Norma Masson

C. elegans EGL-9 and Mammalian Homologs Define a Family of Dioxygenases that Regulate HIF by Prolyl Hydroxylation

HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.

Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein.

The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen-dependent proteolysis of α subunits of hypoxia inducible factor-1 (HIF). Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and up-regulation of hypoxia inducible genes. Here we investigate the mechanisms underlying these processes and show that extracts from VHL-deficient renal carcinoma cells have a defect in HIF-α ubiquitylation activity which is complemented by exogenous pVHL. This defect was specific for HIF-α among a range of substrates tested. Furthermore, HIF-α subunits were the only pVHL-associated proteasomal substrates identified by comparison of metabolically labeled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-α interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-α molecules sufficient for recognition by pVHL. In contrast, while full-length pVHL and the p19 variant interact with HIF-α, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the β-domain of pVHL and loss of interaction was associated with defective HIF-α ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis. The findings indicate that pVHL regulates HIF-α proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its β domain interacts with short recognition sequences in HIF-α subunits.

Independent function of two destruction domains in hypoxia-inducible factor-α chains activated by prolyl hydroxylation

Oxygen‐dependent proteolytic destruction of hypoxia‐inducible factor‐α (HIF‐α) subunits plays a central role in regulating transcriptional responses to hypoxia. Recent studies have defined a key function for the von Hippel–Lindau tumour suppressor E3 ubiquitin ligase (VHLE3) in this process, and have defined an interaction with HIF‐1α that is regulated by prolyl hydroxylation. Here we show that two independent regions within the HIF‐α oxygen‐dependent degradation domain (ODDD) are targeted for ubiquitylation by VHLE3 in a manner dependent upon prolyl hydroxylation. In a series of in vitro and in vivo assays, we demonstrate the independent and non‐redundant operation of each site in regulation of the HIF system. Both sites contain a common core motif, but differ both in overall sequence and in the conditions under which they bind to the VHLE3 ligase complex. The definition of two independent destruction domains implicates a more complex system of pVHL–HIF‐α interactions, but reinforces the role of prolyl hydroxylation as an oxygen‐dependent destruction signal.

Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH)

Studies on hypoxia-sensitive pathways have revealed a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the IκB family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and IκBα were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARD-containing proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes.

HIF prolyl and asparaginyl hydroxylases in the biological response to intracellular O(2) levels.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that plays a crucial role in mediating cellular responses to oxygen. Oxygen availability influences multiple steps in HIF activation and recent studies have indicated that at least two steps in this process are governed by a novel mode of signal transduction involving enzymatic hydroxylation of specific amino acid residues in HIF-α subunits by a series of 2-oxoglutarate (2-OG)-dependent oxygenases. These enzymes are non-haem iron enzymes that use dioxygen in the hydroxylation reaction and therefore provide a direct link between the availability of molecular oxygen and regulation of HIF. Prolyl hydroxylation regulates proteolytic destruction of HIF-α by the von Hippel-Lindau ubiquitin ligase complex, whereas HIF-α asparaginyl hydroxylation regulates recruitment of transcriptional coactivators. The involvement of at least two distinct types of 2-OG-dependent oxygenase in oxygen-regulated transcription suggests that these enzymes may be well suited to a role in cellular oxygen sensing.

Peptide blockade of HIFα degradation modulates cellular metabolism and angiogenesis

Hypoxia-inducible factor-1 (HIF) is a transcription factor central to oxygen homeostasis. It is regulated via its α isoforms. In normoxia they are ubiquitinated by the von Hippel-Lindau E3 ligase complex and destroyed by the proteasome, thereby preventing the formation of an active transcriptional complex. Oxygen-dependent enzymatic hydroxylation of either of two critical prolyl residues in each HIFα chain has recently been identified as the modification necessary for targeting by the von Hippel-Lindau E3 ligase complex. Here we demonstrate that polypeptides bearing either of these prolyl residues interfere with the degradative pathway, resulting in stabilization of endogenous HIFα chains and consequent up-regulation of HIF target genes. Similar peptides in which the prolyl residues are mutated are inactive. Induction of peptide expression in cell cultures affects physiologically important functions such as glucose transport and leads cocultured endothelial cells to form tubules. Coupling of these HIFα sequences to the HIV tat translocation domain allows delivery of recombinant peptide to cells with resultant induction of HIF-dependent genes. Injection of tat-HIF polypeptides in a murine sponge angiogenesis assay causes a markedly accelerated local angiogenic response and induction of glucose transporter-1 gene expression. These results demonstrate the feasibility of using these polypeptides to enhance HIF activity, opening additional therapeutic avenues for ischemic diseases.

