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The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin β and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Å crystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2–ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117–196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

Hypoxia-inducible factors (HIFs) are transcription factors in the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) protein family that contain internal hydrophobic cavities within their PAS-A and PAS-B domains. Among HIFs, the HIF-2α PAS-B domain contains a relatively large cavity exploited for the development of specific artificial ligands such as PT2399. Administration of PT2399 could suppress HIF-2α target gene expression without affecting HIF-1 activity in mice under hypoxia conditions. A single mutation (S305M) within the HIF-2α PAS-B domain suppressed HIF-2α activity while conferring resistance to PT2399 in vivo, indicating the vital role of PAS-B domain in HIF-2α hypoxia response. In contrast, the mutant mice did not phenocopy PT2399 intervention in wild-type mice under metabolic stress. Under a high-fat diet (HFD), the mutant mice exert enhanced adipogenesis and obtain larger adipose mass and body weight gain compared to wild type. However, administration of PT2399 along with HFD feeding sufficiently suppressed HFD-induced body weight and adipose mass increase through suppression of adipogenesis and lipogenesis. The accompanying decreased lipid accumulation in the liver and improved glucose tolerance in wild-type mice were not observed in the mutant mice indicating negative regulation of HIF-2α on obesity and a complex role for the PAS-B domain in metabolic regulation. Notably, short-term administration of PT2399 to obese mice decreased adipose mass and improved metabolic condition. These results indicate a regulatory role for HIF-2α in obesity progression and suggest a therapeutic opportunity for PT2399 in obesity and associated metabolic disorders.

Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer and there is great interest in applying inhibitors of HIF as anti-cancer therapeutics. The most advanced of these are small molecules that target the HIF-2 isoform through binding the PAS-B domain of HIF-2α. These molecules are undergoing clinical trials with promising results in renal and other cancers where HIF-2 is considered to be driving growth. Nevertheless, a central question remains as to whether such inhibitors impact on physiological responses to hypoxia at relevant doses. Here we show that pharmacological HIF-2α inhibition with PT2385, at doses similar to those reported to inhibit tumour growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatisation and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerisation with HIF-1β, did not respond to PT2385 indicating that these effects are on target. Furthermore, the finding of a hypomorphic ventilatory phenotype in untreated HIF-2α S305M mutant mice suggests a function for the HIF-2α PAS-B domain beyond heterodimerisation with HIF-1β. Although PT2385 was well-tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.

This study aimed to assess the efficacy of acute subanesthetic dosages of xenon inhalation to cause erythropoiesis and determine the effect of chronic xenon dosing on hematological parameters and athletic performance. To assess the acute effects, seven subjects breathed three subanesthetic concentrations of xenon: 30% fraction of inspired xenon (FiXe) for 20 min, 50% FiXe for 5 min, and 70% FiXe for 2 min. Erythropoietin (EPO) was measured at baseline, during, and after xenon inhalation. To determine the chronic effects, eight subjects breathed 70% FiXe for 2 min on 7 consecutive days, and EPO, total blood, and plasma volume were measured. Phase II involved assessment of 12 subjects for EPO, total blood volume, maximal oxygen uptake, and 3-km time before and after random assignment to 4 wk of xenon or sham gas inhalation. FiXe 50% and 70% stimulated an increase in EPO at 6 h [+2.3 mIU/mL; 95% confidence interval (CI) 0.1–4.5; P = 0.038] and at 192 h postinhalation (+2.9 mIU/mL; 95% CI 0.6–5.1; P = 0.017), respectively. Seven consecutive days of dosing significantly elevated plasma volume (+491 mL; 95% CI 194–789; P = 0.002). Phase II showed no significant effect on EPO, hemoglobin mass, plasma volume, maximal oxygen uptake, or 3-km time. Acute exposure to subanesthetic doses of xenon caused a consistent increase in EPO, and 7 consecutive days of xenon inhalation significantly expanded plasma volume. However, this physiological response appeared to be transient, and 4 wk of xenon inhalation did not stimulate increases in plasma volume or erythropoiesis, leaving cardiorespiratory fitness and athletic performance unchanged. NEW & NOTEWORTHY This is the first study to examine each element of the cascade by which xenon inhalation is purported to take effect, starting with measurement of the hypoxia-inducible factor effector, erythropoietin, to hemoglobin mass and blood volume and athletic performance. We found that acute exposure to xenon increased serum erythropoietin concentration, although major markers of erythropoiesis remained unchanged. While daily dosing significantly expanded plasma volume, no physiological or performance benefits were apparent following 4 wk of dosing.

This study aimed to quantify the sedative effects, detection rates, and cardiovascular responses to xenon. On 3 occasions, participants breathed xenon (FiXe 30% for 20 min; FiXe 50% for 5 min; FiXe 70% for 2 min) in a nonblinded design. Sedation was monitored by a board-certified anesthesiologist. During 70% xenon, participants were also verbally instructed to operate a manual value with time-to-task failure being recorded. Beat-by-beat hemodynamics were measured continuously by ECG, photoplethysmography, and transcranial Doppler. Over 48 h postadministration, xenon was measured in blood and urine by gas chromatography-mass spectrometry. Xenon caused variable levels of sedation and restlessness. Task failure of the self-operating value occurred at 60–90 s in most individuals. Over the first minute, 50% and 70% xenon caused a substantial reduction in total peripheral resistance ( P < 0.05). All dosages caused an increase in cardiac output ( P < 0.05). By the end of xenon inhalation, slight hypertension was observed after all three doses ( P < 0.05), with an increase in middle cerebral artery velocity ( P < 0.05). Xenon was consistently detected, albeit in trace amounts, up to 3 h after all three doses of xenon inhalation in blood and urine with variable results thereafter. Xenon inhalation caused sedation incompatible with self-operation of a breathing apparatus, thus causing a potential life-threatening condition in the absence of an anesthesiologist. Yet, xenon can only be reliably detected in blood and urine up to 3 h postacute dosing. NEW & NOTEWORTHY Breathing xenon in dosages conceivable for doping purposes (FiXe 30% for 20 min; FiXe 50% for 5 min; FiXe 70% for 2 min) causes an initial rapid fall in total peripheral resistance with tachycardia and thereafter a mild hypertension with elevated middle cerebral artery velocity. These dose duration intervals cause sedation that is incompatible with operating a breathing apparatus and can only be detected in blood and urine samples with a high probability for up to ~3 h.

