Characterization of functionally deficient SIM2 variants found in patients with neurological phenotypes.

Single-minded 2 (SIM2) is a neuron-enriched basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH/PAS) transcription factor essential for mammalian survival. SIM2 is located within the Down syndrome critical region (DSCR) of chromosome 21, and manipulation in mouse models suggests Sim2 may play a role in brain development and function. During the screening of a clinical exome sequencing database, nine SIM2 non-synonymous mutations were found which were subsequently investigated for impaired function using cell-based reporter gene assays. Many of these human variants attenuated abilities to activate transcription and were further characterized to determine the mechanisms underpinning their deficiencies. These included impaired partner protein dimerization, reduced DNA binding, and reduced expression and nuclear localization. This study highlighted several SIM2 variants found in patients with disabilities and validated a candidate set as potentially contributing to pathology.

Asparagine Hydroxylation is a Reversible Post-translational Modification.

Amino acid hydroxylation is a common post-translational modification, which generally regulates protein interactions or adds a functional group that can be further modified. Such hydroxylation is currently considered irreversible, necessitating the degradation and re-synthesis of the entire protein to reset the modification. Here we present evidence that the cellular machinery can reverse FIH-mediated asparagine hydroxylation on intact proteins. These data suggest that asparagine hydroxylation is a flexible and dynamic post-translational modification akin to modifications involved in regulating signaling networks, such as phosphorylation, methylation and ubiquitylation.

Power to see-Drivers of aerobic glycolysis in the mammalian retina: A review.

The mammalian retina converts most glucose to lactate rather than catabolizing it completely to carbon dioxide via oxidative phosphorylation, despite the availability of oxygen. This unusual metabolism is known as aerobic glycolysis or the Warburg effect. Molecules and pathways that drive aerobic glycolysis have been identified and thoroughly studied in the context of cancer but remain relatively poorly understood in the retina. Here, we review recent research on the molecular mechanisms that underly aerobic glycolysis in the retina, focusing on key glycolytic enzymes including hexokinase 2 (HK2), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). We also discuss the potential involvement of cell signalling and transcriptional pathways including phosphoinositide 3-kinase (PI3K) signalling, fibroblast growth factor receptor (FGFR) signalling, and hypoxia-inducible factor 1 (HIF-1), which have been implicated in driving aerobic glycolysis in the context of cancer.

Characterization of the novel spontaneously immortalized rat Müller cell line SIRMu-1.

Müller cells (MCs) play a crucial role in the retina, and cultured MC lines are an important tool with which to study MC function. Transformed MC lines have been widely used; however, the transformation process can also lead to unwanted changes compared to the primary cells from which they were derived. To provide an alternative experimental tool, a novel monoclonal spontaneously immortalized rat Müller cell line, SIRMu-1, was derived from primary rat MCs and characterized. Immunofluorescence, western blotting and RNA sequencing demonstrate that the SIRMu-1 cell line retains similar characteristics to cultured primary MCs in terms of expression of the MC markers cellular retinaldehyde-binding protein, glutamine synthetase, S100, vimentin and glial fibrillary acidic protein at both the mRNA and protein levels. Both the cellular morphology and overall transcriptome of the SIRMu-1 cells are more similar to primary rat MCs than the commonly used rMC-1 cells, a well-described, transformed rat MC line. Furthermore, SIRMu-1 cells proliferate rapidly, have an effectively indefinite life span and a high transfection efficiency. The expression of Y chromosome specific genes confirmed that the SIRMu-1 cells are derived from male MCs. Thus, the SIRMu-1 cell line represents a valuable experimental tool to study roles of MCs in both physiological and pathological states.

FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1.

The asparagine hydroxylase, factor inhibiting HIF (FIH), confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However, the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, in cultured cells, overexpression of N22A mutant OTUB1 impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH. Additionally, we propose that our results provide new insight into the regulation of cellular energy metabolism during hypoxic stress and the potential for targeting hydroxylases for therapeutic benefit.

Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway.

