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.

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.

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.

Outgrowth of spoilage yeasts and moulds and post-processing acidification can limit the shelf-life of some fermented dairy products including fresh lactic curd cheeses. The possibility of using high pressure processing (HPP) for controlling these problems was investigated in a commercially manufactured fresh lactic curd cheese (pH 4.3-4.4) and fermented milk models (pH 4.3-6.5). The effects of HPP at 300 and 600 MPa on inactivation of glycolytic enzymes of lactic acid bacteria were also evaluated. Fresh cheeses made from pasteurised bovine milk using a commercial Lactococcus starter preparation were treated with high pressures ranging from 200 to 600 MPa (less than or equal to 22°C, 5 min) under vacuum packaging conditions and subsequently stored at 4°C for 8 weeks. Treatment at greater than or equal to 300 MPa substantially reduced the viable count of Lactococcus and effectively prevented the outgrowth of yeasts and moulds for 6 to 8 weeks without adversely affecting the sensory and textural attributes of the product. However, it had no significant effects (p less than 0.01) on variation of titratable acidity during storage. Fermented milk models were prepared by individually growing Lactococcus lactis subsp. lactis C10, Lactococcus lactis subsp. cremoris BK5, Streptococcus thermophilus TS1, Lactobacillus acidophilus 2400 and Lactobacillus delbrueckii subsp. bulgaricus 2517 in UHT skim milk and diluting the resulting fermented milk with UHT skim milk up to pH 6.5. Pressure treatment of the milk models at pH 5.2 resulted in substantial inhibition of post-processing acidification during storage and markedly reduced the viable count of Lactococcus at both 300 and 600 MPa and other bacteria only at 600 MPa. Treatment of the milk model at 600 MPa decreased the viable counts of Candida zeylanoides and Candida lipolytica (wildtype spoilage yeasts of lactic curd cheese, added as challenge cultures) from 105 CFU mL-1 to below the detection limit (log 0 CFU mL-1) at all pH levels tested (pH 4.3-6.5) and effectively controlled their outgrowth for 8 weeks. Treatment of milk model at 300 MPa had a similar effect only on C. zeylanoides. The viable count of C. lipolytica was reduced by 2.6, 2.4 and 2.3 logs by treatment at 300 MPa at pH levels of 4.3, 5.2 and 6.5, respectively, which subsequently recovered by 2.9, 2.8 and 3.2 logs within 3 weeks. Glycolytic enzymes of various starter bacteria showed different responses to pressure treatment. The lactate dehydrogenase in L. lactis subsp. lactis and Lb. acidophilus was quite resistant to pressures up to 600 MPa, but it was almost completely inactivated in S. thermophilus at pressure levels as low as 300 MPa. The â-galactosidase in Lb. acidophilus was more pressure stable than â-galactosidase in S. thermophilus and Phospho-â-galactosidase in L. lactis subsp. lactis. The findings of this study suggests HPP at 300-600 MPa as an effective method for controlling the outgrowth of some spoilage yeasts and moulds in fresh lactic curd cheeses. The results obtained with selected lactic acid bacteria in fermented milk models can be used to assist in establishing HPP operating parameters for development of new generation cultured dairy products, of reduced acidity and extended shelf-life.