Objective In an earlier study wherein we induced acute reflux by interrupting proton pump inhibitor (PPI) therapy in patients with reflux oesophagitis (RO) healed by PPIs, we refuted the traditional concept that RO develops as an acid burn. The present study explored our alternative hypothesis that RO results from reflux-stimulated production of pro-inflammatory molecules mediated by hypoxia-inducible factors (HIFs). Design Using oesophageal biopsies taken from patients in our earlier study at baseline and at 1 and 2 weeks off PPIs, we immunostained for HIF-1α, HIF-2α and phospho-p65, and measured pro-inflammatory molecule mRNAs. We exposed human oesophageal squamous cell lines to acidic bile salts, and evaluated effects on HIF activation, p65 function, pro-inflammatory molecule production and immune cell migration. Results In patient biopsies, increased immunostaining for HIF-2α and phospho-p65, and increased pro-inflammatory molecule mRNA levels were seen when RO redeveloped 1 or 2 weeks after stopping PPIs. In oesophageal cells, exposure to acidic bile salts increased intracellular reactive oxygen species, which decreased prolyl hydroxylase function and stabilised HIF-2α, causing a p65-dependent increase in pro-inflammatory molecules; conditioned media from these cells increased T cell migration rates. HIF-2α inhibition by small hairpin RNA or selective small molecule antagonist blocked the increases in pro-inflammatory molecule expression and T cell migration induced by acidic bile salts. Conclusions In patients developing RO, increases in oesophageal HIF-2α correlate with increased pro-inflammatory molecule expression. In oesophageal epithelial cells, acidic bile salts stabilise HIF-2α, which mediates expression of pro-inflammatory molecules. HIF-2α appears to have a role in RO pathogenesis. Trial registration number NCT01733810; Results.

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1β) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1β, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

The PI(3)K-Akt-mTORC1 pathway is a highly dynamic network that is balanced and stabilized by a number of feedback inhibition loops. Specifically, activation of mTORC1 has been shown to lead to the inhibition of its upstream growth factor signalling. Activation of the growth factor receptors is triggered by the binding of their cognate ligands in the extracellular space. However, whether secreted proteins contribute to the mTORC1-dependent feedback loops remains unclear. We found that cells with hyperactive mTORC1 secrete a protein that potently inhibits the function of IGF-1. Using a large-scale, unbiased quantitative proteomic platform, we comprehensively characterized the rapamycin-sensitive secretome in TSC2(-/-) mouse embryonic fibroblasts, and identified IGFBP5 as a secreted, mTORC1 downstream effector protein. IGFBP5 is a direct transcriptional target of HIF1, which itself is a known mTORC1 target. IGFBP5 is a potent inhibitor of both the signalling and functional outputs of IGF-1. Once secreted, IGFBP5 cooperates with intracellular branches of the feedback mechanisms to block the activation of IGF-1 signalling. Finally, IGFBP5 is a potential tumour suppressor, and the proliferation of IGFBP5-mutated cancer cells is selectively blocked by IGF-1R inhibitors.

Hypoxia inducible factor (HIF) transcription factors reside at the center of signaling pathways used by mammalian cells to sense and respond to low oxygen levels. While essential to maintain oxygen homeostasis, misregulation of HIF protein activity correlates with tumor development and metastasis. To provide artificial routes to target misregulated HIF activity, we identified small molecule antagonists of the HIF-2 transcription factor that bind an internal cavity within the C-terminal PAS domain of the HIF-2α subunit. Here we describe a new class of chiral small molecule ligands that provide the highest affinity binding, the most effective, isoform-selective inhibition of HIF-2 in cells, and trigger the largest protein conformation changes reported to date. The current results further illuminate the molecular mechanism of HIF-2 antagonism and suggest additional routes to develop higher affinity and potency HIF-2 antagonists.

Though essential for many vital biological processes, excess iron results in the formation of damaging reactive oxygen species (ROS). Therefore, iron metabolism must be tightly regulated. F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, regulates cellular and systemic iron homeostasis by facilitating iron regulatory protein 2 (IRP2) degradation. FBXL5 possesses an N-terminal hemerythrin (Hr)-like domain that mediates its own differential stability by switching between two different conformations to communicate cellular iron availability. In addition, the FBXL5-Hr domain also senses O2 availability, albeit by a distinct mechanism. Mice lacking FBXL5 fail to sense intracellular iron levels and die in utero due to iron overload and exposure to damaging levels of oxidative stress. By closely monitoring intracellular levels of iron and oxygen, FBLX5 prevents the formation of conditions that favor ROS formation. These findings suggest that FBXL5 is essential for the maintenance of iron homeostasis and is a key sensor of bioavailable iron. Here, we describe the iron and oxygen sensing mechanisms of the FBXL5 Hr-like domain and its role in mediating ROS biology.

