Matthew E. Cockman

The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis

Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The α subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor,. Here we demonstrate a critical role for the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF α-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored oxygen-dependent instability. pVHL and HIF α-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and thatit is necessary for the oxygen-dependent degradation of HIF α-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumours. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing.

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.

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.

Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor.

The stability and activity of hypoxia-inducible factor (HIF) are regulated by the post-translational hydroxylation of specific prolyl and asparaginyl residues. We show that the HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also catalyzes hydroxylation of highly conserved asparaginyl residues within ankyrin repeat (AR) domains (ARDs) of endogenous Notch receptors. AR hydroxylation decreases the extent of ARD binding to FIH while not affecting signaling through the canonical Notch pathway. ARD proteins were found to efficiently compete with HIF for FIH-dependent hydroxylation. Crystallographic analyses of the hydroxylated Notch ARD (2.35Å) and of Notch peptides bound to FIH (2.4–2.6Å) reveal the stereochemistry of hydroxylation on the AR and imply that significant conformational changes are required in the ARD fold in order to enable hydroxylation at the FIH active site. We propose that ARD proteins function as natural inhibitors of FIH and that the hydroxylation status of these proteins provides another oxygen-dependent interface that modulates HIF signaling.

Proteomics-based Identification of Novel Factor Inhibiting Hypoxia-inducible Factor (FIH) Substrates Indicates Widespread Asparaginyl Hydroxylation of Ankyrin Repeat Domain-containing Proteins

Post-translational hydroxylation has been considered an unusual modification on intracellular proteins. However, following the recognition that oxygen-sensitive prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor hypoxia-inducible factor (HIF), interest has centered on the possibility that these enzymes may have other substrates in the proteome. In support of this certain ankyrin repeat domain (ARD)-containing proteins, including members of the IκB and Notch families, have been identified as alternative substrates of the HIF asparaginyl hydroxylase factor inhibiting HIF (FIH). Although these findings imply a potentially broad range of substrates for FIH, the precise extent of this range has been difficult to determine because of the difficulty of capturing transient enzyme-substrate interactions. Here we describe the use of pharmacological “substrate trapping” together with stable isotope labeling by amino acids in cell culture (SILAC) technology to stabilize and identify potential FIH-substrate interactions by mass spectrometry. To pursue these potential FIH substrates we used conventional data-directed tandem MS together with alternating low/high collision energy tandem MS to assign and quantitate hydroxylation at target asparaginyl residues. Overall the work has defined 13 new FIH-dependent hydroxylation sites with a degenerate consensus corresponding to that of the ankyrin repeat and a range of ARD-containing proteins as actual and potential substrates for FIH. Several ARD-containing proteins were multiply hydroxylated, and detailed studies of one, Tankyrase-2, revealed eight sites that were differentially sensitive to FIH-catalyzed hydroxylation. These findings indicate that asparaginyl hydroxylation is likely to be widespread among the ∼300 ARD-containing species in the human proteome.

Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans

The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.

Hydroxylation of the eukaryotic ribosomal decoding center affects translational accuracy

Significance The processing of DNA sequences into proteins is fine-tuned to meet the conflicting demands of accuracy and speed. DNA mutations can introduce premature stop codons, leading to inactive proteins. We report that oxygen-dependent posttranslational modification of the ribosomal decoding center affects stop codon readthrough in an mRNA sequence-dependent manner. Our work demonstrates that oxygenases catalyzing RPS23 hydroxylation are conserved in eukaryotes, including yeasts, flies, and humans. In basal eukaryotes, RPS23 undergoes two hydroxylations, whereas in animals we only observe one hydroxylation. Yeast ribosomes lacking hydroxylation manifest altered stop codon readthrough up to ∼10-fold. The results reveal oxygen-dependent modifications that regulate translational accuracy and suggest unprecedented approaches to modulating ribosomal accuracy for medicinal application. The mechanisms by which gene expression is regulated by oxygen are of considerable interest from basic science and therapeutic perspectives. Using mass spectrometric analyses of Saccharomyces cerevisiae ribosomes, we found that the amino acid residue in closest proximity to the decoding center, Pro-64 of the 40S subunit ribosomal protein Rps23p (RPS23 Pro-62 in humans) undergoes posttranslational hydroxylation. We identify RPS23 hydroxylases as a highly conserved eukaryotic subfamily of Fe(II) and 2-oxoglutarate dependent oxygenases; their catalytic domain is closely related to transcription factor prolyl trans-4-hydroxylases that act as oxygen sensors in the hypoxic response in animals. The RPS23 hydroxylases in S. cerevisiae (Tpa1p), Schizosaccharomyces pombe and green algae catalyze an unprecedented dihydroxylation modification. This observation contrasts with higher eukaryotes, where RPS23 is monohydroxylated; the human Tpa1p homolog OGFOD1 catalyzes prolyl trans-3-hydroxylation. TPA1 deletion modulates termination efficiency up to ∼10-fold, including of pathophysiologically relevant sequences; we reveal Rps23p hydroxylation as its molecular basis. In contrast to most previously characterized accuracy modulators, including antibiotics and the prion state of the S. cerevisiae translation termination factor eRF3, Rps23p hydroxylation can either increase or decrease translational accuracy in a stop codon context-dependent manner. We identify conditions where Rps23p hydroxylation status determines viability as a consequence of nonsense codon suppression. The results reveal a direct link between oxygenase catalysis and the regulation of gene expression at the translational level. They will also aid in the development of small molecules altering translational accuracy for the treatment of genetic diseases linked to nonsense mutations.

FIH-dependent asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.

Studies on hypoxia‐sensitive pathways have identified a series of Fe(II)‐dependent dioxygenases that regulate hypoxia‐inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The asparaginyl hydroxylase factor inhibiting HIF (FIH) targets a conserved asparaginyl residue in the C‐terminal transactivation domain of HIF‐α. This modification suppresses HIF transcriptional activity by inhibiting co‐activator recruitment. Recent work has demonstrated that FIH targets an alternative class of substrate. Proteins containing a common interaction motif known as the ankyrin repeat domain (ARD) have been shown to be efficiently hydroxylated by FIH. This review aims to summarize what is currently known regarding ARD hydroxylation, including the kinetics and determinants of FIH‐mediated ARD hydroxylation, the structural and functional consequences of ARD hydroxylation, and the potential for cross‐talk between ARD proteins and HIF signaling.

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.

Factor‐inhibiting hypoxia‐inducible factor (FIH) catalyses the post‐translational hydroxylation of histidinyl residues within ankyrin repeat domains

Factor‐inhibiting hypoxia‐inducible factor (FIH) is an Fe(II)/2‐oxoglutarate‐dependent dioxygenase that acts as a negative regulator of the hypoxia‐inducible factor (HIF) by catalysing β‐hydroxylation of an asparaginyl residue in its C‐terminal transcriptional activation domain (CAD). In addition to the hypoxia‐inducible factor C‐terminal transcriptional activation domain (HIF‐CAD), FIH also catalyses asparaginyl hydroxylation of many ankyrin repeat domain‐containing proteins, revealing a broad sequence selectivity. However, there are few reports on the selectivity of FIH for the hydroxylation of specific residues. Here, we report that histidinyl residues within the ankyrin repeat domain of tankyrase‐2 can be hydroxylated by FIH. NMR and crystallographic analyses show that the histidinyl hydroxylation occurs at the β‐position. The results further expand the scope of FIH‐catalysed hydroxylations.