James S. O. McCullagh

Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans

The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.

Single amino acid radiocarbon dating of Upper Paleolithic modern humans

Archaeological bones are usually dated by radiocarbon measurement of extracted collagen. However, low collagen content, contamination from the burial environment, or museum conservation work, such as addition of glues, preservatives, and fumigants to “protect” archaeological materials, have previously led to inaccurate dates. These inaccuracies in turn frustrate the development of archaeological chronologies and, in the Paleolithic, blur the dating of such key events as the dispersal of anatomically modern humans. Here we describe a method to date hydroxyproline found in collagen (∼10% of collagen carbon) as a bone-specific biomarker that removes impurities, thereby improving dating accuracy and confidence. This method is applied to two important sites in Russia and allows us to report the earliest direct ages for the presence of anatomically modern humans on the Russian Plain. These dates contribute considerably to our understanding of the emergence of the Mid-Upper Paleolithic and the complex suite of burial behaviors that begin to appear during this period.

Determination of underivatized amino acid δ13C by liquid chromatography/isotope ratio mass spectrometry for nutritional studies: the effect of dietary non‐essential amino acid profile on the isotopic signature of individual amino acids in fish

This study provides data for the effect of dietary non-essential amino acid composition on the delta(13)C values of individual amino acids in rainbow trout (Oncorhynchus mykiss) using liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS). In this experiment, trout were reared either on a control diet or on three experimental diets, differing in the composition of non-essential/conditionally essential amino acids, for a period of 6 weeks. The control diet was a commercial trout starter feed with fish meal as the main protein source. The experimental diets contained no protein, only synthetic amino acids. Diet 1 resembled the composition of fish meal in both essential and non-essential amino acids, Diet 2 had all essential amino acids, but cysteine, glycine, proline and tyrosine were replaced by the corresponding amounts of their precursors, and in Diet 3 all non-essential amino acids were replaced by glutamate. LC/IRMS was used for the determination of delta(13)C values of individual amino acids from diets and tissues without derivatization. Diet affected the delta(13)C of individual amino acids in fish. For fish on Diets 1-3 amino acid delta(13)C values showed a similar trend: phenylalanine showed very little change from diet to body tissue. Arginine, lysine, tyrosine and proline showed strong depletion from diet to body tissue and glycine, alanine, aspartate and serine all showed variable but strong enrichment in (13)C. Improvements are necessary before all amino acid delta(13)C values can be determined; however, this study demonstrates that measuring amino acid isotopic signatures by LC/IRMS is a promising new technique for nutritional physiologists.

Review: Current applications and challenges for liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS)

High-precision isotope analysis is recognized as an essential research tool in many fields of study. Until recently, continuous flow isotope ratio mass spectrometry (CF-IRMS) was available via an elemental analyzer or a gas chromatography inlet system for compound-specific analysis of light stable isotopes. In 2004, however, an interface that coupled liquid chromatography with IRMS (LC/IRMS) became commercially available for the first time. This brought the capability for new areas of application, in particular enabling compound-specific δ(13)C analysis of non-volatile, aqueous soluble, compounds from complex mixtures. The interface design brought with it several analytical constraints, however, in particular a lack of compatibility with certain types of chromatography as well as limited flow rates and mobile phase compositions. Routine LC/IRMS methods have, however, been established for measuring the δ(13)C isotopic ratios of underivatized individual compounds for application in archeology, nutrition and physiology, geochemistry, hydrology, soil science and food authenticity. Seven years after its introduction, we review the technical advances and constraints, methodological developments and new applications of liquid chromatography coupled to isotope ratio mass spectrometry.

Optimal Translational Termination Requires C4 Lysyl Hydroxylation of eRF1

Summary Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation.

Noninvasive Quantification of 2-Hydroxyglutarate in Human Gliomas with IDH1 and IDH2 Mutations.

Mutations in the isocitrate dehydrogenase genes (IDH1/2) occur often in diffuse gliomas, where they are associated with abnormal accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). Monitoring 2-HG levels could provide prognostic information in this disease, but detection strategies that are noninvasive and sufficiently quantitative have yet to be developed. In this study, we address this need by presenting a proton magnetic resonance spectroscopy ((1)H-MRS) acquisition scheme that uses an ultrahigh magnetic field (≥ 7T) capable of noninvasively detecting 2-HG with quantitative measurements sufficient to differentiate mutant cytosolic IDH1 and mitochondrial IDH2 in human brain tumors. Untargeted metabolomics analysis of in vivo (1)H-MRS spectra discriminated between IDH-mutant tumors and healthy tissue, and separated IDH1 from IDH2 mutations. High-quality spectra enabled the quantification of neurochemical profiles consisting of at least eight metabolites, including 2-HG, glutamate, lactate, and glutathione in both tumor and healthy tissue voxels. Notably, IDH2 mutation produced more 2-HG than IDH1 mutation, consistent with previous findings in cell culture. By offering enhanced sensitivity and specificity, this scheme can quantitatively detect 2-HG and associated metabolites that may accumulate during tumor progression, with implications to better monitor patient responses to therapy.

Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases

Accumulation of (R)-2-hydroxyglutarate in cells results from mutations to isocitrate dehydrogenase that correlate with cancer. A recent study reports that (R)-, but not (S)-2-hydroxyglutarate, acts as a co-substrate for the hypoxia-inducible factor prolyl hydroxylases via enzyme-catalysed oxidation to 2-oxoglutarate. Here we investigate the mechanism of 2-hydroxyglutarate-enabled activation of 2-oxoglutarate oxygenases, including prolyl hydroxylase domain 2, the most important human prolyl hydroxylase isoform. We observe that 2-hydroxyglutarate-enabled catalysis by prolyl hydroxylase domain 2 is not enantiomer-specific and is stimulated by ferrous/ferric ion and reducing agents including L-ascorbate. The results reveal that 2-hydroxyglutarate is oxidized to 2-oxoglutarate non-enzymatically, likely via iron-mediated Fenton-chemistry, at levels supporting in vitro catalysis by 2-oxoglutarate oxygenases. Succinic semialdehyde and succinate are also identified as products of 2-hydroxyglutarate oxidation. Overall, the results rationalize the reported effects of 2-hydroxyglutarate on catalysis by prolyl hydroxylases in vitro and suggest that non-enzymatic 2-hydroxyglutarate oxidation may be of biological interest.

The 2-oxoglutarate-dependent oxygenase JMJD6 catalyses oxidation of lysine residues to give 5S-hydroxylysine residues.

Amino acid analyses reveal that JMJD6-catalysed hydroxylation of RNA-splicing regulatory protein fragments occurs to give hydroxylysine products with 5S stereochemistry. This contrasts with collagen lysyl hydroxylases, which give 5R-hydroxylated products. The work suggests that more than one subfamily of lysyl hydroxylases has evolved and illustrates the importance of stereochemical assignments in proteomic analyses.

Investigations on the Oxygen Dependence of a 2-Oxoglutarate Histone Demethylase

Histone N(ϵ)-methyl lysine demethylases are important in epigenetic regulation. KDM4E (histone lysine demethylase 4E) is a representative member of the large Fe(II)/2-oxoglutarate- dependent family of human histone demethylases. In the present study we report kinetic studies on the reaction of KDM4E with O2. Steady-state assays showed that KDM4E has a graded response to O2 over a physiologically relevant range of O2 concentrations. Pre-steady state assays implied that KDM4E reacts slowly with O2 and that there are variations in the reaction kinetics which are dependent on the methylation status of the substrate. The results demonstrate the potential for histone demethylase activity to be regulated by oxygen availability.

RADIOCARBON DATING OF INDIVIDUAL AMINO ACIDS FROM ARCHAEOLOGICAL BONE COLLAGEN

Since the development of accelerator mass spectrometry (AMS) for radiocarbon dating in the late 1970s, its ability to date small samples of bone has been of huge importance in archaeology and Quaternary paleoecology. The conventional approach to sample preparation has been to extract and gelatinize protein, which is then combusted and graphitized for analysis. However, this ?bulk protein? can contain a heterogeneous mixture of non-collagenous molecules, including humic acids and other soil components that may be of a different age than the bone and therefore affect the accuracy of its 14C date. Sample pretreatment methods have been an important area of development in recent years but still show inadequacies for the dating of severely contaminated bone. The idea of isolating and dating individual compounds such as single amino acids, to improve dating accuracy, has been discussed in the literature since the 1960s. Hydroxyproline, for example, makes up over 10% of bone collagen but is extremely rare in most other animal proteins, increasing the chances of its presence being endogenous to the individual being dated. Its successful isolation has therefore been considered a potential ?gold standard? for dating archaeological bone; however, extracting and suitably purifying single amino acids from bone has proved a challenging task. This paper presents a novel method for the compound-specific 14C dating of individual amino acids, including hydroxyproline, from archaeological bone protein. It is based on a preparative, mixed-mode liquid chromatography separation of underivatized amino acids, entirely in aqueous solution and free of organic solvents. The method is presented here in detail including application to standard bone samples establishing its accuracy and background carbon contribution. Results from 14C dating hydroxyproline and other individual amino acids, from both historical and archaeological bone, are shown to provide AMS dates that are statistically indistinguishable from those of the bulk protein.