Abstract HIF prolyl 4-hydroxylases (HIF-P4Hs) and HIF asparaginyl hydroxylase (FIH) are novel members of the 2-oxoglutarate dioxygenase family that play key roles in the regulation of the hypoxia-inducible transcription factor (HIF). They hydroxylate specific proline and asparagine residues in HIF-α, leading to its proteasomal degradation and inhibition of its transcriptional activity, respectively. These enzymes are inhibited in hypoxia, and as a consequence HIF-α becomes stabilized, forms a dimer with HIF-β, attains its maximal transcriptional activity and induces expression of many genes that are important for cell survival under hypoxic conditions. The three HIF-P4Hs and FIH were expressed here as recombinant proteins in insect cells and purified to near homogeneity. All these enzymes were found to require long peptide substrates. The three HIF-P4Hs and FIH acted differently on the various potential hydroxylation sites in the HIF-α isoforms. The HIF-P4Hs acted well on sequences with cores distinctly different from the core motif -Leu-X-X-Leu-Ala-Pro-, suggested based on sequence analysis studies, the alanine being the only relatively strict requirement in addition to the proline itself. Acidic residues around the hydroxylation site also played a distinct role. These results together with those of others provide evidence that there is no conserved core motif for the hydroxylation by HIF-P4Hs. The Km values of the HIF-P4Hs for O2 were slightly above its atmospheric concentration, while the Km of FIH was about one-third of these values but still 2.5 times that of the type I collagen P4H. The HIF-P4Hs are thus effective oxygen sensors, as even small decreases in the amount of O2 affect their activities, while a more severe decrease is required to inhibit FIH activity. Small molecule inhibitors of the collagen P4Hs also inhibited the HIF-P4Hs and FIH but with distinctly different Ki values, indicating that it should be possible to develop specific inhibitors for the HIF-P4Hs and FIH. The HIF-P4Hs were found to bind the iron cosubstrate more tightly than FIH and the collagen P4Hs, and the chelator desferrioxamine was an ineffective inhibitor of the HIF-P4Hs in vitro. Several metals were effective competitive inhibitors of FIH but they were ineffective inhibitors of the HIF-P4Hs. The well-known stabilization of HIF-1α by cobalt and nickel is thus not due to a simple competitive inhibition of the HIF-P4Hs, and is probably at least in part due to HIF-P4H-independent mechanisms. The effective inhibition of FIH by these metals nevertheless indicates that the stabilized HIF-1α is transcriptionally fully active.

Abstract Oxygen homeostasis is critical to all animals, as both excess (hyperoxia) and reduced (hypoxia) levels of oxygen can result in pathological changes and ultimately in the loss of cellular and organismal viability. Complex systems have evolved to sense and adapt to changes in cellular oxygen availability, and the hypoxia-inducible factor HIF plays a pivotal role in this elaborate molecular network. In normoxic conditions the α-subunit of HIF becomes hydroxylated by HIF prolyl 4-hydroxylases (HIF-P4Hs 1-3), earmarking HIF-α for proteasomal degradation. Additionally, in the presence of oxygen the hydroxylation of an asparagine residue by the HIF asparaginyl hydroxylase FIH inhibits the transactivation of HIF-target genes by blocking the interaction of HIF-α with a transcriptional coactivator. In addition to being a feature of an organism’s normal life, hypoxia is also characteristic of many common diseases such as severe anemia and myocardial infarction, and it notably decreases these hydroxylation reactions, as HIF-P4Hs and FIH have an absolute requirement for oxygen as a cosubstrate. HIF-α thus escapes degradation and translocates into the nucleus, where