1. Cell Biology

Searching for molecular hypoxia sensors among oxygen-dependent enzymes

  1. Li Li  Is a corresponding author
  2. Susan Shen
  3. Philip Bickler
  4. Matthew P Jacobson
  5. Lani F Wu  Is a corresponding author
  6. Steven J Altschuler  Is a corresponding author
  1. Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, United States
  2. Department of Psychiatry, University of California, San Francisco, United States
  3. Hypoxia Research Laboratory, University of California San Francisco, San Francisco, United States
  4. Center for Health Equity in Surgery and Anesthesia, University of California San Francisco, San Francisco, United States
  5. Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, United States
https://doi.org/10.7554/eLife.87705
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Cite this article
  1. Li Li
  2. Susan Shen
  3. Philip Bickler
  4. Matthew P Jacobson
  5. Lani F Wu
  6. Steven J Altschuler
(2023)
Searching for molecular hypoxia sensors among oxygen-dependent enzymes
eLife 12:e87705.
https://doi.org/10.7554/eLife.87705
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3 figures, 6 tables and 1 additional file

Figures

Figure 1
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Three classes of by O2-dependent enzymes (dioxygenases, monooxygenases, and oxidases) and the reactions they catalyze.

Dioxygenases catalyze the insertion of both oxygen atoms of the dioxygen molecule into substrates. Monooxygenases catalyze the insertion of one oxygen atom of the dioxygen molecule into a substrate and the other oxygen atom is reduced to H2O. Oxidases catalyze the reduction of dioxygen to H2O or H2O2.

Figure 2
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Known and candidate sensors for hypoxia inside O2-dependent enzymes.

(A) Known hypoxia sensors and their corresponding cellular responses to hypoxia. Decreased O2 concentration inhibits activities of hypoxia sensors in O2-dependent enzyme category and results in changes downstream signaling pathway as the cellular response to hypoxia. PHD catalyzes the hydroxylation at two prolyl residues of HIFα, and then the hydroxylated HIFα is recognized and ubiquitylated by pVHL. Following ubiquitilation, HIFα is degraded by proteasome. During hypoxia, activity of PHD is diminished and HIFα is stabilized. Accumulated HIFα translocates to the nucleus, and in dimerization with HIF1β, recruits other transcriptional coactivators (p300, CBP), binds with the hypoxia response elements (HREs) and activates the transcription of HIF target genes. The products of these genes participate in adaptation to hypoxia including metabolic shift, EPO production, vasculogenesis, etc. FIH catalyzes the asparaginyl hydroxylation of HIFα, and this hydroxylation inhibits HIFα from recruiting transcriptional coactivators. Compared with PHD, FIH is inhibited by more severe hypoxia. KDM3A catalyzes the demethylation of K244 monomethylation of PGC-1α, which is a transcriptional coactivator and regulates mitochondrial biogenesis. Under normoxia, PGC-1α binds with transcriptional factor NRF1/2 and activates the transcription of nucleus-encoded mitochondrial genes. Under hypoxia, the inhibited activity of KDM3A leads to accumulation of K224 monomethylation at PGC-1α. The maintained monomethylation at K224 of PGC-1α reduces its binding ability with NRF1/2 and results in decreased mitochondrial biogenesis. KDM5A catalyzes the demethylation at Lys4 of histone H3 (H3K4). Hypoxia inhibits its activity and results in the hypermethylation at H3K4, which is responsible for the gene activation. Similarly, hypoxia also inhibits KDM6A, and results in the hypermethylation at its target site H3K27 and gene repression. TET methylcytosine dioxygenases (TET1, TET2, and TET3) catalyze conversion of DNA 5-methylcytosine (5-mC) to the 5-hydroxymethylcytosine (5hmC) and mediates DNA demethylation. Hypoxia reduces TET activity and causes DNA hypermethylation. Together, these proteins sense hypoxia and lead to transcription alteration by chromatin reprogramming. KDM5C catalyzes the demethylation of ULK1 R170me2s, which regulates ULK1 activity. Under normoxia, R170me2s of ULK1 is removed by KDM5C and ULK1 remains inactive. Under hypoxia, the inhibited activity of KDM5C leads to accumulation of ULK1 R170me2s, and results in ULK1 activation and autophagy induction. ADO catalyzes the thiol oxidation at the N terminal Cys of a protein, which then triggers its degradation through N-degron pathway. Hypoxia inhibits the activity of ADO and leads to the stabilization of its substrates. One of the identified ADO substrates is RSG4/5, regulators of the G protein signaling. Stabilization of RGS4/5 results in the modulation of G-protein-coupled calcium ion signaling. (B) Candidate O2 sensors with reduced enzymatic activities in hypoxia. Hypoxia leads to: inhibition of KDM4A and KDM4B and accumulated hypermethylation at H3K9; inhibition of SCD and increased cellular fatty acid saturation; inhibition of IDO and changes of immunoregulation; inhibition of PAM and reduced protein amidation; in vitro inhibition of RIOX1 and RIOX2 which are responsible for ribosome hydroxylation; in vitro inhibition of AOC3; RNA hypermethylation possibly through inhibition of FTO/ALKBH5; potential inhibition of DUOX1 and DUOX2. PHD: prolyl hydroxylase domain-containing protein; HIF: hypoxia-inducible factor; pVHL: von Hippel-Lindau protein E3 ligase; CBP, cyclic-AMP response element binding protein binding protein; EPO: erythropoietin; FIH: factor inhibiting HIF1; KDM: JmjC (Jumonji C) domain lysine demethylase; PGC: peroxisome proliferator-activated receptor gamma coactivator; NRF: nuclear respiratory factor; TET: ten-eleven translocation methylcytosine dioxygenases; ADO: cysteamine (2-aminoethanethiol) dioxygenase; RGS: regulators of G protein signalling; SCD: stearoyl-CoA desaturases; IDO: indoleamine 2,3-dioxygenase; AOC: amine oxidase, copper containing; PAM: peptidylglycine α-amidating monooxygenase; RIOX: ribosomal oxygenase, FTO: fat mass and obesity-associated protein; ALKBH: AlkB homolog; DUOX: dual oxidase.

