The conservation and functionality of the oxygen-sensing enzyme Factor Inhibiting HIF (FIH) in non-vertebrates.

The asparaginyl hydroxylase, Factor Inhibiting HIF (FIH), is a cellular dioxygenase. Originally identified as oxygen sensor in the cellular response to hypoxia, where FIH acts as a repressor of the hypoxia inducible transcription factor alpha (HIF-α) proteins through asparaginyl hydroxylation, FIH also hydroxylates many proteins that contain ankyrin repeat domains (ARDs). Given FIH's promiscuity and the unclear functional effects of ARD hydroxylation, the biological relevance of HIF-α and ARD hydroxylation remains uncertain.

Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation.

Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components.

Factor inhibiting HIF (FIH) recognizes distinct molecular features within hypoxia-inducible factor-α (HIF-α) versus ankyrin repeat substrates.

Factor Inhibiting HIF (FIH) catalyzes the β-hydroxylation of asparagine residues in HIF-α transcription factors as well as ankyrin repeat domain (ARD) proteins such as Notch and Gankyrin. Although FIH-mediated hydroxylation of HIF-α is well characterized, ARDs were only recently identified as substrates, and less is known about their recognition and hydroxylation by FIH. We investigated the molecular determinants of FIH substrate recognition, with a focus on differences between HIF and ARD substrates.

Consequences of IkappaB alpha hydroxylation by the factor inhibiting HIF (FIH).

The factor inhibiting HIF-1 (FIH-1) hydroxylates many ankyrin repeat-containing proteins including IκBα. It is widely speculated that hydroxylation of IκBα has functional consequences, but the effects of hydroxylation have not been demonstrated. We prepared hydroxylated IκBα and compared it to the unhydroxylated protein.

Characterization of ankyrin repeat-containing proteins as substrates of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible transcription factor.

The hypoxia-inducible transcription factors (HIFs) are essential mediators of the genomic response to oxygen deficiency (hypoxia) in multicellular organisms. The HIFs are regulated by four oxygen-sensitive hydroxylases-three prolyl hydroxylases and one asparaginyl hydroxylase. These hydroxylases are all members of the 2-oxoglutarate (2OG)-dependent dioxygenase superfamily and convey changes in cellular oxygen concentration to the HIF-alpha (alpha) subunit, leading to potent accumulation and activity in hypoxia versus degradation and repression in normoxia.