it dimerizes with HIF-β and recruits transcriptional coactivators to the hypoxia-response elements of target genes, inducing their transcription and triggering the hypoxia response aimed at restoring cellular oxygen homeostasis. In this study we generated a genetically modified HIF-P4H-2 hypomorphic mouse line that expresses only 8% of the wild-type HIF-P4H-2 mRNA in the heart. We showed that chronic cardiac HIF-P4H-2 deficiency leads to stabilization of HIF-1α and HIF-2α and protects the heart against acute ischemia-reperfusion injury without causing any adverse effects. Furthermore, we identified and cloned a novel human transmembrane prolyl 4-hydroxylase P4H-TM and showed that it regulates HIF-1α protein levels in cellulo and hydroxylates HIF-1α in vitro similarly to the HIF-P4Hs, but may also have other physiological substrates. Using forward genetic tools we showed that lack of P4H-TM during development leads to basement membrane defects and compromised kidney function in zebrafish embryos. Finally, we demonstrated that FIH displays substrate selectivity in terms of hydroxylation and binding of HIF-1α and novel substrates Notch1-3. We showed that FIH has higher affinity for oxygen with Notch1 than with HIF-1α as a substrate, implying that FIH-mediated hydroxylation of Notch can continue in oxygen concentrations where HIF-1α hydroxylation would be markedly reduced. / Tiivistelmä Happitasapainon ylläpito on edellytys elimistön normaalille toiminnalle, koska sekä liian korkea (hyperoksia) että liian matala (hypoksia) happipitoisuus ovat elimistölle stressitiloja ja johtavat pitkittyessään haitallisiin seurauksiin. Happipitoisuuden muutosten havaitsemiseksi ja niihin reagoimiseksi onkin elimistössä kehittynyt monimutkainen säätelyjärjestelmä, jossa avainasemassa on hypoksia-indusoituva tekijä HIF. Solun happipitoisuuden ollessa normaali yksi kolmesta HIF prolyyli 4-hydroksylaasi-isoentsyymistä (HIF-P4Ht 1-3) katalysoi kahden proliinitähteen hydroksylaation HIF-α-alayksikössä. 4-hydroksiproliini toimii signaalina HIF-α:n nopealle proteasomaaliselle hajotukselle. Lisäksi HIF asparaginyyli hydroksylaasi FIH:n katalysoima HIF-α:n asparagiinitähteen hydroksylaatio estää transaktivaatiovaikutuksen. Koska HIF-P4Ht ja FIH tarvitsevat kosubstraatikseen happea, nämä hydroksylaatioreaktiot vähenevät happipitoisuuden laskiessa, jolloin HIF-α stabiloituu ja siirtyy solun tumaan, jossa se muodostaa kompleksin HIF-β-alayksikön kanssa ja houkuttelee paikalle tarvittavat kofaktorit. HIF-kompleksi tehostaa hypoksiavasteessa tarvittavien geenien luentaa sitoutumalla tumassa niiden promoottoreihin ja pyrkii näin palauttamaan solun happipitoisuuden normaaliksi. Tässä työssä luotiin geneettisesti muunneltu HIF-P4H-2 hypomorfi-hiirilinja, jonka sydämissä tuottuu vain 8 % normaalista HIF-P4H-2 lähetti-RNA:n määrästä. HIF-P4H-2:n puutoksen havaittiin johtavan HIF-1α:n ja HIF-2α:n stabiloitumiseen sydämessä ja suojaavan sydäntä kudosvaurioilta iskemian ja reperfuusion aikana aiheuttamatta haitallisia vaikutuksia. Tässä väitöskirjassa karakterisoitiin aiemmin tuntematon ihmisen transmembraaninen prolyyli 4-hydroksylaasi, P4H-TM. Sen osoitettiin säätelevän HIF-1α:n määrää soluissa ja katalysoivan HIF-1α:n kahden proliinitähteen hydroksylaatiota in vitro-olosuhteissa HIF-P4H-entsyymien tavoin. Seeprakalamallin avulla näytettiin, että P4H-TM:n puutos kalan kehityksen aikana aiheuttaa tyvikalvopoikkeavuuksia ja johtaa vakavaan munuaisen toiminnan häiriintymiseen seeprakalan poikasissa. FIH:n katalysoiman hydroksylaatioreaktion kineettisiä ominaisuuksia verrattiin tässä tutkimuksessa ensimmäistä kertaa aiemmin tunnetun HIF-α substraatin ja uusien Notch substraattien kesken. Tulokset osoittivat, että substraatin sitomisessa ja hydroksylaatiossa on merkittäviä eroja eri substraattien välillä.