ß-lapachone regulates mammalian inositol pyrophosphate levels in an NQO1- and oxygen-dependent manner.

Inositol pyrophosphates (PP-InsPs) are energetic signaling molecules with important functions in mammals. As their biosynthesis depends on ATP concentration, PP-InsPs are tightly connected to cellular energy homeostasis. Consequently, an increasing number of studies involve PP-InsPs in metabolic disorders, such as type 2 diabetes, aspects of tumorigenesis, and hyperphosphatemia. Research conducted in yeast suggests that the PP-InsP pathway is activated in response to reactive oxygen species (ROS). However, the precise modulation of PP-InsPs during cellular ROS signaling is unknown. Here, we report how mammalian PP-InsP levels are changing during exposure to exogenous (H2O2) and endogenous ROS. Using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that PP-InsP levels decrease upon exposure to oxidative stressors in HCT116 cells. Application of quinone drugs, particularly ß-lapachone (ß-lap), under normoxic and hypoxic conditions enabled us to produce ROS in cellulo and to show that ß-lap treatment caused PP-InsP changes that are oxygen-dependent. Experiments in MDA-MB-231 breast cancer cells deficient of NAD(P)H:quinone oxidoreductase-1 (NQO1) demonstrated that ß-lap requires NQO1 bioactivation to regulate the cellular metabolism of PP-InsPs. Critically, significant reductions in cellular ATP concentrations were not directly mirrored in reduced PP-InsP levels as shown in NQO1-deficient MDA-MB-231 cells treated with ß-lap. The data presented here unveil unique aspects of ß-lap pharmacology and its impact on PP-InsP levels. The identification of different quinone drugs as modulators of PP-InsP synthesis will allow the overall impact on cellular function of such drugs to be better appreciated.

Non-canonical nucleosides: Biomimetic triphosphorylation, incorporation into mRNA and effects on translation and structure.

Recent advances in mRNA therapeutics demand efficient toolkits for the incorporation of nucleoside analogues into mRNA suitable for downstream applications. Herein, we report the application of a versatile enzyme cascade for the triphosphorylation of a broad range of nucleoside analogues, including unprotected nucleobases containing chemically labile moieties. Our biomimetic system was suitable for the preparation of nucleoside triphosphates containing adenosine, cytidine, guanosine, uridine and non-canonical core structures, as determined by capillary electrophoresis coupled to mass spectrometry. This enabled us to establish an efficient workflow for transcribing and purifying functional mRNA containing these nucleoside analogues, combined with mass spectrometric verification of analogue incorporation. Our combined methodology allows for analyses of how incorporation of nucleoside analogues that are commercially unavailable as triphosphates affect mRNA properties: The translational fidelity of the produced mRNA was demonstrated in analyses of how incorporated adenosine analogues impact translational recoding. For the SARS-CoV-2 frameshifting site, analyses of the mRNA pseudoknot structure using circular dichroism spectroscopy allowed insight into how the pharmacologically active 7-deazaadenosine destabilises RNA secondary structure, consistent with observed changes in recoding efficiency.

Biomimetic S-Adenosylmethionine Regeneration Starting from Multiple Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes.

S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one-pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems' flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

A Cobalamin-Dependent Radical SAM Enzyme Catalyzes the Unique Cα -Methylation of Glutamine in Methyl-Coenzyme†M Reductase.

Methyl-coenzyme M reductase, which is responsible for the production of the greenhouse gas methane during biological methane formation, carries several unique posttranslational amino acid modifications, including a 2-(S)-methylglutamine. The enzyme responsible for the Cα -methylation of this glutamine is not known. Herein, we identify and characterize a cobalamin-dependent radical SAM enzyme as the glutamine C-methyltransferase. The recombinant protein from Methanoculleus thermophilus binds cobalamin in a base-off, His-off conformation and contains a single [4Fe-4S] cluster. The cobalamin cofactor cycles between the methyl-cob(III)alamin, cob(II)alamin and cob(I)alamin states during catalysis and produces methylated substrate, 5'-deoxyadenosine and S-adenosyl-l-homocysteine in a 1 : 1 : 1 ratio. The newly identified glutamine C-methyltransferase belongs to the class B radical SAM methyltransferases known to catalyze challenging methylation reactions of sp3 -hybridized carbon atoms.

An Oxygen Sensation: Progress in Macromolecule Hydroxylation Triggered by the Elucidation of Cellular Oxygen Sensing.

