An Endoplasmic Reticulum Transmembrane Prolyl 4-Hydroxylase Is Induced by Hypoxia and Acts on Hypoxia-inducible Factor α

Abstract

Prolyl 4-hydroxylases (P4Hs) act on collagens (C-P4Hs) and the oxygen-dependent degradation domains (ODDDs) of hypoxia-inducible factor ␣ subunits (HIF-P4Hs) leading to degradation of the latter. We report data on a human P4H possessing a transmembrane domain (P4H-TM). Its gene is also found in zebrafish but not in flies and nematodes. Its sequence more closely resembles those of the C-P4Hs than the HIF-P4Hs, but it lacks the peptide substrate-binding domain of the C-P4Hs. P4H-TM levels in cultured cells are increased by hypoxia, and P4H-TM is N-glycosylated and is located in endoplasmic reticulum membranes with its catalytic site inside the lumen, a location differing from those of the HIF-P4Hs. Despite this, P4H-TM overexpression in cultured neuroblastoma cells reduced HIF-␣ ODDD reporter construct levels, and its small interfering RNA increased HIF-1␣ protein level, in the same way as those of HIF-P4Hs. Furthermore, recombinant P4H-TM hydroxylated the two critical prolines in HIF-1␣ ODDD in vitro, with a preference for the C-terminal proline, whereas it did not hydroxylate any prolines in recombinant type I procollagen chains.Prolyl 4-hydroxylases (P4Hs) 2 catalyze the formation of 4-hydroxyproline by the hydroxylation of proline residues in peptide linkages. Two animal P4H families are known today: collagen P4Hs (C-P4Hs), endoplasmic reticulum (ER) luminal enzymes that have a central role in the synthesis of all collagens (1-3), and HIF-P4Hs, nuclear and cytoplasmic enzymes that play a key role in the response of cells to hypoxia (4 -7).The C-P4Hs act on Xaa-Pro-Gly sequences in collagens and more than 20 collagen-like proteins, the 4-hydroxyproline residues formed being essential for the assembly of triple-helical molecules (1-3). All vertebrate C-P4Hs are ␣ 2 ␤ 2 tetramers in which the enzyme and chaperone protein disulfide-isomerase (PDI) acts as the ␤ subunit (1-3). Three isoforms of the catalytic ␣ subunit have been cloned and characterized and found to form [␣(I)] 2 ␤ 2 , [␣(II)] 2 ␤ 2 , and [␣(III)] 2 ␤ 2 tetramers with PDI, known as C-P4Hs I, II, and III, respectively (3,8,9).The HIF-P4Hs regulate the hypoxia-inducible factors (HIFs) by hydroxylating proline residues in Leu-Xaa-Xaa-Leu-Ala-Pro sequences at two separate sites in their ␣ subunits (10, 11). The human HIF-P4Hs have three isoenzymes, HIF-P4Hs 1-3 (also known as PHDs 1-3, HPHs 3-1, and EGLNs 2, 1, and 3, respectively), which show a 42-59% sequence identity to each other but essentially no sequence similarity to the C-P4Hs except for the catalytically critical residues (12-14). HIFs are ␣␤ heterodimers that act as master regulators of the transcription of more than 100 hypoxia-regulated genes (5-7). The human HIF-␣ subunit has three isoforms, HIF-1␣-HIF-3␣. HIF-1␣ and HIF-2␣ are synthesized constitutively, and hydroxylation of at least one of two critical proline residues in their oxygendependent degradation domain (ODDD), Pro 402 and Pro 564 in HIF-1␣, generates a binding site for the von Hippel-Lindau E3 ubiquitin ligase complex that tar...

Role and regulation of prolyl hydroxylase domain proteins

“…While these findings are all encouraging, it should be cautioned that PHDs are also potential tumor suppressors and it remains to be determined if long-term exposure to PHD inhibitors may favor tumor development. Nonetheless, given the presence of three PHDs, and now the discovery of a fourth related hydroxylase, P4H-TM, 11 it may be possible to exploit differential tissue expression profiles and HIF-a specificity to achieve the best therapeutic outcomes while minimizing associated side effects.…”

“…45 Interestingly, P4H-TM, which is related to PHDs, is anchored in the ER membrane with its catalytic domain located in the lumen rather than cytosol. 11 In spite of such an orientation, the enzyme hydroxylates HIF-1a and triggers HIF-1a degradation.…”

“…10 While Drosophila and Caenorhabditis elegans each has a single family member, known as dHPH in Drosophila 6 and egg laying nine (EGLN or EGL-9) in C. elegans (for its essential egg-laying function), 7 mammals have four members belonging to this subfamily, including PHD1/EGLN2/HPH3, PHD2/EGLN1/HPH2, PHD3/EGLN3/ HPH1 and a recently characterized protein named as P4H-TM. 6,7,11 Several other names have also been used for mammalian homologs, including SM-20 for rat PHD3 7 and HIF-P4H1, HIF-P4H2 and HIF-P4H3 to indicate that hydroxylation occurs on position 4 of proline residues. 12 Given that the term 'PHD proteins' both reflects the functionality as hydroxylases and avoids committing them as being HIF specific (because non-HIF substrates also exist 13 ), we will refer to mammalian homologs as PHD1, PHD2 and PHD3, however, keeping the name P4H-TM 11 for the fourth member of the subfamily due to the fact that other names have not been used in the literature.…”

HIF Prolyl-4-hydroxylase Interacting Proteins: Consequences for Drug Targeting

“…However, the HIF prolyl-4-hydroxylase enzymes will be referred to as "PHDs" throughout this review. The physiological relevance of a fourth family member, called PH-4, is less clear: HIF seems to be regulated under PH-4 overexpression conditions only [6,7].…”

“…Subsequently, HIF-binding HREs were identified in the regulatory regions of the corresponding genes [42,43]. At least in some cell lines, PH-4 seems to be induced by hypoxia but it is not a proven HIF target gene so far [7]. Up to date, little is known about other gene regulatory elements contributing to tissue-specific and signal-related expression of the genes encoding PHDs.…”