No description available.

No description available.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Erika Rae Geisbrecht / The E3 ubiquitin ligase TRIM32 is a member of tripartite motif (TRIM) family of proteins involved in various processes including differentiation, cell growth, muscle regeneration and cancer. TRIM32 is conserved between vertebrates (humans, mouse) and invertebrates (Drosophila). The N-terminus of this protein is characterized by a RING domain, B-box domain, and Coiled-Coil region, while the C-terminus contains six NHL repeats. In humans, mutations that cluster in the NHL domains of TRIM32 result in the muscle disorders Limb-Girdle Muscular Dystrophy type 2H (LGMD2H) and Sarcotubular Myopathy (STM). Mutations in the B-box region cause Bardet-Biedl Syndrome (BBS), a clinically separate disorder that affects multiple parts of the body. A comprehensive genetic analysis in vertebrate models is complicated by the ubiquitous expression of TRIM32 and neurogenic defects in TRIM32-/- mutant mice that are independent of the muscle pathology associated with LGMD2H. The model organism Drosophila melanogaster possesses a TRIM32 [dTRIM32/Thin (Tn)/Abba] homolog highly expressed in muscle tissue. We previously showed that dTRIM32 is localized to Z-disk of the sarcomere and is required for myofibril stability. Muscles form correctly in Drosophila tn mutants, but exhibit a degenerative muscle phenotype once contraction ensues. Mutant or RNAi knockdown larvae are also defective in locomotion, which mimics clinical features associated with loss of TRIM32 in LGMD2H patients. It is predicted that mutations in the NHL domain either affect protein structure or are involved in protein-protein interactions. However, the molecular mechanism by which these mutations affect the interaction properties of dTRIM32 is not understood. Biochemical pulldown assays using the bait fusion protein GST-dTRIM32-NHL identified numerous dTRIM32 binding proteins in larval muscle tissue. Many key glycolytic enzymes were present in the dTRIM32 pulldowns and not in control experiments. Glycolytic genes are expressed in the developing Drosophila musculature and are required for myoblast fusion. Strikingly, many glycolytic proteins are also found at the Z-disk, consistent with dTRIM32 localization. Our biochemical and genetic studies provide evidence that there is direct interaction between dTRIM32 and glycolytic proteins (Aldolase and PGLYM). dTRIM32 also regulates glycolytic enzyme levels and protein localization at their sites of action. These data together suggest a role for dTRIM32 in coordinating glycolytic enzyme function, possibly for localized ATP production or to maintain muscle mass via glycolytic intermediates.

Cancer cells, unlike normal tissue, frequently rely on glycolysis for the production of energy and the metabolic intermediates required for their growth regardless of cellular oxygenation levels. This metabolic reconfiguration, termed the Warburg effect, provides a potential strategy to preferentially target tumours from a therapeutic perspective. The present study sought to investigate the glycolytic phenotype of breast and ovarian cancer, and assess the possibility of exploiting several glycolytic targets therapeutically. Initially the growth dependency of breast and ovarian cancer cells on the availability of glucose was established. An array of 10 compounds reported to inhibit key enzymes of the glycolytic pathway were investigated and compared against an extended panel of breast and ovarian cancer cell line models. All inhibitors investigated, targeted against multiple points of the pathway, were shown to block the glycolytic pathway as demonstrated by glucose accumulation in the culture media combined with decreased lactate secretion, and attenuated breast and ovarian cancer cell proliferation in a concentration dependent manner. Furthermore their mechanism of action was investigated by flow cytometric analysis and their antiproliferative effect was associated with induction of apoptosis and G0/G1 cell cycle arrest. The glycolytic inhibitors were further assessed in combination strategies with established chemotherapeutic and targeted agents and several synergistic interactions, characterised by low combination index values, were revealed. Among them, 3PO (a novel PFKFB3 inhibitor) enhanced the effect of cisplatin in both platinum sensitive and platinum resistant ovarian cancer cells suggesting a strategy for treatment of platinum resistant disease. Furthermore robust synergy was identified between IOM-1190 (a novel GLUT1 inhibitor) and metformin, an antidiabetic inhibitor of oxidative phosphorylation, resulting in strong inhibition of breast cancer cell growth. This combination is proposed for the treatment of highly aggressive triple negative breast tumours. An additional objective of this research was to investigate the effect of the oxygen level on sensitivity to glycolysis inhibition. Breast cancer cells were found to be more sensitive to glycolysis inhibition in high oxygen conditions. This enhanced resistance at low oxygen levels was associated with upregulation of the targeted glycolytic enzymes as demonstrated at both the mRNA (by gene expression microarray profiling, Illumina BeadArrays) and protein level (by Western blotting). Manipulation of LDHA (Lactate Dehydrogenase A) by siRNA knockdown provided further evidence that downregulation of this target was sufficient to significantly suppress breast cancer cell proliferation. Finally, the expression of selected glycolytic targets was examined in a clinical tissue microarray set of a large cohort of ovarian tumours using quantitative immunofluorescence technology, AQUA. The role of the glycolytic phenotype in ovarian cancer was suggested and interesting associations between the glycolytic profile and clear cell and endometrioid ovarian cancers revealed. Increased PKM2 (Pyruvate kinase isozyme M2) and LDHA expression were demonstrated in clear cell tumours and also low expression of these enzymes was significantly correlated with improved survival of endometrioid ovarian cancer patients. Taken together the findings of this study support the glycolytic pathway as a legitimate target for further investigation in breast and ovarian cancer treatment.

No description available.

No description available.

No description available.