This thesis describes studies directed towards understanding structure-function relationships of triosephosphate isomerase (TIM), from a protozoan parasite Plasmodium falciparum and a thermophilic archaea Methanocaldococcus jannaschii. Triosephosphate isomerase, a ubiquitous glycolytic enzyme, has been the subject of biochemical, enzymatic and structural studies for the last five decades. Studies on TIM have been central to the development of mechanistic enzymology. The present study investigates the role of specific residues in the structure and function of Plasmodium falciparum triosephosphate isomerase (PfTIM). The structure and stability of a tetrameric triosephosphate isomerase from Methanocaldococcus jannaschii (MjTIM) is also presented. Chapter 1 provides a general introduction to the glycolytic enzyme triosephosphate isomerase, conservation of TIM sequences, its fold and three dimensional organization. The isomerisation reaction interconverting dihydroxyacetone phosphate and glyceraldehyde 3phosphate catalyzed by triosephosphate isomerase is an example of a highly stereospecific proton transfer process (Hall & Knowles, 1975; Rieder & Rose, 1959). This chapter briefly reviews mechanistic features and discusses the role of active site residues and the functional flexible loop 6. Triosephosphate isomerase adopts the widely occurring ( β/ α)8 barrel fold and mostly occurs as a dimer (Banner et al., 1975). Protein engineering studies, related to folding, stability and design of monomeric TIM are also addressed. A brief introduction to thermophilic TIMs and higher oligomeric TIMs is given. The role of this enzyme in disease states like hemolytic anemia and neuromuscular dysfunction is surveyed. The production of methylglyoxal, a toxic metabolite, as a byproduct of the TIM reaction is also considered. Many proteins utilize segmental motions to catalyze a specific reaction. The omega loop (loop 6) of triosephosphate isomerase is important for preventing the ene-diol intermediate from forming the cytotoxic byproduct, methylglyoxal. The active site loop-6 of triosephosphate isomerase moves about 7Ǻ on ligand binding. It exhibits a hinged lid motion alternating between two well defined, “open” and “closed”, conformations (Joseph et al., 1990). Though the movement of loop 6 is not ligand gated, in crystals the ligand bound forms invariably reveal a closed loop conformation. Plasmodium falciparum TIM is an exception which predominantly exhibits “open” loop conformations, even in the ligand bound state (Parthasarathy et al., 2002). Phe 96 is a key residue that is involved in contacts between the flexible loop-6 and the protein body in PfTIM. Notably, in all TIM sequences determined thus far, with the exception of plasmodial sequences, this residue is Ser 96. In Chapter 2 the mutants F96S, F96H and F96W are reported. The crystal structures of the mutant enzymes with or without bound ligand are described. In all the ligand free cases, loop-6 adopts an “open” conformation. Kinetic parameters for all the mutants establish that residue 96 does not play an essential role in modulating the loop conformation but may be important for ligand binding. Structural analysis of the mutants along with WT enzyme reveals the presence of a water network which can modulate ligand binding. Subunit interfaces of oligomeric proteins provide an opportunity to understand protein- protein interactions. Chapter 3 describes biochemical and biophysical studies on two separate dimer-interface destabilizing mutants C13E and W11F/W168F/Y74W of PfTIM. The intention was to generate a stable monomer by disrupting the interaction hubs. C13 is a part of a large hydrophobic patch (Maithal et al., 2002a) at the dimer interface. Introduction of a negative charge at position 13 destabilizes the interface and reduces activity. Y74 is a part of an aromatic cluster of the interface (Maithal et al., 2002b). The Y74W triple mutant was designed to disrupt the aromatic cluster by introducing additional atoms. Tryptophan is also a fluorophore, allowing studies of the dimer disruption by fluorescence, after mutating the two inherent tryptophan residues, W11 and W168 to phenylalanine. The mutants showed reduced activity and were more sensitive than the wild type enzyme to chemical denaturants as well as thermal denaturation. Evidenced for monomer formation is presented. These studies together with previous work reveal that the interface is important for both activity and stability. In order to develop a model for understanding the relationship between protein stabilization and oligomeric status, characterization of the TIM from Methanocaldococcus jannaschii (MjTIM) has been undertaken. Chapter 4 describes the purification and characterization of MjTIM. The MjTIM gene was cloned and expressed in pTrc99A and protein was isolated from AA200 E. coli cells. Hyperexpressed protein was purified to homogeneity and relevant kinetic parameters have been determined. The tetrameric nature of MjTIM is established by gel filtration studies. Circular dichroism (CD) studies establish the stability of the overall fold, even at temperatures as high as 95ºC. A surprising loss of enzyme activity upon prolonged incubation at high temperature was observed. ESI-MS studies establish that oxidation of thiol groups of the protein may be responsible for the thermal inactivation. Chapter 5 describes the molecular structure of MjTIM, determined in collaboration with Prof. MRN Murthy’s group at the Indian Institute of Science (Gayathri et al., 2007). The crystal structure of the recombinant triosephosphate isomerase (TIM) from the archaeabacteria Methanocaldococcus jannaschii has been determined at a resolution of 2.3 Å. MjTIM is tetrameric, as suggested by solution studies and from the crystal structure, as in the case of two other structurally characterised archaeal TIMs. The archaeabacterial TIMs are shorter compared to the dimeric TIMs, with the insertions in the dimeric TIMs occurring in the vicinity of the putative tetramer interface, resulting in a hindrance to tetramerization in the dimeric TIMs. The charge distribution on the surface of archaeal TIMs also facilitates tetramerization. Analysis of the barrel interactions in TIMs suggests that these interactions are unlikely to account for the thermal stability of archaeal TIMs. A feature of the unliganded structure of MjTIM is the complete absence of electron density for the loop 6 residues. The disorder of the loop may be ascribed to a missing salt bridge between residues at the N- and C- terminal ends of the loop in MjTIM. Chapter 6 is a follow up of an interesting observation made by Vogel and Chmielewski (1994), who noticed that subtilisin cleaved rabbit muscle triosephosphate isomerase religated spontaneously upon addition of organic solvents. Further extension of this nicking and religation process with PfTIM emphasizes the importance of tertiary interactions in contributing to the stability of the (β/α)8 barrel folds (Ray et al., 1999). This chapter establishes that subtilisin nicking and religation is also facile in thermophilic MjTIM. Fragments generated by subtilisin nicking were identified using MALDI mass spectrometry at early and late stages of the cleavage for both the dimeric PfTIM and tetrameric MjTIM. This chapter also describes the comparative thermal and denaturant stability of both the enzymes. The accessibility of the Cys residues of MjTIM has been probed by examining the rates of labeling of thiol groups by iodoacetamide. The differential labeling of Cys residues has been demonstrated by mass spectrometry. Chapter 7 summarizes the main results and conclusions of the studies described in this thesis.