Figure 3 with 3 supplements
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Enzymatic reactions catalyzed by discussed O2-dependent enzymes.

(A) Examples of hydroxylation reactions catalyzed by 2-OG-dependent dioxygenases. (B) Examples of demethylation reactions catalyzed by 2-OG-dependent dioxygenases. (C–K) Reactions catalyzed by indoleamine 2,3-dioxygenase (IDO)/tryptophan 2,3-dioxygenase (TDO) (C), arachidonate lipoxygenases (ALOXs) (D), (2-aminoethanethiol) dioxygenase (ADO) (E), heme oxygenases (HOs) (F), nitric oxide synthases (NOSs) (G), tyrosine 3-hydroxylase (TH) (H), peptidylglycine α-amidating monooxygenase (PAM) (I), stearoyl-CoA desaturase 1 (SCD1), (J) and copper amine oxidases (CAOs) (K).

Figure 3—figure supplement 1
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Catalytic mechanism for 2-OG-dependent dioxygenases.

2-OG-dependent dioxygenases share consensus mechanisms for the catalyzed hydroxylation (Islam et al., 2018; Fletcher and Coleman, 2020; Rose et al., 2011): the Fe(II) at the catalytic center is initially coordinated by 2 His side chains and a carboxylate from Glu or Asp, plus three additional H2O molecules to complete the octahedral coordination geometry. Then, bidentate coordination of 2-OG to Fe(II) replaces 2 H2O molecules, and the third Fe(II)-bound H2O molecule leaves after the binding of the primary substrate into the active site, making a vacant coordination site for O2. After the binding and activation of O2 at the Fe(II) center, one of the O2 atoms inserts into the primary substrate for hydroxylation, while the other O2 atom facilitates the oxidative decarboxylation of 2-OG, forming succinate and CO2 as co-products.

Figure 3—figure supplement 2
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O2-binding sites for dioxygenases using heme.