The 2019 Nobel Prize in Physiology or Medicine honours three scientists that devoted their careers to pursuing an audacious basic science question: by what mechanisms do animals sense oxygen, and how can cells adapt to a lack of oxygen? The identification of the human hypoxia inducible factor pathway has enabled new approaches for the therapy of related diseases including cancer, cardiovascular disease, anaemia, and stroke. The intricate molecular details of oxygen sensing broadened interest in the family of iron- and 2-oxoglutarate-dependent oxygenases known from elaborate natural product chemistry, and catalysed major progress in macromolecule hydroxylation. The laureates' work enables numerous avenues for molecular scientists, from C-H activation chemistry to PROTAC technology, medicinal chemistry, and epigenetics.

Studies on the Substrate Selectivity of the Hypoxia-Inducible Factor Prolyl Hydroxylase†2 Catalytic Domain.

In animals, the response to chronic hypoxia is mediated by upregulation of the α,ß-heterodimeric hypoxia-inducible factors (HIFs). Levels of HIFα isoforms, but not HIFß, are regulated by their post-translational modification as catalysed by prolyl hydroxylase domain enzymes (PHDs). Different roles for the human HIF-1/2α isoforms and their two oxygen-dependent degradation domains (ODDs) are proposed. We report kinetic and NMR analyses of the ODD selectivity of the catalytic domain of wild-type PHD2 (which is conserved in nearly all animals) and clinically observed variants. Studies using Ala scanning and "hybrid" ODD peptides imply that the relatively rigid conformation of the (hydroxylated) proline plays an important role in ODD binding. They also reveal differential roles in binding for the residues on the N- and C-terminal sides of the substrate proline. The overall results indicate how the PHDs achieve selectivity for HIFα ODDs and might be of use in identifying substrate-selective PHD inhibitors.

A mechanism for induction of a hypoxic response by vaccinia virus.

Viruses have evolved sophisticated strategies to exploit host cell function for their benefit. Here we show that under physiologically normal oxygen levels (normoxia) vaccinia virus (VACV) infection leads to a rapid stabilization of hypoxia-inducible factor (HIF)-1α, its translocation into the nucleus and the activation of HIF-responsive genes, such as vascular endothelial growth factor (VEGF), glucose transporter-1, and pyruvate dehydrogenase kinase-1. HIF-1α stabilization is mediated by VACV protein C16 that binds the human oxygen sensing enzyme prolyl-hydroxylase domain containing protein (PHD)2 and thereby inhibits PHD2-dependent hydroxylation of HIF-1α. The binding between C16 and PHD2 is direct and specific, and ectopic expression of C16 alone induces transcription of HIF-1α responsive genes. Conversely, a VACV strain lacking the gene for C16, C16L, is unable to induce HIF-1α stabilization. Interestingly, the N-terminal region of C16 is predicted to have a PHD2-like structural fold but lacks the catalytic active site residues of PHDs. The induction of a hypoxic response by VACV is reminiscent of the biochemical consequences of solid tumor formation, and illustrates a poxvirus strategy for manipulation of cellular gene expression and biochemistry.

Photoactivable peptides for identifying enzyme-substrate and protein-protein interactions.

Photoactivated cross-linking of peptides to proteins is a useful strategy for identifying enzyme-substrate and protein-protein interactions in cell lysates as demonstrated by studies on the human hypoxia inducible factor system.

The hypoxia-inducible transcription factor pathway regulates oxygen sensing in the simplest animal, Trichoplax adhaerens.

The hypoxic response in humans is mediated by the hypoxia-inducible transcription factor (HIF), for which prolyl hydroxylases (PHDs) act as oxygen-sensing components. The evolutionary origins of the HIF system have been previously unclear. We demonstrate a functional HIF system in the simplest animal, Trichoplax adhaerens: HIF targets in T. adhaerens include glycolytic and metabolic enzymes, suggesting a role for HIF in the adaptation of basal multicellular animals to fluctuating oxygen levels. Characterization of the T. adhaerens PHDs and cross-species complementation assays reveal a conserved oxygen-sensing mechanism. Cross-genomic analyses rationalize the relative importance of HIF system components, and imply that the HIF system is likely to be present in all animals, but is unique to this kingdom.

Chemical basis for the selectivity of the von Hippel Lindau tumor suppressor pVHL for prolyl-hydroxylated HIF-1alpha.

In animals, the post-translational hydroxylation of hypoxia inducible factor (HIF) is a central mechanism for regulating gene expression in an oxygen-dependent manner. The oxygenase-catalyzed trans-4-prolyl hydroxylation of HIF-alpha increases its affinity for the von Hippel Lindau protein elongin B/C (VCB) ubiquitin ligase complex, leading to HIF-alpha degradation. The level of binding of HIF-alpha to VCB is increased by approximately 1000-fold upon addition of a single oxygen atom to a conserved proline residue. Here, we describe computational studies on the chemical basis of this "switchlike" signaling event. The results support crystallographic analyses showing the importance of hydrogen bonding in the binding of hydroxylated HIF-alpha to VCB and suggest that trans 4-hydroxylation "preorganizes" the proline residue to adopt the C(4)-exo conformation, via operation of the stereoelectronic gauche effect.

