Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-α subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Å resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded β-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1–3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

Myosin binding protein-C (MyBP-C) is a thick filament–associated protein localized to the crossbridge-containing C zones of striated muscle sarcomeres. The cardiac isoform is composed of eight immunoglobulin I–like domains and three fibronectin 3–like domains and is known to be a physiological substrate of cAMP-dependent protein kinase. MyBP-C contributes to thick filament structure via interactions at its C-terminus with the light meromyosin section of the myosin rod and with titin. The protein also has a role in the regulation of contraction, due to the binding of its N-terminus to the subfragment-2 portion of myosin, which reduces actomyosin ATPase activity; phosphorylation abolishes this interaction, resulting in release of the “brake” on crossbridge cycling. Several structural models of the interaction of MyBP-C with myosin have been proposed, although its precise arrangement on the thick filament remains to be elucidated. Mutations in the gene encoding cardiac MyBP-C are a common cause of hypertrophic cardiomyopathy, and this has led to increased interest in the protein’s function. Investigation of disease-causing mutations in domains with unknown function has led to further insights into the mechanism of cMyBP-C action. This Review aims to collate the published data on those aspects of MyBP-C that are well characterized and to consider new and emerging data that further define its structural and regulatory roles and its arrangement in the sarcomere. We also speculate on the mechanisms by which hypertrophic cardiomyopathy–causing truncation and missense mutations affect the normal functioning of the sarcomere.

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture.

Oxygen sensing across kingdoms: The ability to sense and respond to changes in oxygen levels is critical for most forms of life. To date, mechanistic studies of this process in mammals have focused on the oxygen-sensitive stability of a transcription factor called hypoxia-inducible factor. Masson et al. discovered an enzymatic oxygen sensor in humans that is functionally identical to plant cysteine oxidases, enzymes that control responses to hypoxia in plants. The human and plant enzymes convert the N-terminal cysteine in substrate proteins to cysteine sulfinic acid, a modification that ultimately targets the proteins for degradation. Oxygen sensing is impaired in many human diseases, and further study of the human enzyme could help in the development of strategies for therapeutic intervention. Science , this issue p. 65

Inhibition of the human 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (human PHD1–3) causes upregulation of HIF, thus promoting erythropoiesis and is therefore of therapeutic interest. We describe cellular, biophysical, and biochemical studies comparing four PHD inhibitors currently in clinical trials for anaemia treatment, that describe their mechanisms of action, potency against isolated enzymes and in cells, and selectivities versus representatives of other human 2OG oxygenase subfamilies. The ‘clinical’ PHD inhibitors are potent inhibitors of PHD catalyzed hydroxylation of the HIF-α oxygen dependent degradation domains (ODDs), and selective against most, but not all, representatives of other human 2OG dependent dioxygenase subfamilies. Crystallographic and NMR studies provide insights into the different active site binding modes of the inhibitors. Cell-based results reveal the inhibitors have similar effects on the upregulation of HIF target genes, but differ in the kinetics of their effects and in extent of inhibition of hydroxylation of the N- and C-terminal ODDs; the latter differences correlate with the biophysical observations.