The heme Fe(II) is coordinated by the four N atoms from the porphyrin and one N atom from one histidyl residue in the catalytic pocket, leaving the vacant coordination site for O2 binding and activation (Singleton et al., 2014).

Figure 3—figure supplement 3
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Mitochondrial electron transport chain (ETC).

The ETC consists of NADH ubiquinone oxireductase (Complex I), succinate dehydrogenase (Complex II), CoQH2-cytochrome c reductase (Complex III), and cytochrome c oxidase (Complex IV). In the ETC, electrons are transported from the NADH or FADH2 to ubiquinone at Complex I or II, then to cytochrome c at Complex III, and finally to O2 at Complex IV. This process is coupled with ATP generation at ATP synthase (Complex V) to form the oxidative phosphorylation (OxPhos) process, which is the major source of energy production.

Tables

Table 1
Physiological O2 distribution in different organs/tissues*.
Organ/tissue%O2pO2 (mmHg)Concentration(μM)
Ambient air21160206
Alveoli14104134
Arterial blood13100129
Kidney4–9.530–7339–94
Liver4–730–5439–69
Heart2–615–4619–59
Brain3–523–3929–50
Small intestine2–915–6919–89
Large intestine0–60–460–59
Bone marrow1.5–711–5414–69
  1. *

    The O2 levels in different organs are adjusted from references Burmester and Hankeln, 2014; Lecomte et al., 2005; Hatefi, 1985; Zaccara et al., 2019; Ball et al., 2014 and the partial pressure and concentration are calculated according to references Ortiz-Prado et al., 2019; Carreau et al., 2011; Jagannathan et al., 2016; Cigognini et al., 2016; Donovan et al., 2010; Mas-Bargues et al., 2019; Place et al., 2017.

Table 2
Categories of O2-dependent enzymes.
CategorySubcategory by catalytic centerMetal species at catalytic centerLigands for the metal species at catalytic center (cofactor/substrate and enzyme residues)Number of enzymes
Dioxygenase2-OG-dependent dioxygenaseFe2-OG, His, His, Asp/Glu59
Heme-dependent dioxygenaseFeHeme, His5
LipoxygenaseFeHis, His, His, Ile, His/Asa/Asn/none6
OthersFeHis, His, His/Asp/Glu*10
MonooxygenaseHeme-dependent monooxygenaseFeHeme, Cys/His/Glu61
Non-Heme Fe-dependent monooxygenaseFeHis, His, His/Asp/Glu*9
Cu-dependent monooxygenaseCuHis, His, Met5
Flavin-dependent monooxygenaseNone (uses flavin)N/A12
Others†N/AN/A2
OxidaseHeme-copperFe and CuHis, His, His for Cu; Heme and His for Fe1
Fe-dependent oxidaseFeVaries14
Cu-dependent oxidaseCuVaries7
Flavin-dependent oxidaseNone (uses flavin)N/A25
Others†N/AN/A5
  1. *

    Substrates/cofactor ligands for this category varies for each member depending on the reaction it catalyzes.

  2. †

    Members in this category are not fully studied.

Table 3
Direct HIF modulator in 2-OG-dependent dioxygenases.
Gene symbolProtein nameType of reactionHydroxylation sites in HIFαNon-HIF substrate examples
EGLN1PHD2Prolyl hydroxylationHIF1α Pro402, Pro564;
HIF2α Pro405, Pro531;
HIF3α Pro492		FLNA, Akt
EGLN2PHD1Prolyl hydroxylationHIF1α Pro402, Pro564;
HIF2α Pro405, Pro531;
HIF3α Pro492		FOXO3, Cep192, TP53
EGLN3PHD3Prolyl hydroxylationHIF1α Pro564;
HIF2α Pro405, Pro531;
HIF3α Pro492		ATF-4, ADRB2, TP53
HIF1ANFIH1Asparaginyl hydroxylationHIF1α Asn803,
HIF2α Asn847		IκBα, Notch, OTUB1, RIPK4
Table 4
Reported Km values of O2-dependent enzymes.

