HBO1 is required for the maintenance of leukaemia stem cells.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings

A double-emulsion microfluidic platform for in vitro green fluorescent protein expression

Microfluidic droplet technology has gained popularity due to the advantages over conventional emulsion techniques and capabilities for a wide range of applications. In this paper, the development of a simple microfluidic-based double-emulsion system is reported. Such a system could be potentially used for in vitro protein synthesis. The system involves a two-step process to make water-in-oil-in-water (W/O/W) emulsions. A PMMA microchip is used for the formation of water-in-oil (W/O) single-emulsion droplets. Then, the single-emulsion droplets are transported to a PDMS/glass microchip to make the W/O/W double-emulsion droplets. The system was first characterized by detecting fluorescein sodium salt as a model dye in the internal aqueous droplets using laser-induced fluorescence. The effect of the flow rates of the internal aqueous phase and outer continuous aqueous phase on the formation of the double-emulsion droplets is investigated to provide information for system optimization. On-chip storage of double-emulsion droplets is also investigated to allow for protein synthesis from a PCR-generated DNA template using either commercial in vitro transcription and translation kits or crude Escherichia coli S30 extracts. In vitro expression of the green fluorescent protein is successfully demonstrated in this system

Enzyme synthesis and activity assay in microfluidic droplets on a chip

There is growing demand for high-throughput measurement of biochemical reactions in drug discovery and directed evolution programs. To meet this need, a powerful platform based on droplet-based bioreactors manipulated by microfluidic systems is being developed, which can overcome the limitations of scale and power encountered in conventional screening methods. This paper reports our progress in the synthesis of enzymes and assay of their activity within a microfluidic droplet system. The model system we use involves the organophosphorus hydrolase enzyme OpdA from Agrobacterium radiobacter and a robust microchip made from polymethyl methacrylate (PMMA). Synthesis of OpdA from cognate DNA within water-in-oil droplets was tested using both in-house and commercial in vitro transcription and translation (IVTT) kits. OpdA activity was measured using coumaphos as substrate and by monitoring the fluorescence released by its product, chlorferone. OpdA was demonstrated to be synthesized and assayed within the droplets using the commercial in vitro transcription and translation kit, although the activity measured within the droplets diminished over time, apparently due to leakage of chlorferone out of the droplets

Determination of the Structure of the Catabolic <em>N-</em>Succinylornithine Transaminase (AstC) from <em>Escherichia coli</em>

<div><p><i>Escherichia coli</i> possesses two acyl ornithine aminotransferases, one catabolic (AstC) and the other anabolic (ArgD), that participate in L-arginine metabolism. Although only 58% identical, the enzymes have been shown to be functionally interchangeable. Here we have purified AstC and have obtained X-ray crystal structures of apo and holo-AstC and of the enzyme complexed with its physiological substrate, succinylornithine. We compare the structures obtained in this study with those of ArgD from <i>Salmonella typhimurium</i> obtained elsewhere, finding several notable differences. Docking studies were used to explore the docking modes of several substrates (ornithine, succinylornithine and acetylornithine) and the co-substrate glutamate/α-ketogluterate. The docking studies support our observations that AstC has a strong preference for acylated ornithine species over ornithine itself, and suggest that the increase in specificity associated with acylation is caused by steric and desolvation effects rather than specific interactions between the substrate and enzyme.</p> </div

Modeled structures of AstC with substrates and products.