Background: FBXL5 is an iron-responsive E3 ubiquitin ligase. Results: FBXL5-null mice die during embryogenesis, whereas Fbxl5 heterozygotes perform better than wild type littermates when fed a low iron diet due to enhanced iron absorption. Conclusion: FBXL5 plays an essential role in the in vivo maintenance of cellular and systemic iron homeostasis. Significance: FBXL5 is an essential physiological iron sensor., Maintenance of cellular iron homeostasis requires post-transcriptional regulation of iron metabolism genes by iron regulatory protein 2 (IRP2). The hemerythrin-like domain of F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, senses iron and oxygen availability and facilitates IRP2 degradation in iron replete cells. Disruption of the ubiquitously expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, although viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Mice containing a single functional Fbxl5 allele behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. The responsiveness of IRP2 to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the divalent metal transporter-1. These results confirm the role of FBXL5 in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity.

The transcription factor HIF-2, an important driver of clear cell renal cell carcinoma, has been called 'undruggable'. However, in this issue of Nature, two groups report on the development and testing of a novel HIF-2 inhibitor, termed PT2399. William Kaelin and colleagues show that PT2399 causes tumour regression in preclinical mouse models of primary and metastatic pVHL-defective clear cell renal cell carcinoma. James Brugarolas and colleagues tested the compound in mice with tumour grafts generated from human renal cell cancers. PT2399 decreased tumour growth in 10 out of 18 cell lines and was well tolerated. The authors identify potential markers of sensitivity and provide initial characterization of the effects and mechanisms of resistance acquisition in vivo. Both groups report variable sensitivity to PT2399 in some pVHL-defective cell lines, highlighting a need for predictive biomarkers to be developed for use of this approach in the clinic.

For several well-documented reasons, it has been challenging to develop artificial small molecule inhibitors of protein/protein complexes. Such reagents are of particular interest for transcription factor complexes given links between their misregulation and disease. Here we report parallel approaches to identify regulators of a hypoxia signaling transcription factor complex, involving the ARNT subunit of the HIF (Hypoxia Inducible Factor) activator and the TACC3 (Transforming Acidic Coiled Coil Containing Protein 3) coactivator. In one route, we used in vitro NMR and biochemical screening to identify small molecules that selectively bind within the ARNT PAS (Per-ARNT-Sim) domain that recruits TACC3, identifying KG-548 as an ARNT/TACC3 disruptor. A parallel, cell-based screening approach previously implicated the small molecule KHS101 as an inhibitor of TACC3 signaling. Here, we show that KHS101 works indirectly on HIF complex formation by destabilizing both TACC3 and the HIF component HIF-1α. Overall, our data identify small molecule regulators for this important complex and highlight the utility of pursuing parallel strategies to develop protein/protein inhibitors.

Regulation of both systemic and cellular iron homeostasis requires the capacity to sense iron levels and appropriately modify the expression of iron metabolism genes. These responses are coordinated through the efforts of several key regulatory factors including F-box and Leucine-rich Repeat Protein 5 (FBXL5), Iron Regulatory Proteins (IRPs), Hypoxia Inducible Factor (HIF), and ferroportin. Notably, the stability of each of these proteins is regulated in response to iron. Recent discoveries have greatly advanced our understanding of the molecular mechanisms governing iron-sensing and protein degradation within these pathways. It has become clear that iron's privileged roles in both enzyme catalysis and protein structure contribute to its regulation of protein stability. Moreover, these multiple pathways intersect with one another in larger regulatory networks to maintain iron homeostasis. This article is part of a Special Issue entitled: Cell Biology of Metals.

Iron regulatory proteins play a principal role in maintaining cellular iron homeostasis by post-transcriptionally regulating factors responsible for iron uptake, utilization, and storage. An E3 ubiquitin ligase complex containing FBXL5 targets IRP2 for proteasomal degradation under iron- and oxygen-replete conditions, whereas FBXL5 itself is degraded when iron and oxygen availability decreases. FBXL5 contains a hemerythrin-like (Hr) domain at its N terminus that mediates its own differential stability. Here, we investigated the iron- and oxygen-dependent conformational changes within FBXL5-Hr that underlie its role as a cellular sensor. As predicted, FBXL5-Hr undergoes substantive structural changes when iron becomes limiting, accounting for its switch-like behavior. However, these same changes are not observed in response to oxygen depletion, indicating that this domain accommodates two distinct sensing mechanisms. Moreover, FBXL5-Hr does not behave as a dynamic sensor that continuously samples the cellular environment, assuming conformations in equilibrium with ever-changing cellular iron levels. Instead, the isolated domain appears competent to incorporate iron only at or near the time of its own synthesis. These observations have important implications for mechanisms by which these metabolites are sensed within mammalian cells.

Iron Sensor Intracellular iron is an essential cofactor for many proteins, but can also damage macromolecules, so its levels are carefully controlled. Cellular iron homeostasis is mediated by iron regulatory proteins that regulate the expression of genes involved in iron uptake and storage. However, it is not clear how cells sense iron bioavailability (see the Perspective by Rouault ). Using different approaches, Salahudeen et al. (p. 722 , published online 17 September) and Vashisht et al. (p. 718 , published online 17 September) have identified the F-box protein FBXL5 as a human iron sensor. FBXL5 is part of an E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins and thereby cellular iron levels. It contains a hemerythrin domain that binds iron and acts as an iron-dependent regulatory switch, causing the degradation of FBXL5 under low iron conditions. This alternative pathway for the regulation of iron homeostasis has implications for both normal cellular physiology and disease.