Hypoxia-inducible factor (HIF) is a transcriptional activator with a central role in regulating cellular responses to hypoxia. It is also emerging as a major target for viral manipulation of the cellular environment. Under normoxic conditions, HIF is tightly suppressed by the activity of oxygen-dependent prolyl and asparaginyl hydroxylases. The asparaginyl hydroxylase active against HIF, factor inhibiting HIF (FIH), has also been shown to hydroxylate some ankyrin repeat (ANK) proteins. Using bioinformatic analysis, we identified the five ANK proteins of the parapoxvirus orf virus (ORFV) as potential substrates of FIH. Consistent with this prediction, coimmunoprecipitation of FIH was detected with each of the ORFV ANK proteins, and for one representative ORFV ANK protein, the interaction was shown to be dependent on the ANK domain. Immunofluorescence studies revealed colocalization of FIH and the viral ANK proteins. In addition, mass spectrometry confirmed that three of the five ORFV ANK proteins are efficiently hydroxylated by FIH in vitro While FIH levels were unaffected by ORFV infection, transient expression of each of the ORFV ANK proteins resulted in derepression of HIF-1α activity in reporter gene assays. Furthermore, ORFV-infected cells showed upregulated HIF target gene expression. Our data suggest that sequestration of FIH by ORFV ANK proteins leads to derepression of HIF activity. These findings reveal a previously unknown mechanism of viral activation of HIF that may extend to other members of the poxvirus family. IMPORTANCE: The protein-protein binding motif formed from multiple repeats of the ankyrin motif is common among chordopoxviruses. However, information on the roles of these poxviral ankyrin repeat (ANK) proteins remains limited. Our data indicate that the parapoxvirus orf virus (ORFV) is able to upregulate hypoxia-inducible factor (HIF) target gene expression. This response is mediated by the viral ANK proteins, which sequester the HIF regulator FIH (factor inhibiting HIF). This is the first demonstration of any viral protein interacting directly with FIH. Our data reveal a new mechanism by which viruses reprogram HIF, a master regulator of cellular metabolism, and also show a new role for the ANK family of poxvirus proteins.

Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.

Factor inhibiting HIF (FIH, also known as HIF1AN) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels.

Oxygen-regulated gene expression in murine cumulus cells.

Oxygen is an important component of the environment of the cumulus-oocyte complex (COC), both in vivo within the ovarian follicle and during in vitro oocyte maturation (IVM). Cumulus cells have a key role in supporting oocyte development, and cumulus cell function and gene expression are known to be altered when the environment of the COC is perturbed. Oxygen-regulated gene expression is mediated through the actions of the transcription factors, the hypoxia-inducible factors (HIFs). In the present study, the effect of oxygen on cumulus cell gene expression was examined following in vitro maturation of the murine COC at 2%, 5% or 20% oxygen. Increased expression of HIF-responsive genes, including glucose transporter-1, lactate dehydrogenase A and BCL2/adenovirus E1B interacting protein 3, was observed in cumulus cells matured at 2% or 5%, compared with 20% oxygen. Stabilisation of HIF1α protein in cumulus cells exposed to low oxygen was confirmed by western blot and HIF-mediated transcriptional activity was demonstrated using a transgenic mouse expressing green fluorescent protein under the control of a promoter containing hypoxia response elements. These results indicate that oxygen concentration influences cumulus cell gene expression and support a role for HIF1α in mediating the cumulus cell response to varying oxygen.

Characterization of human variants in obesity-related SIM1 protein identifies a hot-spot for dimerization with the partner protein ARNT2.

The bHLH (basic helix-loop-helix) PAS (Per/Arnt/Sim) transcription factor SIM1 (single-minded 1) is important for development and function of regions of the hypothalamus that regulate energy homoeostasis and the feeding response. Low-activity SIM1 variants have been identified in individuals with severe early-onset obesity, but the underlying molecular causes of impaired function are unknown. In the present study we assess a number of human SIM1 variants with reduced activity and determine that impaired function is frequently due to defects in dimerization with the essential partner protein ARNT2 (aryl hydrocarbon nuclear translocator 2). Equivalent variants generated in the highly related protein SIM2 (single-minded 2) produce near-identical impaired function and dimerization defects, indicating that these effects are not unique to the structure of SIM1. On the basis of these data, we predict that other select SIM1 and SIM2 variants reported in human genomic databases will also be deficient in activity, and identify two new low-activity SIM1 variants (V290E and V326F) present in the population. The cumulative data is used in homology modelling to make novel observations about the dimerization interface between the PAS domains of SIM1 and ARNT2, and to define a mutational 'hot-spot' in SIM1 that is critical for protein function.

