principalmente à disponibilidade de oxigênio intracelular, por exemplo, é em grande parte mediada pelo fator de transcrição induzido por hipóxia 1 (HIF-1). HIF-1 é um heterodímero composto pelas subunidades ? e ?, que respondem a sequência consenso (5'-RCGTG-3') e ativam a transcrição de mais de 100 genes envolvidos em diversos aspectos cruciais da biologia tumoral, incluindo angiogênese, metabolismo de glicose, diferenciação celular, apoptose e resistência a radio e quimioterapias. São conhecidas três isoformas da subunidade ? (1 a 3) e todas se heterodimerizam com a subunidade ?. No geral, HIFs são constituídas de diferentes domínios funcionais, como de ligação ao DNA, de heterodimerização, transativação e degradação. Atualmente, pouco se sabe sobre os mecanismos estruturais e funcionais dos domínios da HIF- 1, deste modo este trabalho objetivou o estudo estrutural destes domínios. Os domínios bHLH, Pas-1 e Pac de HIF-1? e HIF-? em diferentes combinações entre si e o domínio Pac da HIF-3? foram clonados, as proteínas foram expressas em sistema bacteriano e purificadas por diferentes técnicas cromatográficas. Diversas destas construções se mostraram insolúveis ou suscetíveis a degradação, enquanto outras foram purificadas com sucesso. As construções Pac, um exemplo de sucesso na produção, foram submetidas a ensaios de anisotropia de fluorescência e ressonância magnética nuclear, o que nos permitiu a caracterização dos perfis de interação entre as várias combinações de heterodimerização. Neste contexto, os resultados mostram que o equilíbrio dinâmico da interação entre Pac-1? com a subunidade -1? é alcançado imediatamente, enquanto que para a interação entre Pac-3? e -1?, são necessários pelo menos 30 horas de incubação. O mesmo pode ser extraído da caracterização da interação direta entre Pac-1? e Pac-3?. Nos experimentos de RMN, foi possível identificar a região de interação entre as subunidades -1? e -3? com a subunidade ?, separadamente. Ambas as subunidades ? interagem com a Pac-1? na região das fitas-beta 1 e 5 e no loop entre as fitas 4 e 5. Em conjunto, estes resultados impactam no mecanismo de antagonização de HIF-3? na atividade transcricional de HIF-1?. Houve ainda a formação de monocristais da subunidade Pac-3?, que foram submetidos a experimentos preliminares de difração de raios X, que apesar de resultar em dados anisotrópicos e insuficientes para resolução estrutural, permitiram a caracterização dos parâmetros cristalinos, incluindo a presença de um alto conteúdo de solvente. Adicionalmente, são também apresentados os resultados obtidos visando a expressão e cristalização das Prolilhidroxilases (PHDs) isoformas 1 a 4, durante estágio de seis meses no Structural Genomics Consortium (SGC), da Universidade de Oxford, na Inglaterra. Foram expressas de maneira solúvel e purificadas, diversas construções das isoformas 1 e 3 das PHDs humanas. Cristais foram obtidos, porém estes foram determinados como sendo de compostos inorgânicos presentes na condição de cristalização. Como resultado final, está sendo estabelecida uma colaboração entre o nosso grupo e o SGC para que os estudos estruturais com PHDs se estendam e sejam realizados em nosso laboratório aqui no Brasil / Abstract: The adaptation process of cancer cells to the microenvironment is the central point leading to the invasive and metastatic phenotypes, and is guaranteed mainly through the precise control of gene expression. The cell response to the energetic and biosynthetic needs and especially to the availability of intracellular oxygen is mediated by the hypoxia inducible transcription factor 1, or HIF-1. HIF-1 functions as a heterodimer composed by subunits ? and ?, binding to responsive elements with the consensus sequence 5'-RCGTG-3 ', thus activating the transcription of more than 100 genes involved in many crucial aspects of tumor biology, including angiogenesis, metabolism glucose, cell differentiation, apoptosis, and resistance to radiotherapy and chemotherapy. There are three known isoforms of the ? subunit (1, 2 and 3) and all heterodimerize with the ? subunit. HIFs are composed of different functional domains, such as the DNA binding domain, the heterodimerization, transactivation and the oxygen-dependent degradation domains. Currently, little is known about the mechanisms of structural and functional domains of HIF-1, thus this work was to study these structural domains. The