Identification of genes regulated by HIF-3±9 isoform in response to the interaction with lipids

• Grant number: 15/25880-9
• Support Opportunities: Scholarships in Brazil - Master
• Start date: July 01, 2016
• End date: May 31, 2018
• 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: Graziele Izalina Vasconcelos Bento
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil

Abstract

The hypoxia-inducible transcription factors (HIF), closely linked to disorders that lead top the progression of cancer, consist of two multidomain subunits including the constitutively expressed HIF-1² and an oxygen-labile HIF-1± (or paralogs HIF-2± and -3±). The heterodimeric complex controls the homeostatic and systemic responses to oxygen availability within the cell. In the presence of oxygen, proline residues in HIF-± are hydroxylated, which leads to subsequent ubiquitination and degradation by the proteasome. In hypoxic conditions (low cellular oxygen levels) the ± subunit is stabilized, and HIF complex increases transcription of genes whose products increase the O2 availability and delivery of nutrients through angiogenesis (growth of new blood vessels) and erythropoiesis (erythrocytes), thus influencing the metabolic aspects of the cell. The gene encoding for HIF-3± (HIF3A) is exclusively susceptible to post-transcriptional processing in humans, with six isoforms shown to exist at mRNA levels and three already confirmed as functional protein. HIF-3±9, the longest (669 aa) and canonical isoform3, is a potent oxygen-labile transcription factor, tightly regulating the expression of a unique set of genes. HIF-3±4 (363 aa), on the other hand, while insensitive to intracellular oxygen levels and incapable of transactivation, is a dominant-negative regulator of HIF-1±. Due to the use of different transcription initiation sites and a combination of splicing events in HIF3A, the C-terminal Per-Arnt-Sim sensor domain (PASb) is the only common architectural feature among the HIF-3± isoforms3. The PASb domains, which are extensively found across the three domains of life, in both HIF-± and -² subunits have a crucial role in forming active HIF heterodimers and recruiting co-regulators. Our group has recently identified by the presence of a fatty acid molecule, of bacterial nature, bound to an extensive hydrophobic C-shaped cavity in the PAS domain of HIF-3±9. Experiments previously performed by us have identified that the protein binds to neutral lipids and phospholipids, and the presence of such a ligand is essential for the correct folding and stability of the HIF-3±, as well as increases the stability of interaction with PAS and may potentially regulate tits transcriptional activity. In this context, this project mainly aims to identify which lipids interact with HIF-3± in mammalian cells. Then the identified lipids will be confirmed in biophysical experiments with recombinant PASB domain. In addition, we want to characterize the transcriptome (by RNAseq and qPCR) of tumor cells that wild-type or mutant HIF-3±9 overexpression which prevents the interaction with lipids, as well as conditions to knock down the SCD1 desaturase, FAS or FABP and culture in medium supplemented with delipidated serum (or adding lipid mixture). This study has important implications for the understanding of HIF-3± function and future development of small molecule therapeutics having specifically target this isoform. (AU)