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Hexokinase has been found in a soluble and a bound form in homogenates of Ascaris suum muscle. Cellulose acetate electrophoresis, isoelectric focusing, and ion exchange chromatography confirmed the presence of only one molecular form of hexokinase in this muscle. A procedure for purifying hexokinase from Ascaris muscle has been developed. The physical and catalytic properties of the enzyme are comparable to mammalian hexokinases. The enzyme is a monomer with a molecular weight of 100,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The Michaelis constants for glucose and ATP are 4.7 x 10-3 M and 2.2 x 10-4 M, respectively. Ascaris' hexokinase also exhibits end-product inhibition by glucose 6-phosphate and ADP. It is postulated that the kinetic para meters of the enzyme are the results of its function, that of generating glucose 6-phosphate primarily for glycogen synthesis.

Hexokinase is the first enzyme in glycolysis, a major pathway for the generation of energy in all eukaryotes. Mammalian cells have four isoforms (I, II, III, IV) that have different tissue distribution and kinetic properties. Among all isoforms, human hexokinase II (hHKII) has been found to be implicated in many cancers with an increased expression which serves a dual role. First, it maintains the high glycolytic rate of malignant cells (Warburg effect) and second it prevents apoptosis when is bound to mitochondria. Trypanosoma brucei is a parasite that causes Human African Trypanosomiasis (HAT) and has two isoforms with extensive sequence similarity (98%), TbHKI (active form) and TbHK2 (inactive form). The bloodstream-form parasites (BSF) depend exclusively on glycolysis for their survival. The enzyme from both organisms is a validated target for drug-discovery against both cancer and HAT. The aim of the present study is the discovery of novel and specific inhibitors of the enzymes based on their structure. Structure-based drug discovery is commonly used in pharmaceutical companies to aid in the discovery of potent lead compounds. In silico studies were performed in this project using the known crystal structure of human hexokinase I and a model of TbHKI generated by the protein modelling tool Phyre2. The docking programs, AutoDock (AD) and AutoDock Vina (Vina), were chosen to perform the docking of ~3 million compounds to the target molecules and scoring functions calculated the predicted binding affinities of each compound. In total, 28 compounds were purchased to test on the target molecules. In the experimental part of the project, the two enzymes were cloned, expressed and purified. hHKII was successfully purified giving a high yield of active and pure protein. The protein was characterised using many biophysical methods to establish the oligomeric state, the homogeneity and the secondary structure. Crystallisation trials failed and for this reason, N and C domains of the hHKII were purified separately. Unfortunately, the domains also failed to crystallise thus SAXS data were collected and analysed to gain information of their shape at low resolution. A novel inhibition assay was developed and used to identify four weak inhibitors against full length hHKII. TbHKI was difficult to express in a soluble form as most of the protein was expressed in inclusion bodies. The purification resulted in a small amount of active protein that was used entirely for biochemical assays. Four compounds were purchased from the docking of the TbHKI model and one was found to inhibit the enzyme over 65% at 100 μM. Because the active site of both enzymes (hHKII, TbHKI) is well conserved the compounds from hHKII docking were also screened against the TbHKI. Four compounds were found to inhibit this enzyme while one of them was also an inhibitor for human isoform. The remaining three were specific for inhibition of TbHKI.

Würzburg, Univ., Diss., 2008 / Zsfassung in engl. Sprache

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 4 uppsatser.

Proliferating tissues, including embryonic and tumour tissues, preferentially employ aerobic glycolysis to support cell growth. This reliance on glycolysis even in the presence of oxygen, referred to as the “Warburg Effect”, may confer a proliferative, survival and invasive advantage and be exploited therapeutically. In this thesis, we demonstrate that the glycolytic enzyme Hexokinase 2 (HK2) is crucial for the “Warburg Effect” in human Glioblastoma Multiforme (GBM), the most common and therapeutically resistant malignant brain tumour. In contrast to normal brain and low-grade gliomas, GBMs exhibited a marked increase in HK2 expression, but not HK1, particularly in perinecrotic, hypoxic regions and its expression predicted poor overall survival of GBM patients. Stable loss of HK2 in GBM cells restored oxidative phosphorylation (OXPHOS)-mediated glucose metabolism, with increased oxygen consumption and decreased lactic acid production, an effect not seen with loss of glycolytic enzymes HK1 or PKM2. Furthermore, HK2 depletion resulted in decreased proliferation in vitro and in vivo and increased sensitivity to apoptotic inducers such as radiation and chemotherapy, both common adjuvant therapies of GBMs. Intracranial xenografts of GBM cells with reduced HK2 demonstrated significantly increased survival with decreased proliferation and angiogenesis yet enhanced invasiveness. In contrast, exogenous HK2 expression in GBM cells promoted proliferation, therapeutic resistance and intracranial growth. This was dependent partly on the PI3K/AKT dependent translocation of HK2 to the mitochondrial membrane. Stable loss of glycolytic enzymes HK2, HK1 and PKM2 reduced GBM proliferation but differentially altered the PI3K/AKT/mTOR and AMPK signaling pathways, the extent to which may influence whether a cell preferentially undergoes autophagy or apoptosis as the primary mode of cell death. Collectively, targeting enzymes employed by the tumour to modulate its energy metabolism, such as HK2 in GBMs, may favourably alter its therapeutic sensitivity to radiation and both classical and novel chemotherapeutic agents.

