Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation

Significance The Fe(II)- and 2-oxoglutarate (2OG)-dependent hypoxia-inducible transcription factor prolyl-hydroxylases play a central role in human oxygen sensing and are related to other prolyl-hydroxylases involved in eukaryotic collagen biosynthesis and ribosomal modification. The finding that a PHD-related prolyl-hydroxylase in Pseudomonas spp. regulates pyocyanin biosynthesis supports prokaryotic origins for the eukaryotic prolyl-hydroxylases. The identification of the switch I loop of elongation factor Tu (EF-Tu) as a Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) substrate provides evidence of roles for 2OG oxygenases in both translational and transcriptional regulation. A structure of the PPHD:EF-Tu complex, the first to the authors' knowledge of a 2OG oxygenase with its intact protein substrate, reveals that major conformational changes occur in both PPHD and EF-Tu and will be useful in the design of new prolyl-hydroxylase inhibitors. </jats:p

Structure of human RNA N(6)-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation

ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N6-methyladenine in RNA. ALKBH5 is upregulated under hypoxia and plays a role in spermatogenesis. We describe a crystal structure of human ALKBH5 (residues 66–292) to 2.0 Å resolution. ALKBH566–292 has a double-stranded β-helix core fold as observed in other 2OG and iron-dependent oxygenase family members. The active site metal is octahedrally coordinated by an HXD…H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other NAOXs. A large loop (βIV–V) in ALKBH5 occupies a similar region as the L1 loop of the fat mass and obesity-associated protein that is proposed to confer single-stranded RNA selectivity. Unexpectedly, a small molecule inhibitor, IOX3, was observed covalently attached to the side chain of Cys200 located outside of the active site. Modelling substrate into the active site based on other NAOX–nucleic acid complexes reveals conserved residues important for recognition and demethylation mechanisms. The structural insights will aid in the development of inhibitors selective for NAOXs, for use as functional probes and for therapeutic benefit

Structure of the ribosomal oxygenase OGFOD1 provides insights into the regio- and stereoselectivity of prolyl hydroxylases

Post-translational ribosomal protein hydroxylation is catalyzed by 2-oxoglutarate (2OG) and ferrous iron dependent oxygenases, and occurs in prokaryotes and eukaryotes. OGFOD1 catalyzes trans-3 prolyl hydroxylation at Pro62 of the small ribosomal subunit protein uS12 (RPS23) and is conserved from yeasts to humans. We describe crystal structures of the human uS12 prolyl 3-hydroxylase (OGFOD1) and its homolog from Saccharomyces cerevisiae (Tpa1p): OGFOD1 in complex with the broad-spectrum 2OG oxygenase inhibitors; N-oxalylglycine (NOG) and pyridine-2,4-dicarboxylate (2,4-PDCA) to 2.1 and 2.6 Å resolution, respectively; and Tpa1p in complex with NOG, 2,4-PDCA, and 1-chloro-4-hydroxyisoquinoline-3-carbonylglycine (a more selective prolyl hydroxylase inhibitor) to 2.8, 1.9, and 1.9 Å resolution, respectively. Comparison of uS12 hydroxylase structures with those of other prolyl hydroxylases, including the human hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs), reveals differences between the prolyl 3- and prolyl 4-hydroxylase active sites, which can be exploited for developing selective inhibitors of the different subfamilies

Uma experiência de tradução do conto “Marcha Fúnebre” de Machado de Assis para o japonês

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Pharmacological Inhibition of FTO

<div><p>In 2007, a genome wide association study identified a SNP in intron one of the gene encoding human FTO that was associated with increased body mass index. Homozygous risk allele carriers are on average three kg heavier than those homozygous for the protective allele. FTO is a DNA/RNA demethylase, however, how this function affects body weight, if at all, is unknown. Here we aimed to pharmacologically inhibit FTO to examine the effect of its demethylase function <i>in vitro</i> and <i>in vivo</i> as a first step in evaluating the therapeutic potential of FTO. We showed that IOX3, a known inhibitor of the HIF prolyl hydroxylases, decreased protein expression of FTO (in C2C12 cells) and reduced maximal respiration rate <i>in vitro</i>. However, FTO protein levels were not significantly altered by treatment of mice with IOX3 at 60 mg/kg every two days. This treatment did not affect body weight, or RER, but did significantly reduce bone mineral density and content and alter adipose tissue distribution. Future compounds designed to selectively inhibit FTO’s demethylase activity could be therapeutically useful for the treatment of obesity.</p></div

Oxygen Consumption Rate (OCR), Extracellular Acidification Rate (ECAR) of C2C12, and wild-type and FTO knockout MEFs treated with 1 μM IOX3 or an equivalent amount of vehicle control for 16 hours.

<p>A) OCR and, B) basal ECAR measured in C2C12 cells treated with vehicle (n = 10) and 1 μM IOX3 (n = 10) at baseline and after Oligomycin, FCCP and Rotenone treatment, data normalised to live stain. C) OCR and, D] ECAR measured in <i>Fto</i>^<i>+/+</i> and <i>Fto</i>^<i>-/-</i> MEFs cells treated with vehicle and 1 μM IOX3 (n = 5 per group) at baseline and after Oligomycin, FCCP and Rotenone treatment, data normalised to live stain. Data were analysed using a 2 way ANOVA with Bonferroni post-hoc test. Data is of readings following each compound injection and are expressed as mean ± SEM. E) Expression of FTO, phosphorylated-AMPKα and HIF-1α with representative ACTIN in cells treated with vehicle, 1uM IOX3, control scrambled siRNA or <i>Fto</i> siRNA for 24 hours. N = 3 biological replicates per condition.</p

DEXA and organ weights of vehicle and 60 mg/kg every two days IOX3-treated mice.

<p>A) Bone Mineral Density (BMD), B) Bone Mineral Content (BMC), C) Liver mass, D) Epigonadal white adipose tissue (WAT), E) Abdominal WAT, F) Peri-renal WAT, G) Brown adipose tissue (BAT), H) Calf muscle weight of vehicle (n = 20) and 60 mg/kg every two days IOX3 (n = 20) treated mice. Data analysed by student’s t-test *P<0.05, **P<0.01, ***P<0.001. Data are expressed as mean ± SEM and individual data points are shown.</p

Chemical structure of IOX3 and IC₅₀ values for FTO and PHD2.

<p>Chemical structure of IOX3 and IC₅₀ values for FTO and PHD2.</p

Plasma EPO levels in vehicle and 60 mg/kg/2days IOX3 treated mice and the effect on FTO protein levels.

<p>A) Plasma EPO levels after one week of dosing in vehicle (n = 20) and 60 mg/kg every two days IOX3 (n = 20) treated mice. B) Terminal plasma EPO levels at 40 days after beginning of the trial, vehicle (n = 20) and 60 mg/kg every two days IOX3 (n = 20) treated mice. C) Weekly body weight of Vehicle (n = 20) and 60 mg/kg every two days IOX3 (n = 20). D) Area under the curve calculated from blood glucose curves. Data were analysed using a student’s t-test and time course data were analysed by 2-way ANOVA with Bonferroni post-hoc test *P<0.05, **P<0.01, ***P<0.001. Data are expressed as mean ± SEM.</p