A conversão enzimática da sacarose pela ação sucessiva da invertase e da glicose oxidase (GOD), permite obter produtos de maior valor agregado, a saber, frutose e o ácido glicônico, dois produtos de amplo uso na indústria farmacêutica, alimentícia e química. Foi estudada a aplicação da invertase imobilizada em resinas aniônicas do tipo Dowex® (um copolímero de poliestireno-divinilbenzeno) sobre a hidrólise da sacarose bem como a oxidação da glicose pela glicose oxidase solúvel ou imobilizada no mesmo suporte em separado (sistema bifásico), utilizando-se um reator de membrana acoplado à membrana de ultrafiltração (100kDa) ou de microfiltração (5µm). Posteriormente, avaliou-se o desempenho de ambas as formas de enzimas, solúveis ou imobilizadas num sistema monofásico empregando o mesmo reator. A bioconversão executada em sistema bifásico permitiu a obtenção de xarope de frutose da ordem de 70% através da separação de glicose e frutose utilizando-se a resina catiônica 50W:8-100. O rendimento de 96,6% e 67,4% para as formas solúveis e imobilizadas respectivamente foram obtidas em sistema monofásico. O não desprendimento das enzimas dos suportes viabilizou o uso da membrana de microfiltração, trazendo vantagens à operação de biorreator com membrana. / The enzymatic conversion of sucrose through a successive action of invertase and glucose oxidase (GOO) allows the obtainment of products with higher commercial value, fructose and gluconic acid, which are widely used in pharmaceutical, food and chemical industries. Invertase and GOO immobilized on Dowex® anionic resin (a polystyrene divinylbenzene copolymer) as well as soluble GOD were used in a membrane bioreactor (MS) for sucrose hydrolysis and glucose oxidation. The MB was coupled with a UF-membrane (100kDa) or a MF-membrane (5µm). The bioconversion was conducted in two steps (biphasic system) as well as in one step (monophasic system). The bioconversion operated in a biphasic system permitted obtaining a fructose syrup with a concentration of about 70% through a separation of glucose and fructose using a cationic resin, 50W:8-100. As for the monophasic system, the yield of 96.6% and 67.4% for soluble and immobilized forms were attained respectively. No leakage of the enzymes from the support allowed the use of a microfiltration membrane, adding advantages to the membrane bioreactor operation.

A conversão enzimática da sacarose pela ação sucessiva da invertase e da glicose oxidase (GOD), permite obter produtos de maior valor agregado, a saber, frutose e o ácido glicônico, dois produtos de amplo uso na indústria farmacêutica, alimentícia e química. Foi estudada a aplicação da invertase imobilizada em resinas aniônicas do tipo Dowex® (um copolímero de poliestireno-divinilbenzeno) sobre a hidrólise da sacarose bem como a oxidação da glicose pela glicose oxidase solúvel ou imobilizada no mesmo suporte em separado (sistema bifásico), utilizando-se um reator de membrana acoplado à membrana de ultrafiltração (100kDa) ou de microfiltração (5µm). Posteriormente, avaliou-se o desempenho de ambas as formas de enzimas, solúveis ou imobilizadas num sistema monofásico empregando o mesmo reator. A bioconversão executada em sistema bifásico permitiu a obtenção de xarope de frutose da ordem de 70% através da separação de glicose e frutose utilizando-se a resina catiônica 50W:8-100. O rendimento de 96,6% e 67,4% para as formas solúveis e imobilizadas respectivamente foram obtidas em sistema monofásico. O não desprendimento das enzimas dos suportes viabilizou o uso da membrana de microfiltração, trazendo vantagens à operação de biorreator com membrana. / The enzymatic conversion of sucrose through a successive action of invertase and glucose oxidase (GOO) allows the obtainment of products with higher commercial value, fructose and gluconic acid, which are widely used in pharmaceutical, food and chemical industries. Invertase and GOO immobilized on Dowex® anionic resin (a polystyrene divinylbenzene copolymer) as well as soluble GOD were used in a membrane bioreactor (MS) for sucrose hydrolysis and glucose oxidation. The MB was coupled with a UF-membrane (100kDa) or a MF-membrane (5µm). The bioconversion was conducted in two steps (biphasic system) as well as in one step (monophasic system). The bioconversion operated in a biphasic system permitted obtaining a fructose syrup with a concentration of about 70% through a separation of glucose and fructose using a cationic resin, 50W:8-100. As for the monophasic system, the yield of 96.6% and 67.4% for soluble and immobilized forms were attained respectively. No leakage of the enzymes from the support allowed the use of a microfiltration membrane, adding advantages to the membrane bioreactor operation.