Prolyl-hydroxylase 3: Evolving Roles for an Ancient Signaling Protein

Abstract

The ability of cells to sense oxygen is a highly evolved process that facilitates adaptations to the local oxygen environment and is critical to energy homeostasis. In vertebrates, this process is largely controlled by three intracellular prolyl-4-hydroxylases (PHD 1-3). These related enzymes share the ability to hydroxylate the hypoxia-inducible transcription factor (HIF), and therefore control the transcription of genes involved in metabolism and vascular recruitment. However, it is becoming increasingly apparent that proline-4-hydroxylation controls much more than HIF signaling, with PHD3 emerging as an exceptionally unique and functionally diverse PHD isoform. In fact, PHD3-mediated hydroxylation has recently been purported to function in such diverse roles as sympathetic neuronal and muscle development, sepsis, glycolytic metabolism, and cell fate. PHD3 expression is also highly distinct from that of the other PHD enzymes, and varies considerably between different cell types and oxygen concentrations. This review will examine the evolution of oxygen sensing by the HIF-family of PHD enzymes, with a specific focus on complex nature of PHD3 expression and function in mammalian cells.

Keywords: EGLN3; HIF-PHD; Hypoxia; Hypoxia Inducible Factor; Oxygen Sensing; PHD3.

Figure 1

The evolution of PHD and HIF in relation to the oxygenation of Earth. Notes: At the top of the figure, the phylogenetic tree of the PHD-containing groups amebozoa and opisthokonta (metazoan and fungi), is depicted with approximate branch points relative to the Earth’s geological age. The arrows indicate the approximate origins of the PHD and HIF genes.,, At the bottom of the figure, the partial pressure of atmospheric O₂ is graphed in relation to time, as adapted from Holland et al. The gray area represents the range of the estimate. In addition, 0.21 atm (the current pO₂) is marked as a dotted line. Holland HD. The oxygenation of the atmosphere and oceans. Philos Trans R Soc Lond B Biol Sci. 2006;361(1470):903–915, by permission of the Royal Society. Abbreviations: PHD, prolyl-4-hydroxylase; HIF, hypoxia-inducible transcription factor; pO₂, partial pressure of oxygen.

Figure 2

The interaction between metabolism and PHD enzymatic activity. Notes: It is apparent that 2-oxoglutarate from the TCA cycle is utilized, along with molecular oxygen, to hydroxylate a proline residue on carbon 4. The reaction produces CO₂ and succinate (left). Hydroxylation is thought to involve a transient OOH molecule, which is stabilized by Fe-2 in the PHD3 catalytic domain. One oxygen atom subsequently becomes incorporated into succinate, and the other in the proline–OH group (right). During oxidative stress, Fe-2 is susceptible to oxidation by reactive oxygen species, which inhibits hydroxylase activity. Fe 3 is reduced back to Fe-2 by ascorbate/Vit C. Abbreviations: TCA, tricarboxylic; PHD, prolyl-4-hydroxylase; Vit, vitamin; ROS, reactive oxygen species; OH, hydroxyl group.

Figure 3

Effectors of PHD3 enzymatic activity. Notes: Proline hydroxylation requires 2-oxoglutarate from the TCA cycle, and O₂ for enzymatic function (blue boxes). The TCA cycle and electron transport chain are the source of many inhibitors of PHD enzymatic activity (yellow boxes). Build-up of fumarate occurs upstream of FH-mut and succinate buildup occurs upstream of SDH-mut. Gain-of-function mutations in the IDH1-mut produce (R)-2-hydroxyglutarate, another PHD inhibitor. Electron leak from complex 3 of the electron transport chain results in superoxide production (O₂⁻) in the presence of O₂, which is converted to H₂O₂ by catalase. ROS, such as H₂O₂ can oxidize Fe-2 to Fe-3. PHD inhibitors commonly used in research include dimethyloxalylglycine, desferrioxamine, and cobalt-chloride (Co-2) (upper right yellow box). Abbreviations: MnSOD, manganese superoxide dismutase; FH, fumarate hydratase; mut, mutations; SDH, succinate dehydrogenase; Co-2, cobalt-chloride; IDH, isocitrate dehydrogenase; IDH1, cytoplasmic isoform if isocitrate dehydrogenase; PHD, prolyl-4-hydroxylase; TCA, tricarboxylic; ROS, reactive oxygen species; OH, hydroxyl group.