Flagellate protozoa from the order Trypanosomatida have developed a range of strategies to survive in their mammalian hosts. A consequence is that the glycolytic pathway has assumed an important role, especially in bloodstream-form Trypanosoma brucei, where it is essential as the sole producer of ATP. The seventh enzyme in the pathway, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is particularly attractive as a drug target because it shares no common properties with the corresponding enzyme in humans. This enzyme catalyses the conversion of 3PGA to 2PGA, with the requirement for metal ions to assist the catalytic function. In this study, two important biochemical and structural aspects of the enzyme were investigated: i) The in vitro and in vivo requirements for biologically relevant metal ions to support the activity of iPGAM, and ii) The ability of trypanosomatid iPGAM to exist in multiple conformations and oligomeric states in solution. The maximum activity of iPGAM in vitro requires Co2+, but this cannot be the case in vivo where ICP-OES analyses confirmed that Co2+ was essentially undetectable in T. brucei cytosolic fractions. The activity of iPGAM in vivo is therefore one of the lowest among the glycolytic enzymes. By contrast, Mg2+ and Zn2+ were found to be the most abundant metals in both cytosolic fractions and in purified bacterially expressed iPGAM. Our newly-developed multimode-plate reader discontinuous assay further revealed that of the biologically relevant metals, only Mg2+ can support iPGAM activity, but at less than 50% of the level of Co2+. By contrast, Zn2+ strongly inhibits iPGAM. This assay which was developed with minimal metal interference on the coupling enzymes, also showed that in solution, the ratio of the concentrations of 3PGA:2PGA (substrate:product) at equilibrium is not 1:1 as observed in the crystal structure, but is in fact 12:1, which may be due to the tighter binding of 2PGA to the enzyme. A series of biophysical analyses, notably by SEC-MALS showed that iPGAM from Leishmania mexicana, another trypanosomatid protozoan parasite exists in different forms and oligomeric states in solution, either as the closed-form monomer, openiii form monomer, or closed/open-form dimer which can be successfully separated by ion-exchange chromatography. The open-form LmiPGAM is particularly relevant for drug development, as the catalytic site in the closed-form structure is poorly inaccessible. Both virtual and high-throughput screening approaches were used to identify novel potential inhibitors. Out of a collection of 11 compounds tested at 1 mM, two showed substantial inhibition with 49% and 14% remaining activity. Taken together, the findings from this study demonstrated the potential of iPGAM to be a key modulator in controlling glycolytic flux in trypanosomes, and thus further validated it as an important drug target.

No description available.

Glycolytic enzymes have been indicated as potential drug targets in trypanosomatid parasites such as Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania spp. Pyruvate kinase (PYK) catalyses the final reaction in the glycolytic pathway to produce ATP and pyruvate from ADP and phosphoenolpyruvate (PEP), and has been validated by RNAi experiments as a suitable drug target in T. brucei. This thesis describes biochemical and structural studies of PYKs from T. cruzi (TcPYK) and T. brucei (TbPYK), providing not only a foundation but also new clues for PYK-specific inhibitor screening and structure-based drug design. Soluble TcPYK and TbPYK (81% sequence identity) have been expressed and purified from E. coli, and their kinetics have been fully characterised. X-ray crystal structures of apoenzyme TcPYK (apo TcPYK), and of TbPYK in complex with fructose 2,6-bisphosphate (F26BP) (TbPYK/F26BP/Mg) have been determined, and each possesses a tetrameric architecture composed of four identical protein chains. Each chain contains four domains which are A-domain, B-domain, C-domain and N-terminal domain. The active site is located in the cleft between the A- and B-domains, while the F26BP-bound effector site is within the C-domain. The conformational transition between inactive T-state and active R-state for both enzymes requires a concerted 8o rigid-body rotation of each of the four AC-cores (Aand C-domains) in the tetramer. During the T- to R-state transition induced by F26BP binding, the side chain of Arg311 is re-orientated to stabilise the short Aα6′ helix at the active site, and the flexible loop at the effector site is stabilised by F26BP. In this active conformation additional salt bridges form across the C-C interface to lock the enzyme in a more stable R-state. TbPYK/F26BP/Mg is the first ‘effector only’ PYK structure and identifies a third Mg2+ binding site (Mg-3) which is distinct from the two canonical Mg2+ binding sites. The substrate PEP was soaked into crystals of TbPYK/F26BP/Mg resulting in an ‘in crystallo’ 23° B-domain rotation forming a partially closed active site. This is accompanied by active site side-chain reorientations, and the movement of Mg2+ from its ‘priming’ position Mg-3 to its canonical position Mg-1. It is plausible that Mg2+ is retained in its ‘priming’ position after product release to act as a co-activator with F26BP to maintain the enzyme in its R-state conformation, as long as F26BP is present. The inherent oxaloacetate decarboxylase activity of PYK was reported over 30 years ago and has been further characterised by 1H NMR studies in this thesis. In addition, a series of TbPYK structures in complex with product (pyruvate), with analogues of the decarboxylase substrate oxaloacetate (D-malate and α-ketoglutarate), or with the competitive inhibitor oxalate have been determined by crystal soaking, and indicate that both decarboxylase activity and kinase activity share a common active site. A proposed mechanism explains the conserved decarboxylase activity of PYK where the active-site Mg2+ and Lys239 in TbPYK (which is conserved between species) play essential roles in the decarboxylation reaction. Three strategies for designing novel inhibitors against trypanosomatid PYKs have been proposed in this thesis. (1) Develop selective modulators to increase the binding affinity of inhibitors. As an example, F16BP has been shown to regulate the inhibitory effect of PEP analogues (oxalate, D-malate, α-ketoglutarate, malonate and L-tartrate) on TbPYK activity. (2) Develop allosteric inhibitors in order to lock trypanosomatid PYKs in an inactive state where the enzyme has low affinity for substrate binding. (3) A third strategy is to combine multiple modulators and inhibitors to increase the inhibition efficiency and selectivity.