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Abstract: Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutate (2OG) to (2R)-hydroxyglutarate (2HG). However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3 and C4 alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of wildtype IDH1/2. Absorbance-based, NMR and electrochemical assays were employed to monitor wildtype IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of wildtype IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates compared to 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl-, 3-butyl-, and 3-benzyl-substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts.

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Abstract: N-Acyl indolines 4 are potent, non-covalent Notum inhibitors developed from a covalent virtual screening hit 2a. The lead compounds were simple to synthesise, achieved excellent potency in a biochemical Notum-OPTS assay and restored Wnt signalling in a cell-based TCF/LEF reporter assay. Multiple high resolution X-ray structures established a common binding mode of these inhibitors with the indoline bound centred in the palmiteolate pocket with key interactions being aromatic stacking and a water mediated hydrogen bond to the oxyanion hole. These N-acyl indolines 4 will be useful tools for use in vitro studies to investigate the role of Notum in disease models, especially when paired with a structurally related covalent inhibitor (e.g. 4w and 2a). Overall, this study highlights the designed switch from covalent to non-covalent Notum inhibitors and so illustrates a complementary approach for hit generation and target inhibition.

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Abstract: The linear ubiquitin chain assembly complex synthesises linear Ub chains which constitute a binding and activation platform for components of the TNF signalling pathway. One of the components of LUBAC is the ubiquitin ligase HOIL-1 which has been shown to generate oxyester linkages on several proteins and on linear polysaccharides. We show that HOIL-1 activity requires linear tetra-Ub binding which enables HOIL-1 to mono-ubiquitylate linear Ub chains and polysaccharides. Furthermore, we describe the crystal structure of a C-terminal tandem domain construct of HOIL-1 comprising the IBR and RING2 domains. Interestingly, the structure reveals a unique bi-nuclear Zn-cluster which substitutes the second zinc finger of the canonical RING2 fold. We identify the C-terminal histidine of this bi-nuclear Zn-cluster as the catalytic base required for the ubiquitylation activity of HOIL-1. Our study suggests that the unique zinc-coordinating architecture of RING2 provides a binding platform for ubiquitylation targets.

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Abstract: Accurately estimating developmental age and life history traits in fossils is crucial for identifying and classifying extinct species and understanding how biological attributes evolved. The evolution of life history traits such as growth pattern is far from clear in birds, and development has been studied in only a handful of modern species. The exceptionally rapid growth of modern birds means ageing methods based on annual incremental growth lines, used in other vertebrates, are inapplicable to birds and robust alternative methods remain to be established. Analysis of avian intracortical bone microstructure, which varies both with age and tissue deposition rate, is a promising approach already used in palaeobiology. However, current thin section-based histological methods are destructive. Moreover, to date, most microstructural studies in avian bone are qualitative, 2D, and involve a limited range of extant species. The objective of this study was to investigate cortical bone microstructure and developmental age and life history traits in living birds, to identify phenotypes which can then be applied to examination of the fossil record, using minimally-destructive high-resolution 3D imaging. First, the necessity of 3D measurement was tested: a combination of idealised, simulated datasets and real synchrotron-based computed tomography (SR CT) datasets were used to compare published methods for measuring key microstructural traits based on 2D sections and 3D volumes. Next, SR CT imaging and quantitative measurements were used to characterise age-related changes in bone microstructure in a range of extant bird species: growth series ducks and pheasants, and a smaller sample size in starlings, rock doves, partridges, and ostrich. The methods tested in modern material were applied to fossils as a proof-of concept. It was found that 3D quantification methods are required for measuring vascular canal orientation and osteocyte lacunar shape and volume, though 2D sections could be used to measure traits such as bone volume fraction (BV/TV) and osteocyte lacunar volume. In all species studied, juvenile, subadult, and adult species could be distinguished by their values of BV/TV, and further information could be added using measured values of vascular canal diameter as well as qualitative assessment. Using a synchrotron-based CT system, high-resolution 3D datasets comparable to modern bone samples were obtained from fossils, and preliminary estimates of developmental age can be made. Further work may reveal more changes within juvenile age stages, and better characterise the variation within extant birds, allowing more accurate interpretation of the fossil record. Therefore developmental studies in a greater number of extant bird species are required using larger sample sizes, to support and add to the results presented in this thesis.

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Abstract: Diffractive optical elements such as periodic gratings are fundamental devices in X-ray imaging – a technique that medical, material science, and security scans rely upon. Fabrication of such structures with high aspect ratios at the nanoscale creates opportunities to further advance such applications, especially in terms of relaxing X-ray source coherence requirements. This is because typical grating-based X-ray phase imaging techniques (e.g., Talbot self-imaging) require a coherence length of at least one grating period and ideally longer. In this paper, the fabrication challenges in achieving high-aspect ratio nanogratings filled with gold are addressed by a combination of laser interference and nanoimprint lithography, physical vapor deposition, metal assisted chemical etching (MACE), and electroplating. This relatively simple and cost-efficient approach is unlocked by an innovative post-MACE drying step with hexamethyldisilazane, which effectively minimizes the stiction of the nanostructures. The theoretical limits of the approach are discussed and, experimentally, X-ray nanogratings with aspect ratios >40 are demonstrated. Finally, their excellent diffractive abilities are shown when exposed to a hard (12.2 keV) monochromatic X-ray beam at a synchrotron facility, and thus potential applicability in phase-based X-ray imaging.