Km values vary based on the assay method and tested substrate. Some enzymes have multiple Km values listed, reflecting measurements from different studies.

CategoryEnzyme*Km for O2Assay detailsReference
DioxygenasePHD2 (EGLN1)250 μMIn vitro radioactivity 2-OG turnover assay with HIF1α (556–574) peptide as substrateHirsilä et al., 2003
1746 μMIn vitro time-resolved fluorescence resonance energy transfer assay with P564-HIF1α peptide (DLEMLAPYIPMDDDFQL) as substrateDao et al., 2009
67 μMIn vitro O2 consumption assay with HIF1α (502–697) peptide as substrateEhrismann et al., 2007
81 μMIn vitro O2 consumption assay with HIF1α (530–698) peptide as substrateEhrismann et al., 2007
PHD1 (EGLN2)230 μMIn vitro radioactivity 2-OG turnover assay with HIF1α (556–574) peptide as substrateHirsilä et al., 2003
PHD3230 μMIn vitro radioactivity 2-OG turnover assay with HIF1α (556–574) peptide as substrateHirsilä et al., 2003
KDM4E197 μMIn vitro O2 consumption assay with ARK(me3)STGGK peptide as substrateCascella and Mirica, 2012
KDM4A173 μMIn vitro MALDI-TOF-MS assay with H31−15K9me3 peptide as substrateHancock et al., 2017
57 μMIn vitro O2 consumption assay with ARK(me3)STGGK peptide substrateCascella and Mirica, 2012
60 μMIn vitro radioactivity 2-OG turnover assay with histone H3(1–19)K9me3 as substrateChakraborty et al., 2019
KDM6A180 μMIn vitro radioactivity 2-OG turnover assay with histone H3(21–44)K27(me3) as substrateChakraborty et al., 2019
KDM4C158 μMIn vitro O2 consumption assay with ARK(me3)STGGK peptide substrateCascella and Mirica, 2012
KDM4B150 μMIn vitro radioactivity 2-OG turnover assay with histone H3(1–19)K9me3 as substrateChakraborty et al., 2019
FIH90 μMIn vitro radioactivity 2-OG turnover assay with HIF1α (788–822) peptide as substrateKoivunen et al., 2004
KDM5A90 μMIn vitro radioactivity 2-OG turnover assay with histone H3(1–21)K4me3 as substrateChakraborty et al., 2019
KDM3A75 μM (7.6% O2) †In vitro demethylation-formaldehyde dehydrogenase-coupled reaction assay with K224-monomethylated PGC-1α peptide as substrateQian et al., 2019
KDM5B40 μMIn vitro radioactivity 2-OG turnover assay with histone H3(1–21)K4me3 as substrateChakraborty et al., 2019
P4HA140 μMStandard P4H activity assay with (Pro-Pro-Gly)10 (Peptide Institute) as a substrateHirsilä et al., 2003
KDM5C35 μMIn vitro radioactivity 2-OG turnover assay with histone H3(1–21)K4me3 as substrateChakraborty et al., 2019
TET130 μMIn vitro radioactivity 2-OG turnover assay with oligonucleotides containing a 5-mC as substrateLaukka et al., 2016
3.0 μM (0.31% O2) †In vitro DNA hydroxymethylation assay with genomic DNA as substrateThienpont et al., 2016
TET230 μMIn vitro radioactivity 2-OG turnover assay with oligonucleotides containing a 5-mC as substrateLaukka et al., 2016
5.2 μM (0.53% O2) *In vitro DNA hydroxymethylation assay with genomic DNA as substrateThienpont et al., 2016