Arginine methylation occurs on a number of proteins involved in a variety of cellular functions. Histone tails are known to be mono- and dimethylated on multiple arginine residues where they influence chromatin remodeling and gene expression. To date, no enzyme has been shown to reverse these regulatory modifications. We demonstrate that the Jumonji domain–containing 6 protein (JMJD6) is a JmjC-containing iron- and 2-oxoglutarate–dependent dioxygenase that demethylates histone H3 at arginine 2 (H3R2) and histone H4 at arginine 3 (H4R3) in both biochemical and cell-based assays. These findings may help explain the many developmental defects observed in the JMJD6 –/– knockout mice.

Endoplasmic reticulum-associated degradation of the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase represents one mechanism by which cholesterol synthesis is controlled in mammalian cells. The key reaction in this degradation is binding of reductase to Insig proteins in the endoplasmic reticulum, which is stimulated by the cholesterol precursor lanosterol. Conversion of lanosterol to cholesterol requires removal of three methyl groups, which consumes nine molecules of dioxygen. Here, we report that oxygen deprivation (hypoxia) slows demethylation of lanosterol and its metabolite 24,25-dihydrolanosterol, causing both sterols to accumulate in cells. In addition, hypoxia increases the amount of Insig-1 and Insig-2 in a response mediated by hypoxia-inducible factor (HIF)-1alpha. Accumulation of lanosterol together with increased Insigs accelerates degradation of reductase, which ultimately slows a rate-determining step in cholesterol synthesis. These results define a novel oxygen-sensing mechanism mediated by the combined actions of methylated intermediates in cholesterol synthesis and the hypoxia-activated transcription factor HIF-1alpha.

A ubiquitous pathway by which mammalian cells sense and respond to changes in oxygen availability relies upon the hypoxic induction of a transcription factor, HIF. HIF in turn activates the expression of an assemblage of genes promoting compensatory shifts in the capacity for anaerobic metabolism, O2 delivery, and other adaptive processes. The stability and activity of HIF are each regulated as a function of O2. Both mechanisms are directly mediated by posttranslational modification of this transcription factor: hydroxylation of proline and asparagine residues, respectively. These modifications are performed by members of the Fe(II)- and 2-oxoglutarate-dependent dioxygenase family whose activities are directly and indirectly dependent on cellular O2 levels. As such, these oxygenases fill a role as environmental and metabolic sensors, a paradigm that may extend to other biological pathways.

The degree of tumor hypoxia correlates with advanced disease stages and treatment resistance. The transcription factor hypoxia-inducible factor-1 (HIF-1) promotes tumor cell adaptation and survival under hypoxic conditions. Therefore, specific HIF-1 inhibitors represent an important new class of potential tumor-selective therapeutic agents. A T47D human breast tumor cell-based reporter assay was used to examine extracts of plants and marine organisms for inhibitors of HIF-1 activation. Bioassay-guided fractionation of the lipid extract of the red alga Laurencia intricata yielded a structurally novel diterpene, laurenditerpenol (1). The structure of 1 was determined spectroscopically. The relative configurations of the substituents of each ring system were assigned on the basis of NOESY correlations. The absolute configuration of position C-1 was determined by the modified Mosher ester procedure (directly in NMR tubes). Compound 1 potently inhibited hypoxia-activated HIF-1 (IC50: 0.4 microM) and hypoxia-induced VEGF (a potent angiogenic factor) in T47D cells. Compound 1 selectively inhibits HIF-1 activation by hypoxia but not iron chelator-induced activation. Further, 1 suppresses tumor cell survival under hypoxic conditions without affecting normoxic cell growth. Compound 1 inhibits HIF-1 by blocking the induction of the oxygen-regulated HIF-1alpha protein. Mitochondrial respiration studies revealed that 1 suppresses oxygen consumption.

Binding of proteins to chloroplast-encoded mRNAs has been shown to be an essential part of chloroplast gene expression. Four nuclear-encoded proteins (38, 47, 55, and 60 kDa) have been identified that bind to the 5'-untranslated region of the Chlamydomonas reinhardtii psbA mRNA with high affinity and specificity. We have cloned a cDNA that represents the 38 kDa protein (RB38) and show that it encodes a novel RNA binding protein that is primarily localized within the chloroplast stroma. RB38 contains four 70 amino acid repeats with a high percentage of basic amino acids, as well as an amino-terminal extension predicted to act as a chloroplast import sequence. We demonstrate that the 38 kDa precursor protein is imported into isolated chloroplasts and interacts with high specificity to uridine-rich regions within the 5'-untranslated region of the psbA mRNA. While database searches have identified hypothetical proteins from several other eukaryotic species with high sequence similarity to the deduced amino acid sequence of RB38, no proteins with homology to RB38 have been biochemically characterized. Bioinformatic analysis of the RB38 sequence, together with structure analysis using circular dichroism and protein modeling, suggests that the 70 amino acid repeats within RB38 are similar in fold to previously identified RNA binding motifs, despite limited sequence homology.

The liver provides for long-term energy needs of the body by converting excess carbohydrate into fat for storage. Insulin is one factor that promotes hepatic lipogenesis, but there is increasing evidence that glucose also contributes to the coordinated regulation of carbohydrate and fat metabolism in liver by mechanisms that are independent of insulin. In this study, we show that the transcription factor, carbohydrate response element-binding protein (ChREBP), is required both for basal and carbohydrate-induced expression of several liver enzymes essential for coordinated control of glucose metabolism, fatty acid, and the synthesis of fatty acids and triglycerides in vivo .

Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix–loop–helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIFα and ARNT. Although this complex involves protein–protein interactions mediated by basic helix–loop–helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2α by NMR spectroscopy. We demonstrate that HIF-2α PAS-B binds the analogous ARNT domain in vitro , showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2α central β-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains, we find that this β-sheet interface is widely used for both intra- and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins.

Oxygen occupies a central role in the maintenance of life as we know it, perhaps most prominently in aerobic metabolism, where O2 serves as the terminal electron acceptor in oxidative phosphorylation. However, oxygen transport by simple diffusion becomes limiting as organisms become larger andmore active. Tomaintain oxygen homeostasis, higher eukaryotes have adopted specialized mechanisms to enhance O2 uptake and distribution. The resulting respiratory and circulatory systems are dynamic, capable of responding to changes in oxygen availability on either a local or organismal level. Though some oxygen-dependent responses rely on the activity of specialized oxygen-sensing cells, conserved oxygen-responsive pathways are expressed in almost every mammalian cell. In fact, this ability of individual cells to sense and respond to changes in oxygen availability is critical for many developmental, physiological, and pathological processes. Unlike the rapid responses to changes in oxygen availability, which can be mediated through posttranslational modifications or membrane depolarization (for review, see Lopez-Barneo et al. 2001), this ubiquitous hypoxic response pathway involves changes in gene expression that occur over several hours. These changes are mediated in part through the induction of hypoxia-inducible transcription factors, the regulatory components of which can also be found in simpler eukaryotes such as fruit flies and nematodes.

Mammalian cells respond to changes in oxygen availability through a conserved pathway that is regulated by the hypoxia-inducible factor (HIF). The alpha subunit of HIF is targeted for degradation under normoxic conditions by a ubiquitin-ligase complex that recognizes a hydroxylated proline residue in HIF. We identified a conserved family of HIF prolyl hydoxylase (HPH) enzymes that appear to be responsible for this posttranslational modification. In cultured mammalian cells, inappropriate accumulation of HIF caused by forced expression of the HIF-1α subunit under normoxic conditions was attenuated by coexpression of HPH. Suppression of HPH in cultured Drosophila melanogaster cells by RNA interference resulted in elevated expression of a hypoxia-inducible gene ( LDH , encoding lactate dehydrogenase) under normoxic conditions. These findings indicate that HPH is an essential component of the pathway through which cells sense oxygen.

The integrated regulation of mRNA stability, processing and translation facilitates the expression of several chloroplast genes, particularly in response to changes in illumination. Nuclear and chloroplast-encoded factors that mediate the expression of specific chloroplast messages have been characterized from green algae and plants. Recent studies suggest that the chloroplast might have recruited eukaryotic proteins, which are usually found in the cytoplasm or the endoplasmic reticulum, to couple the level of photosynthetic activity to gene expression via translational activation. Consequently, elements required for translational initiation of chloroplast messages differ from their prokaryotic ancestors. These results suggest that chloroplast translational regulation is a hybrid between prokaryotic and eukaryotic systems.

The 5′ untranslated region of the chloroplast psbA mRNA, encoding the D1 protein, is processed in Chlamydomonas reinhardtii. Processing occurs just upstream of a consensus Shine-Dalgarno sequence and results in the removal of 54 nucleotides from the 5′ terminus, including a stem-loop element identified previously as an important structure for D1 expression. Examination of this processing event in C. reinhardtii strains containing mutations within the chloroplast or nuclear genomes that block psbA translation reveals a correlation between processing and ribosome association. Mutations within the 5′ untranslated region of the psbA mRNA that disrupt the Shine-Dalgarno sequence, acting as a ribosome binding site, preclude translation and prevent mRNA processing. Similarly, nuclear mutations that specifically affect synthesis of the D1 protein specifically affect processing of the psbA mRNA. In vitro, loss of the stem-loop element does not prohibit the binding of a message-specific protein complex required for translational activation of psbA upon illumination. These results are consistent with a hierarchical maturation pathway for chloroplast messages, mediated by nuclear-encoded factors, that integrates mRNA processing, message stability, ribosome association, and translation.

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors induced in a variety of pathophysiological settings, including cancer. We describe the first detailed structure–activity relationship study of small molecules designed to inhibit HIF-2α–ARNT heterodimerization by binding an internal cavity of the HIF-2α PAS-B domain. Through a series of biophysical characterizations of inhibitor–protein interactions (NMR and X-ray crystallography), we have established the structural requirements for artificial inhibitors of the HIF-2α–ARNT PAS-B interaction. These results may serve as a foundation for discovering therapeutic agents that function by a novel mode of action.