domain (bHLH, Pas-1 and Pac) of HIF-1? and HIF-? in different combinations with each other and Pac domain of HIF-3? were cloned, the proteins were expressed in bacterial system and purified by various chromatographic techniques. Several of these constructs proved insoluble or susceptible to degradation, while others were purified successfully. The constructs Pac, an example of success in production, were tested for fluorescence anisotropy and nuclear magnetic resonance, which allowed us to characterize the profiles of the interaction between the various combinations of heterodimers. In this context, the results show that the dynamic equilibrium of the interaction between the Pac-1? and -1? subunits is achieved immediately, whereas for the interaction between Pac-3? and -1?, it takes at least 30 hours of incubation. The same can be observed from the characterization of direct interaction between Pac-1? and -3?. From the NMR experiments, it was possible to identify the region of interaction between the subunits -1? and -3? with the -1? subunit. Both ? subunits interact with Pac-1? via betastrands 1 and 5 and the loop between the strands 4 and 5. Overall, these results impact in the mechanism of HIF-3? antagonizing the transcriptional activity of HIF-1?. We also obtained single crystals for Pac-3? subunit, which were subjected to preliminary experiments of X-ray diffraction. Although resulting in anisotropic, insufficient data for and structural resolution, it has allowed the characterization of crystalline parameters, including the presence of a high solvent content. Additionally, we also present the results targeting the expression and crystallization of Prolyl-hydroxylases (PHDs) human isoforms 1-4, during the six-month period at the Structural Genomics Consortium (SGC), the University of Oxford in England. Several construct from of isoforms 1 and 3 were successfully expressed and purified in the soluble form. Likewise, crystals were obtained, but these were determined to be composed by inorganic compounds present in the crystallization conditions. At the end, a collaboration was established between our and the SGC group for the structural studies with the PHDs to extend and carry out the experiments in our lab here in Brazil / Mestrado / Clinica Medica / Mestra em Clínica Médica

Hypoxia-inducible factor (HIF) is the master regulator of genes involved in adaptation to hypoxia. The stability and transcriptional activity of HIF are regulated by post-translational hydroxylations: prolyl hydroxylation by the prolyl hydroxylase domain-containing enzymes PHD1 – 3 earmarks HIF for proteasomal degradation, whilst asparaginyl hydroxylation by factor inhibiting HIF (FIH) blocks the interaction of HIF with the transcriptional coactivators p300/CBP. The PHDs and FIH hydroxylate HIF directly from molecular oxygen and are therefore oxygen sensors. Recent literature shows that FIH also hydroxylates a number of proteins containing an ankyrin-repeat domain (ARD). Together with reports suggesting that the PHDs are involved in HIF-independent pathways, this suggests that the HIF hydroxylases may have a wide range of non-HIF targets. This thesis describes my investigations into novel substrates of the HIF hydroxylases. This work has characterized the FIH-dependent hydroxylation of the ARD-containing protein Notch1, and defined a consensus sequence for hydroxylation that corresponds to the ankyrin-repeat consensus. Using this consensus potential sites of hydroxylation in a novel ARD FIH substrate, myosin phosphatase targeting subunit 1 (MYPT1), were identified then subsequently confirmed and characterized. Notch1 competes with HIF for FIH hydroxylation. My experiments show that this occurs because Notch1 is a more efficient substrate than HIF, whilst studies on MYPT1 and other proteins indicate that competitive inhibition of FIH may be a general property of ARDs. There are more than 300 ARD proteins in the human genome, and this thesis demonstrates that FIH may hydroxylate a significant percentage of these. In addition to the analysis of ARD hydroxylation a proteomic investigation into novel PHD3 substrates has identified two candidate proteins, suggesting that the PHDs may also have multiple targets. These results have important implications for oxygen sensing, and indicate that post-translational hydroxylation is likely to be a widespread modification in cell biology.