Figure 4

Models of human PHD1–3 and rat PHD3 (SM-20) proteins. Notes: PHD1, PHD2, and three proteins all share highly homologous P4Hc-containing sequences in their N-terminus. PHD2 is the longest PHD and contains a MYND. Rat PHD3 (SM-20) is unique in that it contains a Mito. Abbreviations: PHD, prolyl-4-hydroxylase; EGLN, egg-laying-defective nine; MYND, C-terminal MYND-type zinc finger domain; P4Hc, prolyl-4-hydroxylase; SM-20, smooth muscle-20; Mito, N-terminal mitochondrial localization signal.

Figure 5

HIF-1α regulation. Notes: In the presence of O₂, HIF-1α is hydroxylated by PHD1, PHD2, or PHD3 on two proline residues in the O₂-dependent domain. These hydroxylated proline residues serve as binding sites for VHL, which binds and Ub HIF-1α. Hydroxylation on asparagine 803 in the C-TAD by FIH blocks p300/CPB recruitment, which further inhibits HIF transcriptional transactivation. In the absence of O₂, HIF is not hydroxylated and VHL does not bind (bottom of figure). HIF-1α protein levels are subsequently stabilized and HIF-1α becomes localized to the nucleus where it dimerizes with HIF-1β via the PAS domain and interacts with DNA through the bHLH domain. P300/CBP is recruited through binding to the C-TAD. Abbreviations: HIF, hypoxia-inducible transcription factor; bHLH, basic helix–loop–helix; DNA, deoxyribonucleic acid; PAS, Per-aryl hydrocarbon receptor nuclear translocator-Sim; VHL, Von Hippel–Lindau tumor suppressor; NODD, nonoxygen-dependent domain; CODD, C-terminal oxygen-dependent degradation domain Ub, ubiquitinates; FIH, factor-inhibiting hypoxia-inducible transcription factor; OH, hydroxide; PHD, prolyl-4-hydroxylase, C-TAD, C-terminal activation domain; CPB, creb-binding protein.

Figure 6

HIF pathway regulation by the PHD enzymes. Notes: HIF-1 can be a substrate for any of the three PHD proteins; however, PHD3, which is the most inducible PHD under hypoxic conditions, has broader substrate specificity and can hydroxylate a number of non-HIF-1α targets. Abbreviations: PHD, prolyl-4-hydroxylase; HIF, hypoxia-inducible transcription factor; OH, hydroxyl group; gDNA, genomic deoxyribonucleic acid; HRE, hypoxia response elements; Neg, negative.

Figure 7

Relative expression of PHD1, PHD2, and PHD3 in ten human cancer cell lines. Notes: The relative expression of PHD enzymes was determined by Appelhoff et al in ten human cancer cell lines: U2-OS, BxPC3, MCF7, ZR751, MDA-435, BT-474, HEP3B, HeLa, 833K, and SuSa cell lines under normoxic and hypoxic (1% O₂) conditions for 16 hours. (A) Appelhoff et al’s data were recompiled and averaged to demonstrate general trends in the relative expression of PHD1, PHD2, and PHD3. Error bars represent one standard deviation. (B) The average fold hypoxic-induction of each PHD is re-graphed from the data above ^40. Abbreviation: PHD, prolyl-4-hydroxylase.

Figure 8

PHD3 regulatory elements. Notes: The PHD3 gene with mammalian conservation is depicted in relation to its position on chromosome 14 q13.1 (modified from the UCSC Genome Browser hg19 [http://genome.ucsc.edu/]). The highly conserved region at ∼+12 kb upstream of exon 1 is expanded (bottom), and putative HRE are indicated (gray box), along with conserved TF binding sites, as predicted by the UCSC hg19 “TFBS conserved” track. The black arrow indicates the direction of 3′–5′ sequence orientation. *Represents highly conserved sequences located in intron 1 and 2 that may represent important regulatory regions. Abbreviations: Chr, chromosome; PHD, prolyl-4-hydroxylase; HRE, hypoxia response elements; NF-1, nuclear factor-1; TF, transcription factor; UCSC, University of California Santa Cruz; TFBS, transcription factor binding sites.

Figure 9

Polymerase 2 interaction with +12 kb region of the PHD3 gene. Notes: The PHD3 locus is represented with mammalian conservation (modified from the UCSC Genome Browser hg19 [http://genome.ucsc.edu/]). ChIP-Seq signal data for polymerase 2 in H1-hESC, HeLa-S3, and HCT-116 cells were mined from several ENCODE data tracks (as indicated) using the UCSC Genome Browser hg19 (http://genome.ucsc.edu/). Abbreviations: PHD, prolyl-4-hydroxylase; hESC, human embryonic stem cells; TFBS, transcription factor binding sites; ENCODE, Encyclopedia of DNA Elements; ChIA-PET, chromatin interaction analysis with paired-end tag sequencing; ChIP-Seq, chromatin immunoprecipitation sequencing.