The pharmacological inhibition of general transcriptional regulators has the potential to block growth through targeting multiple tumorigenic signalling pathways simultaneously. Here, using an innovative cell-based screen, we identify a structurally unique small molecule (named JIB-04) that specifically inhibits the activity of the Jumonji family of histone demethylases in vitro, in cancer cells, and in tumours in vivo. Unlike known inhibitors, JIB-04 is not a competitive inhibitor of α-ketoglutarate. In cancer, but not in patient-matched normal cells, JIB-04 alters a subset of transcriptional pathways and blocks viability. In mice, JIB-04 reduces tumour burden and prolongs survival. Importantly, we find that patients with breast tumours that overexpress Jumonji demethylases have significantly lower survival. Thus, JIB-04, a novel inhibitor of Jumonji demethylases in vitro and in vivo, constitutes a unique potential therapeutic and research tool against cancer, and validates the use of unbiased cellular screens to discover chemical modulators with disease relevance. CA extern

Mammalian cells maintain iron homeostasis by sensing changes in bioavailable iron levels and promoting adaptive responses. FBXL5 is a subunit of an E3 ubiquitin ligase complex that mediates the stability of iron regulatory protein 2, an important posttranscriptional regulator of several genes involved in iron metabolism. The stability of FBXL5 is regulated in an iron- and oxygen-responsive manner, contingent upon the presence of its N-terminal domain. Here we present the atomic structure of the FBXL5 N terminus, a hemerythrin-like α-helical bundle fold not previously observed in mammalian proteins. The core of this domain employs an unusual assortment of amino acids necessary for the assembly and sensing properties of its diiron center. These regulatory features govern the accessibility of a mapped sequence required for proteasomal degradation of FBXL5. Detailed molecular and structural characterization of the ligand-responsive hemerythrin domain provides insights into the mechanisms by which FBXL5 serves as a unique mammalian metabolic sensor.

SummaryMammalian cells express dozens of iron-containing proteins, yet little is known about the mechanism of metal ligand incorporation. Human poly (rC) binding protein 1 (PCBP1) is an iron chaperone that binds iron and delivers it to ferritin, a cytosolic iron storage protein. We have identified the iron-dependent prolyl hydroxylases (PHDs) and asparaginyl hydroxylase (FIH1) that modify hypoxia-inducible factor α (HIFα) as targets of PCBP1. Depletion of PCBP1 or PCBP2 in cells led to loss of PHD activity, manifested by reduced prolyl hydroxylation of HIF1α, impaired degradation of HIF1α through the VHL/proteasome pathway, and accumulation of active HIF1 transcription factor. PHD activity was restored in vitro by addition of excess Fe(II), or purified Fe-PCBP1, and PCBP1 bound to PHD2 and FIH1 in vivo. These data indicated that PCBP1 was required for iron incorporation into PHD and suggest a broad role for PCBP1 and 2 in delivering iron to cytosolic nonheme iron enzymes.

Though iron and oxygen are required to sustain essential biological processes, an excess of either can result in oxidative stress. Therefore, mammals tightly regulate cellular and systemic iron and oxygen homeostasis. At the cellular level, the hypoxia-inducible transcription factors (HIFs) are key mediators of oxygen homeostasis through their regulation of genes involved in anaerobic metabolism and oxygen delivery, among others. Iron regulatory proteins (IRPs) largely govern cellular iron homeostasis through their effects on the translation and stability of mRNAs involved in iron uptake, utilization, export, and storage. Here, we describe regulatory factors for each pathway that sense both iron and oxygen availability and coordinate the maintenance of mammalian iron and oxygen homeostasis at both the cellular and systemic levels.

The hypoxia-inducible factor (HIF) basic helix–loop–helix Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim (bHLH-PAS) transcription factors are master regulators of the conserved molecular mechanism by which metazoans sense and respond to reductions in local oxygen concentrations. In humans, HIF is critically important for the sustained growth and metastasis of solid tumors. Here, we describe crystal structures of the heterodimer formed by the C-terminal PAS domains from the HIF2α and ARNT subunits of the HIF2 transcription factor, both in the absence and presence of an artificial ligand. Unexpectedly, the HIF2α PAS-B domain contains a large internal cavity that accommodates ligands identified from a small-molecule screen. Binding one of these ligands to HIF2α PAS-B modulates the affinity of the HIF2α:ARNT PAS-B heterodimer in vitro. Given the essential role of PAS domains in forming active HIF heterodimers, these results suggest a presently uncharacterized ligand-mediated mechanism for regulating HIF2 activity in endogenous and clinical settings.

Hypoxia-inducible factors (HIFs) are key transcriptional regulators of genes involved in cellular adaptation to reduced oxygen availability through effects on anaerobic metabolism, oxygen delivery, angiogenesis, and cellular survival and proliferation. As such, HIFs contribute to the pathogenesis of diseases in which oxygen availability is compromised, notably ischemia and tumorigenesis. Though tremendous progress has been made in elucidating the mechanisms underlying O(2)-dependent regulation of HIF by Fe(II)- and 2-oxoglutarate-dependent dioxygenases, HIF induction can be uncoupled from these modes of regulation in diseases such as cancer. Consequently, renewed interest has developed in understanding the structure/function relationships of individual P(er)/ARNT/S(im) (PAS) domains that are important for maintaining transcriptionally active HIF complexes, regardless of the manner by which HIF is induced. This review highlights strategies for the biophysical and biochemical characterization of the PAS domains found within both HIF subunits and provides a platform for future efforts to exploit these domains in therapeutic settings.

Members of the Fe(II)- and 2-oxoglutarate–dependent family of dioxygenases have long been known to oxidize several amino acids in various protein targets to facilitate protein folding. However, in recent years investigators have characterized several such hydroxylation modifications that serve a regulatory, rather than structural, purpose. Furthermore, the responsible enzymes seem to function directly as sensors of the cellular environment and metabolic state. For example, a cellular response pathway to low oxygen (hypoxia) is orchestrated through the actions of prolyl and asparaginyl hydroxylases that govern both the oxygen-dependent stability and transcriptional activity of the hypoxia-inducible transcription factor. Recently, a different subfamily of Fe(II)- and 2-oxoglutarate–dependent dioxygenases has been shown to carry out histone demethylation. The discovery of protein regulation via hydroxylation raises the possibility that other Fe(II)- and 2-oxoglutarate–dependent dioxygenases might also serve in a similar capacity.

