Proteomics-based identification of novel factor inhibiting hypoxia-inducible factor (FIH) substrates indicates widespread asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.

Post-translational hydroxylation has been considered an unusual modification on intracellular proteins. However, following the recognition that oxygen-sensitive prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor hypoxia-inducible factor (HIF), interest has centered on the possibility that these enzymes may have other substrates in the proteome. In support of this certain ankyrin repeat domain (ARD)-containing proteins, including members of the IkappaB and Notch families, have been identified as alternative substrates of the HIF asparaginyl hydroxylase factor inhibiting HIF (FIH). Although these findings imply a potentially broad range of substrates for FIH, the precise extent of this range has been difficult to determine because of the difficulty of capturing transient enzyme-substrate interactions. Here we describe the use of pharmacological "substrate trapping" together with stable isotope labeling by amino acids in cell culture (SILAC) technology to stabilize and identify potential FIH-substrate interactions by mass spectrometry. To pursue these potential FIH substrates we used conventional data-directed tandem MS together with alternating low/high collision energy tandem MS to assign and quantitate hydroxylation at target asparaginyl residues. Overall the work has defined 13 new FIH-dependent hydroxylation sites with a degenerate consensus corresponding to that of the ankyrin repeat and a range of ARD-containing proteins as actual and potential substrates for FIH. Several ARD-containing proteins were multiply hydroxylated, and detailed studies of one, Tankyrase-2, revealed eight sites that were differentially sensitive to FIH-catalyzed hydroxylation. These findings indicate that asparaginyl hydroxylation is likely to be widespread among the approximately 300 ARD-containing species in the human proteome.

Hypoxia, hypoxia-inducible factors (HIF), HIF hydroxylases and oxygen sensing.

This article outlines the need for a homeostatic response to alterations in cellular oxygenation. It describes work on erythropoietin control that led to the discovery of the hypoxia-inducible transcription factor (HIF-1) and the parallel recognition that this system was responsive to a widespread oxygen-sensing mechanism. Subsequently, multiple HIF isoforms have been shown to have overlapping but non-redundant functions, controlling expression of genes involved in diverse processes such as angiogenesis, vascular tone, metal transport, glycolysis, mitochondrial function, cell growth and survival. The major role of prolyl and asparaginyl hydroxylation in regulating HIFs is described, as well as the identification of PHD1-3 and FIH as the oxygen-sensing enzymes responsible for these hydroxylations. Current understanding of other processes that modulate overall HIF activity, including influences from other signalling mechanisms such as kinases and nitric oxide levels, and the existence of a variety of feedback loops are outlined. The effects of some mutations in this pathway are documented as is knowledge of other substrates for these enzymes. The importance of PHD1-3 and FIH, and the large family of 2-oxoglutarate and iron(II)-dependent dioxygenases of which they are a part, in biology and medicine are discussed.

MYPT1, the targeting subunit of smooth-muscle myosin phosphatase, is a substrate for the asparaginyl hydroxylase factor inhibiting hypoxia-inducible factor (FIH).

The asparaginyl hydroxylase FIH [factor inhibiting HIF (hypoxia-inducible factor)] was first identified as a protein that inhibits transcriptional activation by HIF, through hydroxylation of an asparagine residue in the CAD (C-terminal activation domain). More recently, several ARD [AR (ankyrin repeat) domain]-containing proteins were identified as FIH substrates using FIH interaction assays. Although the function(s) of these ARD hydroxylations is unclear, expression of the ARD protein Notch1 was shown to compete efficiently with HIF CAD for asparagine hydroxylation and thus to enhance HIF activity. The ARD is a common protein domain with over 300 examples in the human proteome. However, the extent of hydroxylation among ARD proteins, and the ability of other members to compete with HIF-CAD for FIH, is not known. In the present study we assay for asparagine hydroxylation in a bioinformatically predicted FIH substrate, the targeting subunit of myosin phosphatase, MYPT1. Our results confirm hydroxylation both in cultured cells and in endogenous protein purified from animal tissue. We show that the extent of hydroxylation at three sites is dependent on FIH expression level and that hydroxylation is incomplete under basal conditions even in the animal tissue. We also show that expression of MYPT1 enhances HIF-CAD activity in a manner consistent with competition for FIH and that this property extends to other ARD proteins. These results extend the range of FIH substrates and suggest that cross-competition between ARDs and HIF-CAD, and between ARDs themselves, may be extensive and have important effects on hypoxia signalling.

Autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity.

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Continuous, real-time bioimaging of chemical bioavailability and toxicology using autonomously bioluminescent human cell lines.

Bioluminescent imaging is an emerging biomedical surveillance strategy that uses external cameras to detect in vivo light generated in small animal models of human physiology or in vitro light generated in tissue culture or tissue scaffold mimics of human anatomy. The most widely utilized of reporters is the firefly luciferase (luc) gene; however, it generates light only upon addition of a chemical substrate, thus only generating intermittent single time point data snapshots. To overcome this disadvantage, we have demonstrated substrate-independent bioluminescent imaging using an optimized bacterial bioluminescence (lux) system. The lux reporter produces bioluminescence autonomously using components found naturally within the cell, thereby allowing imaging to occur continuously and in real-time over the lifetime of the host. We have validated this technology in human cells with demonstrated chemical toxicological profiling against exotoxin exposures at signal strengths comparable to existing luc systems (~1.33 × 107 photons/second). As a proof-in-principle demonstration, we have engineered breast carcinoma cells to express bioluminescence for real-time screening of endocrine disrupting chemicals and validated detection of 17ß-estradiol (EC50 = ~ 10 pM). These and other applications of this new reporter technology will be discussed as potential new pathways towards improved models of target chemical bioavailability, toxicology, efficacy, and human safety.

Novel insights into the molecular origins and treatment of lung cancer.

Lung cancer is the most common and most deadly cancer worldwide. Because of the aggressive and metastatic nature of many forms of the disease, it is frequently diagnosed late and responds poorly to the therapies currently available. Although our understanding of the molecular origins and evolution of lung cancer is still incomplete, recent research has yielded several developments that may offer opportunities for new, targeted and effective therapy. In this review we first discuss the prevalence and origins of lung cancer, with emphasis on non-small-cell lung cancer and adenocarcinoma, together with current treatments and their efficacy. We then look at a selection of recent papers which between them shed new light on possible therapeutic opportunities, including a novel synthetic interaction with the Kras gene and genomic or proteomic profiling studies that may pave the way for personalized treatment for lung cancer based on specific "signatures" of protein and gene expression. Lung cancer remains the foremost cause of cancer deaths worldwide. Despite advances in both detection and treatment, diagnosis is often late and the prognosis for patients poor. Our understanding of the molecular basis and progression of lung cancer remains incomplete, hampering the design and development of more effective diagnostic tools and therapies for this devastating disease. However, the last twelve months have witnessed the publication of several studies that represent significant advances in our knowledge of lung cancer, and may represent important steps on the road to effective new therapies. In this review we aim to summarize these recent developments, and give our perspectives on the therapeutic possibilities they may offer in the future.

FIH-dependent asparaginyl hydroxylation of ankyrin repeat domain-containing proteins.

Studies on hypoxia-sensitive pathways have identified a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The asparaginyl hydroxylase factor inhibiting HIF (FIH) targets a conserved asparaginyl residue in the C-terminal transactivation domain of HIF-alpha. This modification suppresses HIF transcriptional activity by inhibiting co-activator recruitment. Recent work has demonstrated that FIH targets an alternative class of substrate. Proteins containing a common interaction motif known as the ankyrin repeat domain (ARD) have been shown to be efficiently hydroxylated by FIH. This review aims to summarize what is currently known regarding ARD hydroxylation, including the kinetics and determinants of FIH-mediated ARD hydroxylation, the structural and functional consequences of ARD hydroxylation, and the potential for cross-talk between ARD proteins and HIF signaling.