No description available.

No description available.

No description available.

No description available.

GNE is a bifunctional enzyme responsible for the first committed, rate limiting step in the synthesis of sialic acid, the most common terminal monosaccharide in cellular glycosylation. Mutations in GNE are responsible for two rare human disorders, Hereditary Inclusion Body Myopathy (HIBM) and Sialuria. The hyposialylation of glycoproteins has been a focus in HIBM research based on the assumption that mutations in GNE decrease synthesis of CMP-sialic acid thereby decreasing downstream sialylation. However, glycoproteins are only one class of prevalent, sialylated molecules in mammalian tissues. Analysis of glycosphingolipids revealed aberrant expression in both in vivo and in vitro models of HIBM. Surwisingly, in a mouse model of HIBM with knock-in founder mutation M712T, mutants (GNEM712T/M 12T) display a global increase in all expressed GSL species, including sialylated gangliosides. These findings were confirmed in primary human fibroblasts isolated from four H IBM patients with mutations throughout both enzymatic domains of GNE. All patient cell lines displayed an increase in absolute amounts of all glycosphingolipids when compared to control cell lines. Furthermore an HIBM like GSL phenotype could be induced in control fibroblasts after inhibition of GNE epimerase activity with a novel imino sugar inhibitor. HIBM fibroblasts grown in the presence of this inhibitor displayed dose dependent increases in glycosphingolipid expression above already elevated levels. Treatment with ManNAc and CMP-SA, metabolites downstream of GNE epimerase, had the opposite effect lowering total GSL expression in HIBM cell lines. These results link aberrant GSL expression in HIBM directly to GNE epimerase function. Fibroblasts from patients with COG la, containing mutated phosphmannomutase, also display a global increase in GSL expression. This up-regulation is thought to be a compensatory mechanism for loss of N-linked glycans in an effort to maintain net glycosylation on the cell surface. Similarly, global increases in total GSL expression seen in HIBM may be a compensatory mechanism for decreases in N-linked sialylation.

Stearoyl--CoA desaturase (SCD) plays an important role in converting saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA). Changes in SCD activity and alterations in the balance between MUFA and SFA are implicated in various disease states including diabetes obesity and arteriosclerosis. Furthermore, SCDl is believed to play a role in fat deposition, which is the general focus of the current study. Hence, investigating the mechanism involving SCDl regulation may help the understanding of fat deposition in farm animals and obesity in humans.

Human topoisomerase 11 (topo 11) is an essential enzyme that controls DNA topology by relieving positive and negative supercoiling. It does this by creating a transient double-stranded break in one DNA duplex to allow a second duplex to pass through. The DNA double-stranded break is bridged by the topo 11 enzyme which is attached to each DNA end via a 5' covalent phosphotyrosyl bond. Human cells express two distinct topo JI isoforms, topo JI a and topo JI ~. The anti-cancer drug etoposide is extensively used in the treatment of malignancies. Etoposide traps the topo II homodimer in its normally transient cleavage state, a permanent DNA double stranded break can be produce through cellular processing. Non-homologous end joining (NHEJ) is a major pathway for the repair of to po II- mediated DNA damage, and inhibition of DNA-PK potentiates the cytotoxicity of topo JI poisons. The topo II enzyme remains covalently attached to the broken DNA and must be removed for NHEJ to take place. No mechanism to date has been elucidated for the removal of covalently attached human topo II-l)NA adducts. Inhibition of this process would slow the repair of to po II damage, thus potentiate the effects of to po IJ-targeting drugs. The Irapped in AgaRose DNA [mmunojitaining (TARDIS) assay was successfully adapted to incorporate a protein incubation phase to test candidate proteins for their ability to remove etoposide-stabilised human topo U-DNA adducts. Mrell and Tdp 1 immunoprecipitates from human chronic myelogenous leukaemia cells were identified that were capable of removing topo 11 complexes. Proteomic analysis identified nucleases and DNA repair proteins that were present in the two immunoprecipitates. Results provide evidence for the first time that Mre I I can facilitate the removal of human topo IJ a DNA adducts. In addition, results suggest that human topo II-DNA adducts are also removed by a 5' phosphodiesterase. These results may provide exciting new targets for future leukaemia drugs.

No description available.