PHF8, a gene associated with cleft lip/palate and mental retardation, encodes for an Nepsilon-dimethyl lysine demethylase.

Mutations of human PHF8 cluster within its JmjC encoding exons and are linked to mental retardation (MR) and a cleft lip/palate phenotype. Sequence comparisons, employing structural insights, suggest that PHF8 contains the double stranded beta-helix fold and ferrous iron binding residues that are present in 2-oxoglutarate-dependent oxygenases. We report that recombinant PHF8 is an Fe(II) and 2-oxoglutarate-dependent N(epsilon)-methyl lysine demethylase, which acts on histone substrates. PHF8 is selective in vitro for N(epsilon)-di- and mono-methylated lysine residues and does not accept trimethyl substrates. Clinically observed mutations to the PHF8 gene cluster in exons encoding for the double stranded beta-helix fold and will therefore disrupt catalytic activity. The PHF8 missense mutation c.836C>T is associated with mild MR, mild dysmorphic features, and either unilateral or bilateral cleft lip and cleft palate in two male siblings. This mutant encodes a F279S variant of PHF8 that modifies a conserved hydrophobic region; assays with both peptides and intact histones reveal this variant to be catalytically inactive. The dependence of PHF8 activity on oxygen availability is interesting because the occurrence of fetal cleft lip has been demonstrated to increase with maternal hypoxia in mouse studies. Cleft lip and other congenital anomalies are also linked indirectly to maternal hypoxia in humans, including from maternal smoking and maternal anti-hypertensive treatment. Our results will enable further studies aimed at defining the molecular links between developmental changes in histone methylation status, congenital disorders and MR.

Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases.

The oxygen-dependent hydroxylation of proline residues in the alpha subunit of hypoxia-inducible transcription factor (HIFalpha) is central to the hypoxic response in animals. Prolyl hydroxylation of HIFalpha increases its binding to the von Hippel-Lindau protein (pVHL), so signaling for degradation via the ubiquitin-proteasome system. The HIF prolyl hydroxylases (PHDs, prolyl hydroxylase domain enzymes) are related to the collagen prolyl hydroxylases, but form unusually stable complexes with their Fe(II) cofactor and 2-oxoglutarate cosubstrate. We report crystal structures of the catalytic domain of PHD2, the most important of the human PHDs, in complex with the C-terminal oxygen-dependent degradation domain of HIF-1alpha. Together with biochemical analyses, the results reveal that PHD catalysis involves a mobile region that isolates the hydroxylation site and stabilizes the PHD2.Fe(II).2OG complex. The results will be of use in the design of PHD inhibitors aimed at treating anemia and ischemic disease.

Oxygenase catalyzed 5-methylcytosine hydroxylation.

Recent reports identify the oxygenase catalyzed production of 5-hydroxymethylcytosine as a modification to mammalian DNA (Kriaucionis and Heintz, 2009; Tahiliani et al., 2009). This discovery has potential far-reaching implications for epigenetic regulation and will stimulate efforts to identify new nucleic acid modifications.

Evidence for a stereoelectronic effect in human oxygen sensing.

How PHDs achieve specificity: trans-4-prolyl hydroxylation of the transcription factor HIF occurs with stereochemical retention. Substrate-analogue studies show how the von Hippel Lindau tumor suppressor protein (pVHL) and the oxygen-sensing hydroxylases (PHDs) achieve specificity for hydroxyprolyl/prolyl residues for the C(4)-exo/endo prolyl conformations, respectively.

Regulation of Jumonji-domain-containing histone demethylases by hypoxia-inducible factor (HIF)-1alpha.

The transcription factor HIF (hypoxia-inducible factor) mediates a highly pleiotrophic response to hypoxia. Many recent studies have focused on defining the extent of this transcriptional response. In the present study we have analysed regulation by hypoxia among transcripts encoding human Fe(II)- and 2-oxoglutarate-dependent oxygenases. Our results show that many of these genes are regulated by hypoxia and define two groups of histone demethylases as new classes of hypoxia-regulated genes. Patterns of induction were consistent across a range of cell lines with JMJD1A (where JMJD is Jumonji-domain containing) and JMJD2B demonstrating robust, and JMJD2C more modest, up-regulation by hypoxia. Functional genetic and chromatin immunoprecipitation studies demonstrated the importance of HIF-1alpha in mediating these responses. Given the importance of histone methylation status in defining patterns of gene expression under different physiological and pathophysiological conditions, these findings predict a role for the HIF system in epigenetic regulation.