Reciprocal regulation of the basic helix-loop-helix/Per-Arnt-Sim partner proteins, Arnt and Arnt2, during neuronal differentiation.

Basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) transcription factors function broadly in development, homeostasis and stress response. Active bHLH/PAS heterodimers consist of a ubiquitous signal-regulated subunit (e.g., hypoxia-inducible factors, HIF-1α/2α/3α; the aryl hydrocarbon receptor, AhR) or tissue-restricted subunit (e.g., NPAS1/3/4, Single Minded 1/2), paired with a general partner protein, aryl hydrocarbon receptor nuclear translocator (Arnt or Arnt2). We have investigated regulation of the neuron-enriched Arnt paralogue, Arnt2. We find high Arnt/Arnt2 ratios in P19 embryonic carcinoma cells and ES cells are dramatically reversed to high Arnt2/Arnt on neuronal differentiation. mRNA half-lives of Arnt and Arnt2 remain similar in both parent and neuronal differentiated cells. The GC-rich Arnt2 promoter, while heavily methylated in Arnt only expressing hepatoma cells, is methylation free in P19 and ES cells, where it is bivalent with respect to active H3K4me3 and repressive H3K27me3 histone marks. Typical of a 'transcription poised' developmental gene, H3K27me3 repressive marks are removed from Arnt2 during neuronal differentiation. Our data are consistent with a switch to predominant Arnt2 expression in neurons to allow specific functions of neuronal bHLH/PAS factors and/or to avoid neuronal bHLH/PAS factors from interfering with AhR/Arnt signalling.

Cancer-like metabolism of the mammalian retina.

The retina, like many cancers, produces energy from glycolysis even in the presence of oxygen. This phenomenon is known as aerobic glycolysis and eponymously as the Warburg effect. In recent years, the Warburg effect has become an explosive area of study within the cancer research community. The expanding knowledge about the molecular mechanisms underpinning the Warburg effect in cancer promises to provide a greater understanding of mammalian retinal metabolism and has motivated cancer researchers to target the Warburg effect as a novel treatment strategy for cancer. However, if the molecular mechanisms underlying the Warburg effect are shared by the retina and cancer, treatments targeting the Warburg effect may have serious adverse effects on retinal metabolism. Herein, we provide an updated understanding of the Warburg effect in mammalian retina.

Metabolic reprogramming of the retinal pigment epithelium by cytokines associated with age-related macular degeneration.

The complex metabolic relationship between the retinal pigment epithelium (RPE) and photoreceptors is essential for maintaining retinal health. Recent evidence indicates the RPE acts as an adjacent lactate sink, suppressing glycolysis in the epithelium in order to maximize glycolysis in the photoreceptors. Dysregulated metabolism within the RPE has been implicated in the pathogenesis of age-related macular degeneration (AMD), a leading cause of vision loss. In the present study, we investigate the effects of four cytokines associated with AMD, TNFα, TGF-ß2, IL-6, and IL-1ß, as well as a cocktail containing all four cytokines, on RPE metabolism using ARPE-19 cells, primary human RPE cells, and ex vivo rat eyecups. Strikingly, we found cytokine-specific changes in numerous metabolic markers including lactate production, glucose consumption, extracellular acidification rate, and oxygen consumption rate accompanied by increases in total mitochondrial volume and ATP production. Together, all four cytokines could potently override the constitutive suppression of glycolysis in the RPE, through a mechanism independent of PI3K/AKT, MEK/ERK, or NF-κB. Finally, we observed changes in glycolytic gene expression with cytokine treatment, including in lactate dehydrogenase subunit and glucose transporter expression. Our findings provide new insights into the metabolic changes in the RPE under inflammatory conditions and highlight potential therapeutic targets for AMD.

Factor inhibiting HIF (FIH) recognizes distinct molecular features within hypoxia-inducible factor-α (HIF-α) versus ankyrin repeat substrates.

