Search

YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular proliferation (Ge et al., Nat Chem Biol 12:960-962, 2012). Bioinformatic analysis identified a potential homologue of ycfD in the thermophilic bacterium Rhodothermus marinus (ycfDRM). We describe studies on the characterization of ycfDRM, which is a functional 2OG oxygenase catalysing (2S,3R)-hydroxylation of the ribosomal protein uL16 at R82, and which is active at significantly higher temperatures than previously reported for any other 2OG oxygenase. Recombinant ycfDRM manifests high thermostability (Tm 84 °C) and activity at higher temperatures (Topt 55 °C) than ycfDEC (Tm 50.6 °C, Topt 40 °C). Mass spectrometric studies on purified R. marinus ribosomal proteins demonstrate a temperature-dependent variation in uL16 hydroxylation. Kinetic studies of oxygen dependence suggest that dioxygen availability can be a limiting factor for ycfDRM catalysis at high temperatures, consistent with incomplete uL16 hydroxylation observed in R. marinus cells. Overall, the results that extend the known range of ribosomal hydroxylation, reveal the potential for ycfD-catalysed hydroxylation to be regulated by temperature/dioxygen availability, and that thermophilic 2OG oxygenases are of interest from a biocatalytic perspective. [ABSTRACT FROM AUTHOR]

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N?-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases. [ABSTRACT FROM AUTHOR]

The mechanisms by which gene expression is regulated by oxygen are of considerable interest from basic science and therapeutic perspectives. Using mass spectrometric analyses of Saccharomyces cerevisiae ribosomes, we found that the amino acid residue in closest proximity to the decoding center, Pro-64 of the 40S subunit ribosomal protein Rps23p (RPS23 Pro-62 in humans) undergoes posttranslational hydroxylation. We identify RPS23 hydroxylases as a highly conserved eukaryotic subfamily of Fe(II) and 2-oxoglutarate dependent oxygenases; their catalytic domain is closely related to transcription factor prolyl trans-4-hydroxylases that act as oxygen sensors in the hypoxic response in animals. The RPS23 hydroxylases in S. cerevisiae (Tpa1p), Schizosaccharomyces pombe and green algae catalyze an unprecedented dihydroxylation modification. This observation contrasts with higher eukaryotes, where RPS23 is monohydroxylated; the human Tpa1p homolog OGFOD1 catalyzes prolyl trans-3-hydroxylation. TPA1 deletion modulates termination efficiency up to ∼10-fold, including of pathophysiologically relevant sequences; we reveal Rps23p hydroxylation as its molecular basis. In contrast to most previously characterized accuracy modulators, including antibiotics and the prion state of the S. cerevisiae translation termination factor eRF3, Rps23p hydroxylation can either increase or decrease translational accuracy in a stop codon context-dependent manner. We identify conditions where Rps23p hydroxylation status determines viability as a consequence of nonsense codon suppression. The results reveal a direct link between oxygenase catalysis and the regulation of gene expression at the translational level. They will also aid in the development of small molecules altering translational accuracy for the treatment of genetic diseases linked to nonsense mutations. [ABSTRACT FROM AUTHOR]

