Clonagem e expressão do fator 1 humano induzível por hipóxia (HIF-1) na levedura Saccharomyces cerevisiae

AUTOR(ES)

Túlio César Ferreira

DATA DE PUBLICAÇÃO

2006

RESUMO

The hypoxia-inducible factor-1 (HIF-1) is a central regulator in the response to changes in oxygen concentration and plays an important role in physiological and pathological processes in humans. This transcription factor is expressed when mammalian cells are subjected to hypoxia and activates transcription of over 70 oxygen-regulated target genes. HIF-1 is a heterodimeric protein composed of two members of the basic helix-loop-helix (bHLH)-containing PER-ARNT-SIM (PAS) domain family, HIF-1 (unique to the hypoxia response) and HIF-1 (also known as the aryl hydrocarbon receptor nuclear translocator, ARNT, which is constitutively expressed). HIF-1 stability and activity are regulated by post-translational modifications such as phosphorylation, hydroxylation, nitrosation and acetylation. Under normoxia, HIF-1 is constitutively expressed and subsequently hydroxylated by a HIF prolyl hydroxylase (HPH) causing it to be rapidly targeted for proteasome- mediated degradation. The sequence of events that leads to the full activation of HIF- 1 and the paradoxical effects of HIF-1 (pro- and anti-apoptotic effects) are not completely understood. Neither is clear how oxygen-dependent nor oxygen- independent mechanisms contribute to the post-translational modifications of HIF-1 , nuclear translocation, heterodimerization with ARNT, DNA binding to the cis- regulatory region of target genes and recruitment of cofactors. Therefore, HIF-1 interacting partners and the pos-translational modifications need to be better elucidated. Yeast cells share several aspects of its biochemistry with multicellular organism such as, transcriptional and cellular responses and mechanisms of protein post-transcriptional modifications. In addition they are easy to manipulate under different laboratory conditions. This work aimed to evaluate the viability of HIF-1 expression in the yeast Saccharomyces cerevisiae as an alternative expression model. In yeast cells, HIF-1 may not be targeted to degradation under normoxic conditions, since this microorganism lacks the pathway mediated by the von Hippel Lindau-VHL (a tumor suppressor protein). We intend to used this expression model to study the post-translational modifications and its interaction with other proteins that may affect its dimerization/co-activation. We established a yeast strain over- expressing HIF-1 and ARNT in a constitutive manner under normal conditions of oxygen. We also showed that recombinant HIF-1 was able to form a heterodimer with ARNT and bind to human erythropoietin hypoxia responsive element (HRE) motif. Additionally, we described the presence of this motif in the yeast genome and showed that this organism may harbor HRE motifs.

ASSUNTO(S)

hipóxia hre biologia molecular células saccharomyces cerevisiae expressão eteróloga biologia molecular hif-1 bioquímica clonagem molecular

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