Differential Sensitivity of Hypoxia Inducible Factor Hydroxylation Sites to Hypoxia and Hydroxylase Inhibitors

Abstract

Hypoxia inducible factor (HIF) is regulated by dual pathways involving oxygen-dependent prolyl and asparaginyl hydroxylation of its α-subunits. Prolyl hydroxylation at two sites within a central degradation domain promotes association of HIF-α with the von Hippel-Lindau ubiquitin E3 ligase and destruction by the ubiquitin-proteasome pathways. Asparaginyl hydroxylation blocks the recruitment of p300/CBP co-activators to a C-terminal activation domain in HIF-α. These hydroxylations are catalyzed by members of the Fe(II) and 2-oxoglutarate (2-OG) oxygenase family. Activity of the enzymes is suppressed by hypoxia, increasing both the abundance and activity of the HIF transcriptional complex. We have used hydroxy residue-specific antibodies to compare and contrast the regulation of each site of prolyl hydroxylation (Pro402, Pro564) with that of asparaginyl hydroxylation (Asn803) in human HIF-1α. Our findings reveal striking differences in the sensitivity of these hydroxylations to hypoxia and to different inhibitor types of 2-OG oxygenases. Hydroxylation at the three sites in endogenous human HIF-1α proteins was suppressed by hypoxia in the order Pro402 > Pro564 > Asn803. In contrast to some predictions from in vitro studies, prolyl hydroxylation was substantially more sensitive than asparaginyl hydroxylation to inhibition by iron chelators and transition metal ions; studies of a range of different small molecule 2-OG analogues demonstrated the feasibility of selectively inhibiting either prolyl or asparaginyl hydroxylation within cells.

The asparaginyl hydroxylase factor-inhibiting HIF is essential for tumor growth through suppression of the p53–p21 axis

“…The estimated K m values for oxygen for the PHDs and FIH in vitro are quite different: 250 and 90 mM, respectively (Koivunen et al, 2003). Consistent with this work, a recent study showed in in vivo experiments, using the endogenous human HIF-1a protein, that both prolyl hydroxylation was more sensitive to inhibition to hypoxia (Hx) than asparaginyl hydroxylation (Tian et al, 2011). Thus, the PHD activity should be more sensitive to a moderate decrease in oxygen compared with that of FIH.…”

FIH-1-Mint3 Axis Does Not Control HIF-1α Transcriptional Activity in Nucleus Pulposus Cells

“…It is noteworthy that overexpression of FIH-1 results in a decrease in HIF-1␣ activity even under hypoxic (1% O 2 ) conditions, similar to what was seen previously for PHD2 activity in NP cells (10). Moreover, these results are consistent with previous findings that the in vitro K m for O 2 of FIH-1 is much smaller (90 M) than that of PHD2 (250 M) (32,33) and that substantial levels of hydroxylated Asn 803 can be detected even at 0.1% O 2 (20) in RCC4 and MCF7 cells, suggesting preservation of FIH-1 enzymatic function under hypoxia. Furthermore, a very recent report that FIH-1 can still suppress HIF-dependent GLUT1 and VEGF-A expression in hypoxic glioblastoma in vitro and also in vivo in a xenograft model supports this idea (34).…”

“…This post-translational modification of a specific Asn residue prevents binding to p300/CBP, thereby suppressing HIF-1/2 transcriptional activity (18,19). Noteworthy, because the K m of FIH-1 for oxygen is significantly lower than that of PHD1-3, even under conditions of moderate hypoxia, such as those present in the NP, FIH-1 activity is preserved (20). Thus, controlling expression/activity of FIH-1 is one of the important ways cells control HIF transcriptional activity.…”

MPT0B098, a Novel Microtubule Inhibitor That Destabilizes the Hypoxia-Inducible Factor-1α mRNA through Decreasing Nuclear–Cytoplasmic Translocation of RNA-Binding Protein HuR

“…In the presence of oxygen (normoxia), HIF-1a is hydroxylated by prolyl hydroxylase at prolines 402 and 564 and then recognized by the von Hippel-Lindau (VHL) protein, ubiquitinated by E3 ubiquitin ligase, and degraded rapidly via the proteasome pathway (4). HIF-1a is also hydroxylated by asparaginyl hydroxylase at asparagine 803 and blocks its interaction with the transcriptional coactivators CBP and p300 (5). Both prolyl and asparaginyl hydroxylation are inhibited under the hypoxic condition, which allows HIF1a to accumulate and subsequently leads to the transactivation of hundreds of downstream target genes involved in cell metabolism, survival, angiogenesis, and metastasis (6).…”