Heterologous expression and purification of the hypoxia-induced factor HIF-1 human aiming structural and biochemical studies and structural studies of prolyl-hydroxylases (PHDs) human, isoforms 1 and 3, in complex with inhibitors

Author(s)

Angela Maria Fala

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2013-08-27

Examining board members

Andre Luís Berteli Ambrosio; Artur Torres Cordeiro; Humberto D'Muniz Pereira

• Advisor: Andre Luís Berteli Ambrosio

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 (AU)

• FAPESP's process: 10/13739-6 - Establishing heterologous expression and purification conditions of the human hypoxia-inducible factor 1, HIF-1, for crystallographic studies.
• Grantee: Angela Maria Fala
• Support Opportunities: Scholarships in Brazil - Master