Hypoxia-inducible factors (HIFs) are key transcriptional regulators of genes involved in cellular adaptation to reduced oxygen availability through effects on anaerobic metabolism, oxygen delivery, angiogenesis, and cellular survival and proliferation. As such, HIFs contribute to the pathogenesis of diseases in which oxygen availability is compromised, notably ischemia and tumorigenesis. Though tremendous progress has been made in elucidating the mechanisms underlying O(2)-dependent regulation of HIF by Fe(II)- and 2-oxoglutarate-dependent dioxygenases, HIF induction can be uncoupled from these modes of regulation in diseases such as cancer. Consequently, renewed interest has developed in understanding the structure/function relationships of individual P(er)/ARNT/S(im) (PAS) domains that are important for maintaining transcriptionally active HIF complexes, regardless of the manner by which HIF is induced. This review highlights strategies for the biophysical and biochemical characterization of the PAS domains found within both HIF subunits and provides a platform for future efforts to exploit these domains in therapeutic settings.

The heterodimeric transcription factor hypoxia-inducible factor (HIF) plays an important role in the progression of a number of processes in which O2 availability is compromised and, as such, has become an increasingly attractive therapeutic target. Although tremendous progress has been made in recent years in unraveling the mechanisms underlying O2-dependent regulation of HIF through its O2-dependent degradation domain and C-terminal transactivation domain, our understanding of the contributions of other structural elements, particularly the Per/ARNT/Sim (PAS)-A and PAS-B domains, to the activity of HIF is incomplete. Using insights derived from the recently determined solution structures of the HIF PAS-B domains as a starting point, we have explored the function(s) of the HIF-2alpha PAS domains via mutational analysis. In contrast to recent models, our data reveal that both PAS domains of the HIF-alpha subunit are necessary for heterodimer formation but are not required to mediate other HIF functions in which PAS domains have been implicated. Because disruption of individual PAS domains compromise HIF function independent of the mechanism of HIF induction, these data demonstrate the potential utility of targeting these domains for therapeutic applications.

Many cellular responses to changes in O2 availability are mediated through the hypoxia inducible transcription factor, HIF. HIF regulation is largely dependent on the activity of O2-dependent prolyl hydroxylases. Under normoxic conditions, these enzymes modify HIF to promote its proteasomal degradation. Recently we proposed a second function for the HIF prolyl hydroxylases--recruitment of the candidate tumor suppressor protein ING4 to HIF under hypoxic conditions. Rather than affecting hydroxylase activity or HIF stability, ING4 mediates the ability of HIF to activate transcription of its downstream target genes. This additional mechanism of HIF regulation may serve to fine-tune the magnitude of the cellular hypoxic response under physiological conditions. Furthermore, the identification of a link between ING4 and HIF may shed light on a mechanism by which misregulation of ING4 in tumors promotes angiogenesis, loss of contact inhibition, and changes in cellular proliferation and survival.

Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1alpha protein accumulation without affecting HIF-1alpha mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors.

The hypoxia inducible factor (HIF) plays an important role in the progression of a number of pathophysiological processes including tumorigenesis. In addition to several well characterized oxygen-dependent modes of regulation, the function of the HIF transcription factor can also be influenced through the action of other regulatory pathways. Misregulation of these factors resulting in inappropriate HIF expression or activity can contribute to the progression of human cancers through the induction of genes promoting angiogenesis, glycolysis, cell survival, and metastasis, among other processes. The candidate tumor suppressor protein inhibitor of growth family member 4 (ING4) has recently been implicated as a repressor of angiogenesis and tumor growth through association with NF-κB. Here we demonstrate that suppression of ING4 further induces HIF transcriptional activity as well. ING4 directly associates with the HIF prolyl hydroxylase, an Fe(II)-dependent oxygenase previously shown to mediate HIF stability as a function of oxygen availability. However, rather than affecting HIF's stability, ING4 mediates HIF's activity. These data support a model in which, in addition to regulating HIF stability, HIF prolyl hydroxylases can modulate HIF function through the recruitment of ING4, a likely component of a chromatin-remodeling complex.

Exposure of cells to carcinogenic compounds of nickel(II) and cobalt(II) causes activation of the HIF-1 transcription factor and up-regulates a battery of hypoxia-inducible genes. However, the mechanism of HIF-1 activation by these metals is not known. It was shown recently that hydroxylation of prolines in the HIFalpha subunit of HIF-1 is required for its binding with the von Hippel-Lindau tumor suppressor protein and the subsequent proteasomal destruction. Here we show that responsible prolyl hydroxylases are targets for both nickel(II) and cobalt(II) because degradation of a reporter protein containing the oxygen-dependent degradation domain (Pro-402/564) of HIFalpha was abolished in a von Hippel-Lindau-dependent manner in cells exposed to nickel(II) or cobalt(II). The enzymatic activity of prolyl hydroxylases depends on iron as the activating metal, 2-oxoglutarate as a co-substrate, and ascorbic acid as a cofactor. Hydroxylase activity can be impaired by the depletion of any of these factors. We found that exposure of cells to nickel(II) or cobalt(II) did not affect the level of intracellular iron. Instead, nickel(II) or cobalt(II) exposure greatly depleted intracellular ascorbate. Co-exposure of cells to metals and ascorbate resulted in the increase of intracellular ascorbate and reversed both metal-induced stabilization of HIF-1alpha and HIF-1-dependent gene transcription. Because ascorbate is essential for maintaining iron in prolyl hydroxylases in the active iron(II) state, we suggest that the observed depletion of ascorbate by nickel(II) or cobalt(II) favors iron oxidation and thus inactivation of the enzyme.