Histone lysine demethylases comprise an important family of epigenetic regulatory enzymes. They catalyse the demethylation of tri-, di- and monomethylated lysine residues on histone H3, thus contributing to either silencing or activation of chromatin. Their biological roles are widespread and have just begun to be elucidated. Among other functions, they contribute to establishment and maintenance of pluripotent states in embryonic stem cells, and also to cellular differentiation during development. Abnormal expression or mutation of some demethylases has been linked to diverse diseases, from prostate and oesophageal cancers to X-linked mental retardation. The development of small molecule inhibitors of histone demethylases is therefore of interest, both from the therapeutic perspective, and with the aim of developing chemical probes to understand the diverse functions of the demethylases in vivo. Most histone lysine demethylases belong to the 2-oxoglutarate and ferrous iron dependent dioxygenase superfamily. This family utilises molecular oxygen to catalyse hydroxylation of substrates, together with oxidation/decarboxylation of the 2-oxoglutarate cofactor. In the work outlined in this thesis, the JMJD2 family of histone demethylases was characterised biochemically, with attention given to mechanism, substrate selectivity and the role of eo factors. JMJD2E was identified herein as a novel histone demethylase in H. sapiens, and was shown to be selective for the demethylation of tri-, di- and monomethylated lysine 9 in histone H3. JMJD2E was also found to be particularly amenable to mechanistic and inhibition studies in vitro. A variety of mechanistic investigations established details of the catalytic cycle, its substrate selectivity and the role of iron and ascorbic acid as cofactors. Crystallographic analyses were also employed to compare its substrate selectivity to other JMJD2 family members. Assays suitable for the evaluation of inhibitors of the JMJD2 demethylases were then developed. These included a coupled enzyme assay suitable for kinetic measurements, and two mass spectrometric assays for observing inhibitor binding and catalytic activity. A critical review of the 20G oxygenase inhibitor literature carried out, and was then used as a basis for the identification of inhibitor scaffolds for the JMJD2 demethylases. These were characterised both in vitro (using kinetic assays, mass spectrometry and crystallography), and in cell culture. Some were further developed to achieve selective inhibition of the JMJD2 demethylases over the related prolyl hydroxylase PHD2; crystallography was again employed to understand the mode of inhibition of these potent inhibitors. The kinetic assays developed were optimised for use in a high-throughput screen, and a library of 240 000 compounds was screened against JMJD2E. This was the first instance of high-throughput screening against these promising therapeutic targets. Several hit compounds were identified and characterised further in vitro. Finally, alternative means of inhibiting the JMJD2 demethylases were investigated. Compounds were identified that inhibited JMJD2A by ejection of its unique structural zinc ion, thus demonstrating that selective inhibition of the JMJD2 demethylase family is possible. In summary, this work contains the first detailed investigation of a histone demethylase subfamily, and also the first steps towards identifying potent, selective inhibitors of these epigenetic regulatory enzymes.

2-0xoglutarate/Fe(II)-dependent dioxygenases (ZOG Oxygenases) are a relatively poorly characterised enzyme family that hydroxylate biological macromolecules to regulate a variety of essential cellular processes in mammals, including; chromatin remodeling, extra-cellular matrix formation and oxygen sensing. The work in this th esis focuses on a ZOG Oxygenase termed Myc-Induced Nuclear Antigen (MINAS3). This enzyme has been implicated in ribosome biogenesis and cell proliferation, and observed overexpressed in several tumour types, yet the identity afits substrate(s) and their role in cancer is unknown. The aims of the resea rch that has resulted in this thesis were to; (i) develop a cell model of MINAS3 enzyme activity, (ii) apply this model to study the role of MINAS3 activity in cell transformation and cancer, and (iii) discover novel cellular processes regulated by MINA53 activity. As such, I have created an isogenic cell model consisting of K-Ras-transformed MINAS3 knockout mouse embryonic fibroblasts (MEFs) reconstituted with either wildtype or enzyme-inactive MINAS3. Using this model I have shown that MINAS3 activity maintains normal levels of the large ribosomal subunit (60S), and suppresses anchorage-independent growth, autophagy and gene expression. These observations suggest the existence and involvement of one or more substrates. Indeed, proteomic and biochemical analyses in collaboration with the Schofield laboratory (Chemistry, Oxford) confirmed the identity of a MINA53 substrate, the 60S ribosomal protein Rp127a. Together we have shown that Rpl27a is abundantly hydroxylated, and that MINA53 is a histidinyJ hydroxylase; this represents the first discovery of a ribosomal oxygenase. The model developed here did not support a positive role for MINA53 in the transformation of MEFs. Rather it suggested that MINA53 can suppress transformation in some contexts, This prompted a wider investigation that demonstrated underexpression of MINA53 in several tumour types, and the presence of inactivating mutations in breast. ovarian and colon cancer. This thesis provides data supporting further research to understand the role of Rpl27a hydroxylation in the regulation of 60S biogenesis, autophagy and cancer. 2