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.

Made available in DSpace on 2014-07-29T15:10:33Z (GMT). No. of bitstreams: 1 Tese Wesley de Almeida Brito.pdf: 6161747 bytes, checksum: 6249a08f73180f831c2df168246efa67 (MD5) Previous issue date: 2009-11-18 / Paracoccidioides brasiliensis is a thermal-dimorphic fungus, the causative agent of Paracoccidioidomycosis (PCM), an important endemic mycosis in Latin America. A protein species preferentially expressed in yeast cells with a molecular mass of 80kDa and isoeletric point (pI) of 7.79 was isolated from the proteome of P. brasiliensis and characterized as an aconitase (E.C. 4.2.1.3). Aconitase is an enzyme that catalyzes the isomerization of citrate to isocitrate in both the Krebs cycle (KC) and the glyoxylate cycle (GC). We report the cloning and characterization of the cDNA encoding the aconitase of P. brasiliensis (PbACO). The cDNA showed a 2337 bp open reading frame (ORF) and encoded a predicted protein with 779 amino acids. A polyclonal antibody against the purified recombinant PbACO was obtained in order to analyze the subcellular localization of the molecule in P. brasiliensis. The protein is present in the extracellular fluid, cell wall, mitochondria, cytosol and peroxisomes of yeast cells as demonstrated by western blot and immunocytochemistry analysis. The expression analysis of the Pbaco gene was performed through quantitative real time RT-PCR and results demonstrated increasing expression during differentiation from mycelium to yeast cells. Real time RT-PCR assays was also used to evaluate the Pbaco expression when the fungus grows on media with acetate and ethanol as sole carbon sources and in different iron levels. The results demonstrated that Pbaco transcript is over expressed in acetate and ethanol as sole carbon sources and in highiron conditions. / Paracoccidioides brasiliensis é um fungo termodimórfico, agente causador de uma importante micose sistêmica na América Latina, a paracoccidioidomicose (PCM). Uma proteína apresentando massa molecular de 80kDa e ponto isoelétrico (pI) de 7,79, preferencialmente expressa em células leveduriformes, foi isolada do proteoma de P. brasiliensis e caracterizada como uma aconitase (EC 4.2.1.3). Aconitase é uma enzima que catalisa a isomerização do citrato em isocitrato tanto no ciclo de Krebs (CK) quanto no ciclo glioxalato (CG). No presente estudo reportamos a clonagem e caracterização do cDNA da aconitase de P. brasiliensis (PbACO). O cDNA apresentou um quadro aberto de leitura (ORF) com 2337 pares de bases, codificando uma predita proteína com 779 resíduos de aminoácidos. Objetivando a localização da aconitase de P. brasiliensis foi produzida e purificada uma proteína recombinante a qual foi utilizada para a produção de um anticorpo policlonal anti-PbACO. A proteína foi localizada no fluído extracelular, parede celular, mitocôndria, citosol e nos peroxissomos de células leveduriformes, como demonstrado por ensaios de Western blotting e por imunocitoquímica. A análise da expressão do gene Pbaco foi realizada através RT-PCR em tempo real e os resultados demonstraram um aumento da expressão durante a diferenciação celular de micélio para levedura. A expressão Pbaco também foi avaliada quando o fungo é cultivado em meios de cultura contendo acetato e etanol como única fonte de carbono bem como em diferentes concentrações de ferro. Os resultados demonstraram que o transcrito de Pbaco é mais expresso em acetato e etanol como única fonte de carbono e em altas concentrações de ferro.