Factor Inhibiting HIF (FIH) catalyzes the ß-hydroxylation of asparagine residues in HIF-α transcription factors as well as ankyrin repeat domain (ARD) proteins such as Notch and Gankyrin. Although FIH-mediated hydroxylation of HIF-α is well characterized, ARDs were only recently identified as substrates, and less is known about their recognition and hydroxylation by FIH. We investigated the molecular determinants of FIH substrate recognition, with a focus on differences between HIF and ARD substrates. We show that for ARD proteins, structural context is an important determinant of FIH-recognition, but analyses of chimeric substrate proteins indicate that the ankyrin fold alone is not sufficient to explain the distinct substrate properties of the ARDs compared with HIF. For both substrates the kinetic parameters of hydroxylation are influenced by the amino acids proximal to the target asparagine. Although FIH tolerates a variety of chemically disparate residues proximal to the asparagine, we demonstrate that certain combinations of amino acids are not permissive to hydroxylation. Finally, we characterize a conserved RLL motif in HIF and demonstrate that it mediates a high affinity interaction with FIH in the presence of cell lysate or macromolecular crowding agents. Collectively, our data highlight the importance of residues proximal to the asparagine in determining hydroxylation, and identify additional substrate-specific elements that contribute to distinct properties of HIF and ARD proteins as substrates for FIH. These distinct features are likely to influence FIH substrate choice in vivo and, therefore, have important consequences for HIF regulation.

NanoFIRE: A NanoLuciferase and Fluorescent Integrated Reporter Element for Robust and Sensitive Investigation of HIF and Other Signalling Pathways.

The Hypoxia Inducible Factor (HIF) transcription factors are imperative for cell adaption to low oxygen conditions and development; however, they also contribute to ischaemic disease and cancer. To identify novel genetic regulators which target the HIF pathway or small molecules for therapeutic use, cell-based reporter systems are commonly used. Here, we present a new, highly sensitive and versatile reporter system, NanoFIRE: a NanoLuciferase and Fluorescent Integrated Reporter Element. Under the control of a Hypoxic Response Element (HRE-NanoFIRE), this system is a robust sensor of HIF activity within cells and potently responds to both hypoxia and chemical inducers of the HIF pathway in a highly reproducible and sensitive manner, consistently achieving 20 to 150-fold induction across different cell types and a Z' score > 0.5. We demonstrate that the NanoFIRE system is adaptable via substitution of the response element controlling NanoLuciferase and show that it can report on the activity of the transcriptional regulator Factor Inhibiting HIF, and an unrelated transcription factor, the Progesterone Receptor. Furthermore, the lentivirus-mediated stable integration of NanoFIRE highlights the versatility of this system across a wide range of cell types, including primary cells. Together, these findings demonstrate that NanoFIRE is a robust reporter system for the investigation of HIF and other transcription factor-mediated signalling pathways in cells, with applications in high throughput screening for the identification of novel small molecule and genetic regulators.

Interaction with factor inhibiting HIF-1 defines an additional mode of cross-coupling between the Notch and hypoxia signaling pathways.

Cells adapt to hypoxia by a cellular response, where hypoxia-inducible factor 1alpha (HIF-1alpha) becomes stabilized and directly activates transcription of downstream genes. In addition to this "canonical" response, certain aspects of the pathway require integration with Notch signaling, i.e., HIF-1alpha can interact with the Notch intracellular domain (ICD) to augment the Notch downstream response. In this work, we demonstrate an additional level of complexity in this cross-talk: factor-inhibiting HIF-1 (FIH-1) regulates not only HIF activity, but also the Notch signaling output and, in addition, plays a role in how Notch signaling modulates the hypoxic response. We show that FIH-1 hydroxylates Notch ICD at two residues (N(1945) and N(2012)) that are critical for the function of Notch ICD as a transactivator within cells and during neurogenesis and myogenesis in vivo. FIH-1 negatively regulates Notch activity and accelerates myogenic differentiation. In its modulation of the hypoxic response, Notch ICD enhances recruitment of HIF-1alpha to its target promoters and derepresses HIF-1alpha function. Addition of FIH-1, which has a higher affinity for Notch ICD than for HIF-1alpha, abrogates the derepression, suggesting that Notch ICD sequesters FIH-1 away from HIF-1alpha. In conclusion, the data reveal posttranslational modification of the activated form of the Notch receptor and an intricate mode of cross-coupling between the Notch and hypoxia signaling pathways.