The first Oxygen Homeostasis/Hypoxia Meeting was held on February 12, 2003, at the Sheraton National Hotel, Washington, D.C. The meeting was hosted by Drs. S. Percy Ivy and Giovanni Melillo of the National Cancer Institute, NIH. The purpose of the meeting was to stimulate collaborations among the participants who are engaged in different areas of hypoxia research and application, including basic research on hypoxia, and its induction and consequences; the development of drugs targeting hypoxia and factors involved in pathways leading to (or controlled by)hypoxia; and the development and application of hypoxia imaging techniques and reagents. ispartof: pages:3350-3356 ispartof: Cancer Research vol:64 issue:9 pages:3350-3356 ispartof: Oxygen Homeostasis/Hypoxia Meeting location:Washington, D.C. date:12 Feb - 12 Feb 2003 status: published

Precise regulation of the evolutionarily conserved hypoxia-inducible transcription factor (HIF) ensures proper adaptation to variations in oxygen availability throughout development and into adulthood. Oxygen-dependent regulation of HIF stability and activity are mediated by hydroxylation of conserved proline and asparagine residues, respectively. Because the relevant prolyl and asparginyl hydroxylases use O 2 to effect these posttranslational modifications, these enzymes are implicated as direct oxygen sensors in the mammalian hypoxic response pathway. Here we present the structure of factor-inhibiting HIF-1 (FIH-1), the pertinent asparaginyl hydroxylase involved in hypoxic signaling. Hydroxylation of the C-terminal transactivation domain (CTAD) of HIF by FIH-1 prevents CTAD association with transcriptional coactivators under normoxic conditions. Consistent with other structurally known hydroxylases, FIH-1 is comprised of a β-strand jellyroll core with both Fe(II) and the cosubstrate 2-oxoglutarate bound in the active site. Details of the molecular contacts at the active site of FIH-1 have been elucidated and provide a platform for future drug design. Furthermore, the structure reveals the presence of a FIH-1 homodimer that forms in solution and is essential for FIH activity.

Mammalian cells adapt to hypoxic conditions through a transcriptional response pathway mediated by the hypoxia-inducible factor, HIF. HIF transcriptional activity is suppressed under normoxic conditions by hydroxylation of an asparagine residue within its C-terminal transactivation domain, blocking association with coactivators. Here we show that the protein FIH-1, previously shown to interact with HIF, is an asparaginyl hydroxylase. Like known hydroxylase enzymes, FIH-1 is an Fe(II)-dependent enzyme that uses molecular O2 to modify its substrate. Together with the recently discovered prolyl hydroxylases that regulate HIF stability, this class of oxygen-dependent enzymes comprises critical regulatory components of the hypoxic response pathway.

Carbohydrates mediate their conversion to triglycerides in the liver by promoting both rapid posttranslational activation of rate-limiting glycolytic and lipogenic enzymes and transcriptional induction of the genes encoding many of these same enzymes. The mechanism by which elevated carbohydrate levels affect transcription of these genes remains unknown. Here we report the purification and identification of a transcription factor that recognizes the carbohydrate response element (ChRE) within the promoter of the L-type pyruvate kinase (LPK) gene. The DNA-binding activity of this ChRE-binding protein (ChREBP) in rat livers is specifically induced by a high carbohydrate diet. ChREBP's DNA-binding specificity in vitro precisely correlates with promoter activity in vivo . Furthermore, forced ChREBP overexpression in primary hepatocytes activates transcription from the L-type Pyruvate kinase promoter in response to high glucose levels. The DNA-binding activity of ChREBP can be modulated in vitro by means of changes in its phosphorylation state, suggesting a possible mode of glucose-responsive regulation. ChREBP is likely critical for the optimal long-term storage of excess carbohydrates as fats, and may contribute to the imbalance between nutrient utilization and storage characteristic of obesity.

The ability to sense and respond to changes in oxygen availability is critical for many developmental, physiological, and pathological processes, including angiogenesis, control of blood pressure, and cerebral and myocardial ischemia. Hypoxia-inducible factor-1alpha (HIF-1alpha) is a basic-helix-loop-helix (bHLH)containing member of the PER-ARNT-SIM (PAS) family of transcription factors that plays a central role in the response to hypoxia. HIF-1alpha, and its relatives HIF-2alpha/endothelial PAS domain protein (EPAS) and HIF-3alpha, are induced in response to hypoxia and serve to coordinately activate the expression of target genes whose products facilitate cell survival under conditions of oxygen deprivation. When cells are exposed to chronic hypoxia, the protective response can fail, resulting in apoptosis. This study shows that transcription of the gene encoding Nip3, a proapoptotic member of the Bcl-2 family of cell death factors, is strongly induced in response to hypoxia. The Nip3 promoter contains a functional HIF-1-responsive element (HRE) and is potently activated by both hypoxia and forced expression of HIF-1alpha. Exposure of cultured cells to chronic hypoxia results in the accumulation of a protein recognized by antibodies raised against Nip3. This study demonstrates a direct link between HIF-1alpha and a proapoptotic member of the Bcl-2 family and offers a reasonable physiological function for members of the Bcl-2 subfamily, including Nip3 and its close relative Nix. These observations indicate that Nip3 may play a dedicated role in the pathological progression of hypoxia-mediated apoptosis, as observed after ischemic injury.