The recent clinical success of messenger RNA (mRNA) technology in managing the Covid pandemic has triggered an unprecedented innovation in production and analytical technologies for this therapeutic modality. mRNA is produced by enzymatic transcription of plasmid DNA (pDNA) using polymerase in a cell-free process of in vitro transcription. After transcription, the pDNA is considered a process-related impurity and is removed from the mRNA product enzymatically, chromatographically, or by precipitation. Regulatory requirements are currently set at 10 ng of template pDNA per single human dose, which typically ranges between 30 and 100 µg. Here, we report the development of a generic procedure based on enzymatic digestion and chromatographic separation for the determination of residual pDNA in mRNA samples, with a limit of quantification of 2.3 ng and a limit of detection of less than 0.1 ng. The procedure is based on enzymatic degradation of mRNA and anion exchange HPLC separation, followed by quantification of residual pDNA with a chromatographic method that is already widely adopted for pDNA quality analytics. The procedure has been successfully applied for in-process monitoring of three model mRNAs and a self-amplifying RNA (saRNA) and can be considered a generic substitution for qPCR in mRNA in-process control analytical strategy, with added benefits that it is more cost-efficient, faster, and sequence agnostic. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) is becoming an increasingly important therapeutic modality due to its potential for fast development and platform production. New emerging RNA modalities, such as circular RNA, drive the need for the development of non-affinity purification approaches. Recently, the highly efficient chromatographic purification of mRNA was demonstrated with multimodal monolithic chromatography media (CIM® PrimaS), where efficient mRNA elution was achieved with an ascending pH gradient approach at pH 10.5. Here, we report that a newly developed chromatographic material enables the elution of mRNA at neutral pH and room temperature. This material demonstrates weak anion-exchanging properties and an isoelectric point of 5.3. It enables the baseline separation of mRNA (at least up to 10,000 nucleotides (nt) in size) from parental plasmid DNA (regardless of isoform composition) with both a NaCl gradient and ascending pH gradient approach, while mRNA elution is achieved in a pH range of 5–7. In addition, the basic structure of the novel material is a chromatographic monolith, enabling convection-assisted mass transfer of large RNA molecules to and from the active surface. This facilitates the elution of mRNA in 3–7 column volumes with more than 80% elution recovery and uncompromised integrity. This is demonstrated by the purification of a model mRNA (size 995 nt) from an in vitro transcription reaction mixture. The purified mRNA is stable for at least 34 days, stored in purified H2O at room temperature. [ABSTRACT FROM AUTHOR]

The COVID‐19 pandemic triggered an unprecedented surge in development of mRNA‐based vaccines. Despite the need to increase process productivity and thus decrease the cost of mRNA vaccines, limited scientific literature is available on strategies to increase the yield of in vitro transcription (IVT) reaction, the unit operation with highest cost of goods, which has traditionally been performed as a batch reaction. Single‐use bioreactors are traditionally used for cell‐based production of biopharmaceuticals, but some core functionalities, such as controlled and automated feed addition, are potentially useful for cell‐free mRNA processes. We report the production of 2 g mRNA in an Ambr® 250 Modular bioreactor system with a starting volume of 100 mL, reaching a maximum mRNA concentration of 12 g L−1 by a fed‐batch IVT approach, and demonstrate the feasibility of continuous fed‐batch production, paving the way towards continuous manufacturing of mRNA. [ABSTRACT FROM AUTHOR]

2-Oxoglutarate (2OG) and ferrous iron dependent oxygenases are involved in many biological processes in organisms ranging from humans (where some are therapeutic targets) to plants. These enzymes are of significant biomedicinal interest because of their roles in hypoxic signaling and epigenetic regulation. Synthetic N -oxalylglycine (NOG) has been identified as a broad-spectrum 2OG oxygenase inhibitor and is currently widely used in studies on the hypoxic response and chromatin modifications in animals. We report the identification of NOG as a natural product present in Rheum rhabarbarum (rhubarb) and Spinach oleracea (spinach) leaves; NOG was not observed in Escherchia coli or human embryonic kidney cells (HEK 293T). The finding presents the possibility that NOG plays a natural role in regulating gene expression by inhibiting 2OG dependent oxygenases. This has significance because tricarboxylic acid cycle (TCA) intermediate inhibition of 2OG dependent oxygenases has attracted major interest in cancer research. [ABSTRACT FROM AUTHOR]

• Oligo-dA binding assay was developed for characterization of monolith OdT columns. • Coupling ≥ 2.5 probes of 18-mer OdT per 100 nm2 is optimal for mRNA chromatography. • Increased mRNA residence time is needed to utilize the full capacity of OdT column. • Up to 5 mg/mL mRNA dynamic binding capacity was achieved at 1 min residence time. • Oligo-dT monolith column enables efficient gram-scale mRNA purification. Oligo-deoxythymidilic acid (OdT) probes conjugated to solid-phase supports exhibit high affinity for poly-adenylated mRNA (target) through high-affinity base-pairing interactions. Here we report the development of a OdT-functionalized chromatographic monolith for purification of polyadenylated mRNA and development of purification methods to support large-scale purification of mRNA-based therapeutics. We report the development of a chromatographic assay based on a synthetic oligo-deoxyadenylic acid chain of 10 or 20 nucleotides (OdA10 and OdA20) as a surrogate for polyadenylated mRNA, which was used for optimization of the OdT affinity column (i.e. the amount and structure of OdT immobilized and monolithic channel size). OdA hybridization to OdT monoliths correlated well with the amount of immobilized OdT, while an in-depth analysis revealed that hybridization yield decreased with increasing size of the target, temperature and probe surface coverage. OdA hybridization kinetics was flow rate-independent, confirming convection-based mass transport within the monolith's channels. The demonstrated steep adsorption isotherms enable chromatographic capture of even highly diluted OdA-containing molecules. Dynamic binding capacity for model mRNA was independent of OdT length and amount of immobilized OdT probes above a critical threshold but was highly influenced by the composition of the binding buffer and mRNA residence time. We demonstrated the scalability of the mRNA purification process with OdT monoliths from 0.1 mL to at least 800 mL bed volume, paving the way for manufacturing processes on OdT monoliths with 40 L bed volume. [ABSTRACT FROM AUTHOR]

Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1 mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. [ABSTRACT FROM AUTHOR]

Protein–protein interactions between the hypoxia inducible factor (HIF) and the transcriptional coactivators p300/CBP are potential cancer targets due to their role in the hypoxic response. A natural product based screen led to the identification of indandione and benzoquinone derivatives that reduce the tight interaction between a HIF-1α fragment and the CH1 domain of p300. The indandione derivatives were shown to fragment to give ninhydrin, which was identified as the active species. Both the naphthoquinones and ninhydrin were observed to induce Zn(II) ejection from p300 and the catalytic domain of the histone demethylase KDM4A. Together with previous reports on the effects of related compounds on HIF-1α and other systems, the results suggest that care should be taken in interpreting biological results obtained with highly electrophilic/thiol modifying compounds. [ABSTRACT FROM AUTHOR]

2-Oxoglutarate (2OG) and Fe(II)-dependent oxygenase domaincontaining protein 1 (OGFOD1) is predicted to be a conserved 2OG oxygenase, the catalytic domain of which is related to hypoxia- inducible factor prolyl hydroxylases. OGFOD1 homologs in yeast are implicated in diverse cellular functions ranging from oxygen-dependent regulation of sterol response genes (Ofd1, Schizosaccharomyces pombe) to translation termination/mRNA polyadenylation (Tpa1p, Saccharomyces cerevisiae). However, neither the biochemical activity of OGFOD1 nor the identity of its substrate has been defined. Here we show that OGFOD1 is a prolyl hydroxylase that catalyzes the posttranslational hydroxylation of a highly conserved residue (Pro-62) in the small ribosomal protein S23 (RPS23). Unusually OGFOD1 retained a high affinity for, and forms a stable complex with, the hydroxylated RPS23 substrate. Knockdown or inactivation of OGFOD1 caused a cell type-dependent induction of stress granules, translational arrest, and growth impairment in a manner complemented by wild-type but not inactive OGFOD1. The work identifies a human prolyl hydroxylase with a role in translational regulation. [ABSTRACT FROM AUTHOR]

Genome sequences predict the presence of many 2-oxoglutarate (2OG)-dependent oxygenases of unknown biochemical and biological functions in Drosophila. Ribosomal protein hydroxylation is emerging as an important 2OG oxygenase catalyzed pathway, but its biological functions are unclear. We report investigations on the function of Sudestada1 (Sud1), a Drosophila ribosomal oxygenase. As with its human and yeast homologs, OGFOD1 and Tpa1p, respectively, we identified Sud1 to catalyze prolyl-hydroxylation of the small ribosomal subunit protein RPS23. Like OGFOD1, Sud1 catalyzes a single prolyl-hydroxylation of RPS23 in contrast to yeast Tpa1p, where Pro-64 dihydroxylation is observed. RNAi-mediated Sud1 knockdown hinders normal growth in different Drosophila tissues. Growth impairment originates from both reduction of cell size and diminution of the number of cells and correlates with impaired translation efficiency and activation of the unfolded protein response in the endoplasmic reticulum. This is accompanied by phosphorylation of eIF2a and concomitant formation of stress granules, as well as promotion of autophagy and apoptosis. These observations, together with those on enzyme homologs described in the companion articles, reveal conserved biochemical and biological roles for a widely distributed ribosomal oxygenase. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]