Molecular characterisation of rare loss-of-function NPAS3 and NPAS4 variants identified in individuals with neurodevelopmental disorders.

Aberrations in the excitatory/inhibitory balance within the brain have been associated with both intellectual disability (ID) and schizophrenia (SZ). The bHLH-PAS transcription factors NPAS3 and NPAS4 have been implicated in controlling the excitatory/inhibitory balance, and targeted disruption of either gene in mice results in a phenotype resembling ID and SZ. However, there are few human variants in NPAS3 and none in NPAS4 that have been associated with schizophrenia or neurodevelopmental disorders. From a clinical exome sequencing database we identified three NPAS3 variants and four NPAS4 variants that could potentially disrupt protein function in individuals with either developmental delay or ID. The transcriptional activity of the variants when partnered with either ARNT or ARNT2 was assessed by reporter gene activity and it was found that variants which truncated the NPAS3/4 protein resulted in a complete loss of transcriptional activity. The ability of loss-of-function variants to heterodimerise with neuronally enriched partner protein ARNT2 was then determined by co-immunoprecipitation experiments. It was determined that the mechanism for the observed loss of function was the inability of the truncated NPAS3/4 protein to heterodimerise with ARNT2. This further establishes NPAS3 and NPAS4 as candidate neurodevelopmental disorder genes.

Hypoxia-inducible factor-2 is a novel regulator of aberrant CXCL12 expression in multiple myeloma plasma cells.

BACKGROUND: Multiple myeloma is an incurable malignancy of bone marrow plasma cells. Progression of multiple myeloma is accompanied by an increase in bone marrow angiogenesis. Studies from our laboratory suggest a role for the CXCL12 chemokine in this process, with circulating levels of CXCL12 correlating with bone marrow angiogenesis in patients with multiple myeloma. While the mechanisms responsible for aberrant plasma cell expression of CXCL12 remain to be determined, studies in other systems suggest a role for hypoxia and hypoxia-inducible transcription factors. DESIGN AND METHODS: The expression of hypoxia-inducible factor protein was examined in patients' bone marrow biopsy specimens using immunohistochemistry. The hypoxic regulation of CXCL12 was examined in multiple myeloma plasma cell lines using polymerase chain reaction and western blotting. The role of hypoxia-inducible factors-1 and -2 in the regulation of CXCL12 expression was examined using over-expression and short hairpin RNA knockdown constructs, electrophoretic mobility shift assays and chromatin immunoprecipitation. The contribution of CXCL12 to hypoxia-induced angiogenesis was examined in vivo using a subcutaneous murine model of neovascularization. RESULTS: Strong hypoxia-inducible factor-2 protein expression was detected in CD138(+) multiple myeloma plasma cells in patients' biopsy specimens. Prolonged exposure to hypoxia strongly up-regulated CXCL12 expression in multiple myeloma plasma cells and hypoxia-inducible factor-2 was found to play a key role in this response. Promoter analyses revealed increased hypoxia-inducible factor-2 binding to the CXCL12 promoter under hypoxic conditions. Over-expression of hypoxia-inducible factor in multiple myeloma plasma cells strongly induced in vivo angiogenesis, and administration of a CXCL12 antagonist decreased hypoxia-inducible factor-induced angiogenesis. CONCLUSIONS: Hypoxia-inducible factor-2 is a newly identified regulator of CXCL12 expression in multiple myeloma plasma cells and a major contributor to multiple myeloma plasma cell-induced angiogenesis. Targeting the hypoxic niche, and more specifically hypoxia-inducible factor-2, may represent a viable strategy to inhibit angiogenesis in multiple myeloma and progression of this disease.

When is a target not a target?

Cells rely on prolyl hydroxylase enzymes to sense low levels of oxygen, but they might act on fewer targets than previously thought.