KDM5D25 μMIn vitro radioactivity 2-OG turnover assay with histone H3(1–21)K4me3 as substrateChakraborty et al., 2019
KDM6B20 μMIn vitro radioactivity 2-OG turnover assay with histone H3(21–44)K27(me3) as substrateChakraborty et al., 2019
IDO111.5–24 μMIn vitro O2 consumption assay with L-Trp as substrateKolawole et al., 2015
PTGS110 μM (sheep)In vitro radioactivity label assay with [1-14C]arachidonic acid as substrateJuránek et al., 1999
PTGS213 μM (mouse)In vitro radioactivity label assay with [1-14C]arachidonic acid as substrateJuránek et al., 1999
ALOX513 μM (porcine)In vitro radioactivity label assay with [1-14C]arachidonic acid as substrateJuránek et al., 1999
ALOX1213 μMIn vitro radioactivity label assay with [1-14C]arachidonic acid as substrateJuránek et al., 1999
ALOX1526 μM (porcrine)In vitro radioactivity label assay with [1-14C]arachidonic acid as substrateJuránek et al., 1999
ALOX1526 μM (rabbit)In vitro radioactivity label assay with [1-14C]arachidonic acid as substrateJuránek et al., 1999
ADO>500 μMIn vitro UPLC-MS-TOF assay with RGS4(2–15) peptide as substrateMasson et al., 2019
MonooxygenaseNOS1
(nNOS)		350 μM (rat)In vitro heme-NO complex formation assay with L-arginine as substrateAbu-Soud et al., 1996
NOS2
(iNOS)		130 μM (mouse)In vitro heme-NO complex formation assay with L-arginine as substrateAbu-Soud et al., 2001
NOS3
(eNOS)		4 μM (bovine)In vitro heme-NO complex formation assay with L-arginine as substrateAbu-Soud et al., 2000
25 μM (bovine)In vitro heme-NO complex formation assay with N-hydroxy-L-arginine as substrateAbu-Soud et al., 2000
TH16.2 μM (low-activity state);
46.1 μM (high- activity state);		In vitro radioactivity label assay with 3H-tyrosine as substrateRostrup et al., 2008
12.6–26.7 μM (low-activity state);
28.8–42.9 μM (high-activity state)‡;		In vitro oxygraphic assay with tyrosine as substrateRostrup et al., 2008
2.6–3.9 μM (2–3 mmHg, rat) *In vitro radioactivity label assay with 3H-tyrosine as substrateKatz, 1980
TPH13.9~12.9 μM (3–10 mmHg, rat) †In vitro radioactivity label assay with 3H-tryptophan as substrateKatz, 1980
PAH17 μMIn vitro oxygraphic assay with phenylalanine as substrateRostrup et al., 2008
PAM70 μM (rat)In vitro radioactivity label assay with [α-2H2]-N-acylglycine of different chain length as substratesMcIntyre et al., 2010
OxidaseCytochrome c oxidase<0.1 μM (rat)In vitro O2 consumption assay measuring O2 consumption of purified rat mitochondria at low phosphate potential ([ATP]/[ADP]*[Pi])Bienfait et al., 1975
1–3 μM (rat)In vitro O2 consumption assay measuring O2 consumption of purified rat mitochondria at high phosphate potentialBienfait et al., 1975
0.5 μM (mouse)Cellular assay measuring the ‘apparent K (m)’ for O2 or p 50 of respiration in 32D cells using high-resolution respirometryScandurra and Gnaiger, 2010
AOC338 μMIn vitro enzymatic assay using purified human AOC3Shen et al., 2012
  1. *