Proliferating tissues, including embryonic and tumour tissues, preferentially employ aerobic glycolysis to support cell growth. This reliance on glycolysis even in the presence of oxygen, referred to as the “Warburg Effect”, may confer a proliferative, survival and invasive advantage and be exploited therapeutically. In this thesis, we demonstrate that the glycolytic enzyme Hexokinase 2 (HK2) is crucial for the “Warburg Effect” in human Glioblastoma Multiforme (GBM), the most common and therapeutically resistant malignant brain tumour. In contrast to normal brain and low-grade gliomas, GBMs exhibited a marked increase in HK2 expression, but not HK1, particularly in perinecrotic, hypoxic regions and its expression predicted poor overall survival of GBM patients. Stable loss of HK2 in GBM cells restored oxidative phosphorylation (OXPHOS)-mediated glucose metabolism, with increased oxygen consumption and decreased lactic acid production, an effect not seen with loss of glycolytic enzymes HK1 or PKM2. Furthermore, HK2 depletion resulted in decreased proliferation in vitro and in vivo and increased sensitivity to apoptotic inducers such as radiation and chemotherapy, both common adjuvant therapies of GBMs. Intracranial xenografts of GBM cells with reduced HK2 demonstrated significantly increased survival with decreased proliferation and angiogenesis yet enhanced invasiveness. In contrast, exogenous HK2 expression in GBM cells promoted proliferation, therapeutic resistance and intracranial growth. This was dependent partly on the PI3K/AKT dependent translocation of HK2 to the mitochondrial membrane. Stable loss of glycolytic enzymes HK2, HK1 and PKM2 reduced GBM proliferation but differentially altered the PI3K/AKT/mTOR and AMPK signaling pathways, the extent to which may influence whether a cell preferentially undergoes autophagy or apoptosis as the primary mode of cell death. Collectively, targeting enzymes employed by the tumour to modulate its energy metabolism, such as HK2 in GBMs, may favourably alter its therapeutic sensitivity to radiation and both classical and novel chemotherapeutic agents.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 3 uppsatser.

Human Rhinoviruses (HRVs) utilize Internal Ribosome Entry Sites (IRES) to drive viral protein synthesis. IRESs are specialized RNA elements present within the 5’ UTR of mRNAs that recruit ribosomes independently of the 5’ m7G cap structure. hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1), a multifunctional RNA binding protein, is required for the IRES-dependent translation of many specific RNAs within the cell cytoplasm. The phosphorylation of hnRNP A1 is required for its cytoplasmic accumulation. I have identified and validated the role of HK2 in hnRNP A1 cellular localization by immunofluorescence microscopy, by analysis of HRV infection and by siRNA-based screening. These studies show that decreased HK2 protein levels lead to decreased cytoplasmic accumulation of hnRNPA1 during osmotic shock and HRV infection, to a decrease in HRV-infected cells and to decreased caspase activation in osmotically stressed and HRV-infected cells. Thus, HK2 may regulate hnRNP A1 cytoplasmic localization following HRV infection.

The cytokine, interleukin-7 (IL-7), has essential growth activities that maintain the homeostatic balance of the immune system. Little is known of the mechanism by which IL-7 signaling regulates metabolic activity in support of its vital function in lymphocytes. We observed that IL-7 deprivation caused a rapid decline in ATP levels that were attributable to loss of intracellular glucose retention. To identify the transducer of the IL-7 metabolic signal, we examined the expression of three important regulators of glucose metabolism, the glucose transporter, GLUT-1, and two glycolytic enzymes, Hexokinase II (HXKII) and phosphofructokinase-1 (PFK1), using an IL-7-dependent T-cell line and primary lymphocytes. We found that in lymphocytes deprived of IL-7 loss of glucose uptake correlated with decreased expression of HXKII. Re-addition of IL-7 to cytokine deprived lymphocytes restored the transcription of the HXKII gene within 2 hours, but not that of GLUT-1 or PFK1. IL-7-mediated increases in HXKII, but not GLUT-1 or PFK-1, were also observed at the protein level. Inhibition of HXKII with 3-Bromopyruvate or specific siRNA decreased glucose utilization, as well as ATP levels, in the presence of IL-7, while over-expression of HXKII, but not GLUT-1, restored glucose retention and increased ATP levels in the absence of IL-7. This IL-7 mediated HXKII gene expression was abrogated with inhibition of JNK pathway. IL-7 also increased activation of AP-1 complex and DNA binding of JunD, a transcriptional complex thought to be negative regulator of proliferation. We found that over expression of HXKII caused cell cycle arrest and cell death, indicating that a potent IL-7 signal could produce negative growth signals. We conclude that IL-7 controls glucose utilization by regulating the gene expression of HXKII through activation of JNK-JunD pathway, suggesting a mechanism by which IL-7 supports bioenergetics that control cell fate decisions in lymphocytes.