5-Hydroxymethylcytosine (hmC) was recently detected as the sixth base in mammalian tissue at so far controversial levels. The function of the modified base is currently unknown, but it is certain ...that the base is generated from 5-methylcytosine (mC). This fuels the hypothesis that it represents an intermediate of an active demethylation process, which could involve further oxidation of the hydroxymethyl group to a formyl or carboxyl group followed by either deformylation or decarboxylation. Here, we use an ultra-sensitive and accurate isotope based LC-MS method to precisely determine the levels of hmC in various mouse tissues and we searched for 5-formylcytosine (fC), 5-carboxylcytosine (caC), and 5-hydroxymethyluracil (hmU) as putative active demethylation intermediates. Our data suggest that an active oxidative mC demethylation pathway is unlikely to occur. Additionally, we show using HPLC-MS analysis and immunohistochemistry that hmC is present in all tissues and cell types with highest concentrations in neuronal cells of the CNS.
Mind over matter: LC‐MS has allowed the amount of the post‐replicatively formed DNA base 5‐hydroxymethylcytosine (see picture; left) to be quantified in brain tissue. The nucleoside is most abundant ...in areas that are associated with higher cognitive functions, and its content in mouse hippocampi seems to increase with age. The new method enables hydroxymethylcytosine to be quantified with unprecedented accuracy.
The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report ...cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.
Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the ...molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing.
5-Hydroxymethylcytosine (5-HOMedC) was recently discovered as the sixth base in the mammalian genome. The development of a new phosphoramidite building block is reported, which allows efficient ...synthesis of 5-HOMedC containing DNA. Key steps of the synthesis are a palladium-catalyzed formylation and the simultaneous protection of a hydroxyl and amino group as a cyclic carbamate. DNA synthesis is possible under standard conditions, and deprotection can be carried out with dilute NaOH.
5‐Formylcytosine (fC or 5‐CHOdC) and 5‐carboxylcytosine (caC or 5‐COOHdC) have recently been identified as constituents of mammalian DNA. The nucleosides are formed from 5‐methylcytosine (mC or ...5‐MedC) via 5‐hydroxymethylcytosine (hmC or 5‐HOMedC) and are possible intermediates of an active DNA demethylation process. Here we show efficient syntheses of phosphoramidites which enable the synthesis of DNA strands containing these cytosine modifications based on Pd0‐catalyzed functionalization of 5‐iododeoxycytidine. The first crystal structure of fC reveals the existence of an intramolecular H‐bond between the exocyclic amine and the formyl group, which controls the conformation of the formyl substituent. Using a newly designed in vitro mutagenicity assay we show that fC and caC are only marginally mutagenic, which is a prerequisite for the bases to function as epigenetic control units.
Demethylation: 5‐Hydroxymethylcytosine, 5‐formylcytosine and 5‐carboxylcytosine were efficiently incorporated into DNA. The strands were used to determine the mutagenic properties of the new DNA bases. None of the bases is severely mutagenic, making them perfectly suited nucleosides for epigenetic regulation.
AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses ...hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1-3, 2-4, 5-6 disulfide bonding pattern; an unexpected Cys3-4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.
5‐Hydroxymethylcytosine (hmC) was recently discovered as a new constituent of mammalian DNA. Besides 5‐methylcytosine (mC), it is the only other modified base in higher organisms. The discovery is of ...enormous importance because it shows that the methylation of cytosines to imprint epigenetic information is not a final chemical step that leads to gene silencing but that further chemistry occurs at the methyl group that might have regulatory function. Recent progress in hmC detection—most notably LC‐MS and glucosyltransferase assays—helped to decipher the precise distribution of hmC in the body. This led to the surprising finding that, in contrast to constant mC levels, the hmC levels are strongly tissue‐specific. The highest values of hmC are found in the central nervous system. It was furthermore discovered that hmC is involved in regulating the pluripotency of stem cells and that it is connected to the processes of cellular development and carcinogenesis. Evidence is currently accumulating that hmC may not exclusively be an intermediate of an active demethylation process, but that it functions instead as an important epigenetic marker.
The sixth sense: 5‐Hydroxymethylcytosine (hmC) was recently discovered as a new constituent of mammalian DNA, and besides 5‐methylcytosine it is the only other modified base in higher organisms. This discovery shows that methylation of cyctosine is not a final step in gene silencing, but that further functionalization of the methyl group occurs.
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.
Display omitted
•Jmjd4 hydroxylates translational termination factor eRF1•The C4 lysyl hydroxylase activity of Jmjd4 is unprecedented in animals•Hydroxylation occurs within the eRF1 stop codon recognition domain•Inhibiting eRF1 K63 hydroxylation promotes stop codon readthrough
Accurate termination of protein synthesis at a stop codon is essential in order to maintain protein sequence fidelity. Feng et al. show that translational termination is optimized by lysyl hydroxylation of the eukaryotic release factor eRF1 by Jmjd4, a 2-oxoglutarate- and Fe(II)-dependent oxygenase.
Here, we describe molecular engineering of monovalent ultra-long acting two-chain insulin-Fc conjugates. Insulin-Fc conjugates were synthesized using trifunctional linkers with one amino reactive ...group for reaction with a lysine residue of insulin and two thiol reactive groups used for re-bridging of a disulfide bond within the Fc molecule. The ultra-long pharmacokinetic profile of the insulin-Fc conjugates was the result of concertedly slowing insulin receptor-mediated clearance by (1) introduction of amino acid substitutions that lowered the insulin receptor affinity and (2) conjugating insulin to the Fc element. Fc conjugation leads to recycling by the neonatal Fc receptor and increase in the molecular size, both contributing to the ultra-long pharmacokinetic and pharmacodynamic profiles.
PDF