    O2-dependent enzymes that are known sensors are highlighted in bold; that are reported to be inhibited under hypoxia are highlighted in a light orange background; that are reported to be associated with positive selections in high-altitude populations are highlighted in red (also see Table 6).

  2. †

    Km of these enzyme were reported with units as % O2 or mmHg, and calculated according to Mas-Bargues et al., 2019; Place et al., 2017.

  3. ‡

    Combined data for TH1/3/4 splicing isoforms.

Table 5
JmjC domain-containing histone demethylases and their substrates*.

(A = activating transcription, S = silencing transcription).

KDM classMembers (gene symbol)Histone lysyl residue substratesOther substrates
KDM2KDM2AH3K36me1/me2 (A)p65, NF-κB
KDM2BH3K36me1/me2 (A), H3K4me3 (A)
KDM3KDM3AH3K9me1/me2 (S)PGC-1α K224me
KDM3BH3K9me1/me2 (S)
JJMJD1CH3K9me1/me2 (S)
KDM4KDM4AH3K9me2/me3 (S), H3K36me2 (A), H1.4K26me2/me3WIZ, CDYL1, CSB, and G9a
KDM4BH3K9me2/me3 (S), H3K36me2 (A), H1.4K26me2/me3WIZ, CDYL1, CSB, and G9a
KDM4CH3K9me2/me3 (S), H3K36me2 (A), H1.4K26me2/me3WIZ, CDYL1, CSB, and G9a
KDM4DH3K9me2/me3 (S)
KDM4EH3K9me3 (S)H3R2me2/me1, H3R8me2/me1, H3R26me2/me1, H4R3me2
KDM5KDM5AH3K4me2/me3 (A)
KDM5BH3K4me2/me3 (A)
KDM5CH3K4me2/me3 (A)H3R2me2/me1, H3R8me2, H4R3me2a, ULK1R170me2a
KDM5DH3K4me2/me3 (A)
KDM6KDM6AH3K27me2/me3 (S)
KDM6BH3K27me2/me3 (S)
KDM6C
KDM7KDM7AH3K9me1/me2 (S), H3K27me1/me2 (S)
PHF8H3K27me1/me2 (S), H4K20me1
PHF2H3K9me2/me3 (S)
Jmjc domain onlyNO66H3K4me2/me3 (A), H3K36me2/me3 (A)Rpl8
MINA53H3K9me3 (S)Rpl27a
KDM8H3K36me2 (A)NFATc1
JMJD6H3R2me2,H4R3me2/me1, U2AF2/U2AF65, LUC7L2
  1. *

    Known hydroxylation/demethylation sites are indicated.

Table 6
O2-dependent enzymes encoded by genes associated with positive selection in different high-altitude populations.
PopulationGenes*
TibetanEGLN1 (Lorenzo et al., 2014; Bigham et al., 2010; Yi et al., 2010; Simonson et al., 2010; Xu et al., 2011; Yang et al., 2017; Peng et al., 2011; Wuren et al., 2014), CYP2E1 (Simonson et al., 2010), HMOX2 (Simonson et al., 2010; Peng et al., 2011; Wuren et al., 2014), CYP17A1 (Simonson et al., 2010; Wuren et al., 2014), SCD (Simonson et al., 2010), HIF1AN (Simonson et al., 2010), SC5D (Simonson et al., 2010), KDM5A (Simonson et al., 2010), HPD (Simonson et al., 2010), DOHH (Simonson et al., 2010), XDH (Simonson et al., 2010), CYP20A1 (Simonson et al., 2010), TMEM189 (Simonson et al., 2010), KDM4A (Simonson et al., 2010), PAOX (Simonson et al., 2010)
AndeanEGLN1 (Bigham et al., 2010; Bigham et al., 2009), EGLN2 (Bigham et al., 2009), NOS1 (Bigham et al., 2009), NOS2 (Bigham et al., 2010; Bigham et al., 2009; Crawford et al., 2017), DUOX2 (Jacovas et al., 2018), CYP39A1 (Eichstaedt et al., 2014), KDM2A (Eichstaedt et al., 2014), KMO (Eichstaedt et al., 2014), PLOD3 (Eichstaedt et al., 2014), P3H3 (Eichstaedt et al., 2014), CPOX (Eichstaedt et al., 2014), CYP24A1 (Eichstaedt et al., 2014)
EthiopianPCYOX1 (Scheinfeldt et al., 2012)
  1. *

    Known hypoxia sensors are highlighted in red.

Additional files

Supplementary file 1

Detailed information of 221 oxygen-dependent enzymes in human.

https://cdn.elifesciences.org/articles/87705/elife-87705-supp1-v1.xlsx

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  1. Li Li
  2. Susan Shen
  3. Philip Bickler
  4. Matthew P Jacobson
  5. Lani F Wu
  6. Steven J Altschuler
(2023)
Searching for molecular hypoxia sensors among oxygen-dependent enzymes
eLife 12:e87705.
https://doi.org/10.7554/eLife.87705