The FIH hydroxylase is a cellular peroxide sensor that modulates HIF transcriptional activity

Hypoxic and oxidant stresses can coexist in biological systems, and oxidant stress has been proposed to activate hypoxia pathways through the inactivation of the ‘oxygen‐sensing’ hypoxia‐inducible factor (HIF) prolyl and asparaginyl hydroxylases. Here, we show that despite reduced sensitivity to cellular hypoxia, the HIF asparaginyl hydroxylase—known as FIH, factor inhibiting HIF—is strikingly more sensitive to peroxide than the HIF prolyl hydroxylases. These contrasting sensitivities indicate that oxidant stress is unlikely to signal hypoxia directly to the HIF system, but that hypoxia and oxidant stress can interact functionally as distinct regulators of HIF transcriptional output.

The HIF prolyl hydroxylase PHD3 is a potential substrate of the TRiC chaperonin

Hypoxia‐inducible factor‐1 (HIF) is regulated by oxygen‐dependent prolyl hydroxylation. Of the three HIF prolyl hydroxylases (PHD1, 2 and 3) identified, PHD3 exhibits restricted substrate specificity in vitro and is induced in different cell types by diverse stimuli. PHD3 may therefore provide an interface between oxygen sensing and other signalling pathways. We have used co‐purification and mass spectrometry to identify proteins that interact with PHD3. The cytosolic chaperonin TRiC was found to copurify with PHD3 in extracts from several cell types. Our results indicate that PHD3 is a TRiC substrate, providing another step at which PHD3 activity may be regulated.

Interaction of hydroxylated collagen IV with the von hippel-lindau tumor suppressor.

The von Hippel-Lindau tumor suppressor (pVHL) targets hydroxylated α-subunits of hypoxia-inducible factor (HIF) for ubiquitin-mediated proteasomal destruction through direct interaction with the hydroxyproline binding pocket in its β-domain. Although disruption of this process may contribute to VHL-associated tumor predisposition by up-regulation of HIF target genes, genetic and biochemical analyses support the existence of additional functions, including a role in the assembly of extracellular matrix. In an attempt to delineate these pathways, we searched for novel pVHL-binding proteins. Here we report a direct, hydroxylation-dependent interaction with α-chains of collagen IV. Interaction with pVHL was also observed with fibrillar collagen chains, but not the folded collagen triple helix. The interaction was suppressed by a wide range of tumor-associated mutations, including those that do not disturb the regulation of HIF, supporting a role in HIF-independent tumor suppressor functions.

Heterogeneous Effects of Direct Hypoxia Pathway Activation in Kidney Cancer.

General activation of hypoxia-inducible factor (HIF) pathways is classically associated with adverse prognosis in cancer and has been proposed to contribute to oncogenic drive. In clear cell renal carcinoma (CCRC) HIF pathways are upregulated by inactivation of the von-Hippel-Lindau tumor suppressor. However HIF-1α and HIF-2α have contrasting effects on experimental tumor progression. To better understand this paradox we examined pan-genomic patterns of HIF DNA binding and associated gene expression in response to manipulation of HIF-1α and HIF-2α and related the findings to CCRC prognosis. Our findings reveal distinct pan-genomic organization of canonical and non-canonical HIF isoform-specific DNA binding at thousands of sites. Overall associations were observed between HIF-1α-specific binding, and genes associated with favorable prognosis and between HIF-2α-specific binding and adverse prognosis. However within each isoform-specific set, individual gene associations were heterogeneous in sign and magnitude, suggesting that activation of each HIF-α isoform contributes a highly complex mix of pro- and anti-tumorigenic effects.