<p>6A: Shown here is AO (grey) docked into the active site of SO transaminase. Pyridoxyl phosphate is shown in magenta covalently bound to Lys 252. Hydrogen bonds between Arg 141 and AO are shown in black. This is the highest scoring pose that was correctly oriented for catalysis (6 out of 100). 6B: Shown here is the top scoring conformation of α-ketoglutarate (grey) docked into SO transaminase with pyridoxamine phosphate (magenta). Hydrogen bonds with Arg 141 are shown in black. 6C: Shown here is the top-scoring pose of SO (grey, SO-m) docked into the active site of SO transaminase. Pyridoxyl phosphate is shown in magenta covalently bound to Lys 252. Hydrogen bonds between SO and Arg 141 are shown in black. In cyan is the product succinyl-glutamic semialdehyde (SO-c, derived from the crystal structure by replacing the covalently bound amine group with the appropriate aldehyde). Hydrogen bonds from this ligand to Arg 141 are shown in cyan. In this image the docked structure (grey) has a root-mean-square distance of 1.8Å relative to the crystal structure (cyan). 6D: Shown here is glutamate (grey) docked into the active site of SO transaminase. Pyridoxyl phosphate is shown in magenta covalently bound to Lys 252. Hydrogen bonds between Arg 141 and glutamate are shown in black. This is the highest scoring pose that was correctly oriented for catalysis (2 out of 100).</p

The native AstC structure with PLP.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058298#pone-0058298-g004" target="_blank">Figures 4A and 4B</a> show the native dimer structure as a ribbon Cα trace with the PLP cofactor shown in a magenta colored stick representation. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058298#pone-0058298-g004" target="_blank">Figure 4B</a> is a zoomed image showing how the loop of residues 274–284 comes in from the neighboring protomer to contact the PLP cofactor and form part of the active site of protomer A.</p

The apo-AstC structure.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058298#pone-0058298-g003" target="_blank">Figure 3A</a> shows the superposition of apo-AstC protomer B (burnt orange) on protomer A (cyan) without PLP present in the structure. The N-terminus at the top right and the first 20 residues of protomer B are not modelled due to a lack of electron density for these residues. The other clear difference shown in 3A is loop 142–163, which is missing in protomer B (lower right). An approximate 90 degree rotation in 3B highlights the missing loop of residues 274–283 (lower left side of 3B).</p

Structures of AstC prior and after reaction with substrates.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058298#pone-0058298-g005" target="_blank">Figure 5A</a> shows the 2Fo-Fc density (blue mesh) for the PLP cofactor bound to Lys252 in a Schiff base. The B protomer is in green and the A protomer loop of 277–284 is shown in magenta. The electron density is set at 1.5 sigma. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058298#pone-0058298-g005" target="_blank">Figure 5B</a> shows the density for PLP and Lys252 after the crystals were soaked with ORN. It is clear that the covalent bond between Lys252 and the PLP is broken and we have now modelled the PLP with an additional oxygen atom. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058298#pone-0058298-g005" target="_blank">Figure 5C</a> shows the electron density for Lys252, PLP and the SO found in the SO soaked crystals. There is clean electron density that shows the SO covalently bound to the PLP and a break from Lys252. All density figures are 2Fo-Fc maps and were made with the ligands in the model.</p

Comparison of AstC structure with ArgD from <i>S. typhimurium</i>.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058298#pone-0058298-g007" target="_blank">Figure 7A</a> shows the structure of 2PB0 (in burnt orange) with a dimer of AstC overlayed (cyan). The structures are very similar with the major differences being in the N-terminus (highlighted in 7B) and the loop of 277–286. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058298#pone-0058298-g007" target="_blank">Figure 7B</a> again has the 2PB0 structure in orange and AstC in cyan, but the differences in 2PB0 are highlighted in red. On the top right hand side we have the start of the 2PB0 B chain, Leu18, which does not come into alignment with AstC until residue Phe26. At the top we have the two structures aligned at residue Phe276 where the density falls off for 2PB0 and there is a gap to residue Gly282 (hidden in the helix of the N-terminus of AstC). The 2PB0 model for this part of the chain comes back into alignment with AstC just before the helix at residue Gly287 (bottom of figure).</p

Biochemical data on the activity of <i>E. coli</i> AstC, ArgD and <i>St</i>-ArgD.

1<p><i>E. coli</i> from this study.</p>2<p><i>Salmonella typhimurium</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058298#pone.0058298-Rajaram1" target="_blank">[20]</a>.</p>3<p><i>E. coli</i> ArgD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058298#pone.0058298-Ledwidge1" target="_blank">[19]</a>.</p