Identification of a novel cellular cofactor for the hypoxia-inducible transcription (HIF) and structural studies of the catalytic domains of isoforms 1 and 3 of the human prolyl-hydroxylases (PHD1 and PHD3) in complex with inhibitors

• Grant number: 14/04927-4
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: June 01, 2014
• End date: July 31, 2017
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Andre Luis Berteli Ambrosio
• Grantee: Angela Maria Fala
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM)

Abstract

One of the fundamental goals of cancer research is the understanding of the molecular changes that cause normal cells to evolve into malignant tumors. The adaptation of cancer cells to the microenvironment is the central point which leads to the invasive and metastatic phenotype, and is guaranteed mainly through the precise control of gene expression. The response to energy and biosynthetic requirements and especially the availability of intracellular oxygen, for example, is largely mediated by hypoxia -induced transcription factor (HIF). HIF is a heterodimer composed of subunits ± and ² , which recognizes consensus response element sequences 5'- RCGTG -3 ' and activates the transcription of more than 100 genes involved in several key aspects of tumor biology, including angiogenesis, glucose metabolism, cell differentiation, apoptosis, and resistance to radiotherapy and chemotherapy. Three isoforms of the ± subunit (1 to 3) are known and they all heterodimerize with the ² subunit. Overall, HIFs are composed of several and different functional domains, such as DNA binding, heterodimerization, transactivation and oxygen-dependent (ODD) degradation. Currently, little is known about the mechanisms of structural and functional domains of HIF - 1. The stability and transcriptional activity of HIF is primarily regulated by post-translational modifications (hydroxylation) catalyzed by prolyl-hydroxylase enzymes (PHDs). PHDs are dioxygenases that use 2-oxoglutarate as a cofactor and catalyze the Fe (II) and oxygen dependent hydroxylation of two specific proline residues. Under normoxic conditions, hydroxylation of ODD (at Pro402 and Pro564 in HIF-1) promotes the binding of the tumor suppressor VHL, leading HIF to poly- ubiquitination and proteasomal degradation signaling. Four isoforms of PHDs have been described in humans, being shown as having different preferential affinity or activity by either prolines described above, also distinguishing between isoforms of HIF-1 and -2 . PHD2 prolyl-hydroxylase is the most studied so far, mainly from the structural point of view, however PHDs 1 and 3 have just as important roles in the regulation of HIF stability. During the initial steps of design doctoral candidate scholarship, we identified a possible new and completely unexpected class of intracellular cofactors to HIF-3±: unsaturated fatty acids. Given the unprecedented nature of this observation, many questions are still open, such as what are intracellular relevant fatty acids in humans, as these may interfere with the binding and dimerization transitional activation (or inactivation of HIF) and which are the physiological conditions relevant for such interaction, thereby defining the first objective of this research proposal. As a second objective, in direct collaboration with the laboratory of Prof. Christopher Schofield, University of Oxford, UK, to determine the crystallographic structures of PHD1 and 3, in the presence of representative of the proline - target HIF-1 and HIF-2 peptides, and inhibitors have been identified, providing an understanding of the intrinsic selectivity of isoforms. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

FALA, ANGELA M.; OLIVEIRA, JULIANA F.; ADAMOSKI, DOUGLAS; ARICETTI, JULIANA A.; DIAS, MARILIA M.; DIAS, MARCIO V. B.; SFORCA, MAURCIO L.; LOPES-DE-OLIVEIRA, PAULO S.; ROCCO, SILVANA A.; CALDANA, CAMILA; et al. Unsaturated fatty acids as high-affinity ligands of the C-terminal Per-ARNT-Sim domain from the Hypoxia-inducible factor 3 alpha. SCIENTIFIC REPORTS, v. 5, AUG 3 2015. (12/14298-9, 10/13739-6, 14/04927-4, 14/20673-2, 14/15968-3)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

FALA, Angela Maria. HIF-3alpha: structural studies, identification and characterization of a high-affinity ligand of hypoxia inducible factor (HIF) and structural studies of prolil-hydroxilases (PHDs) from non usual organisms. 2017. Doctoral Thesis - Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia Campinas, SP.