Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

The effect of temperature on the oxidative-decarboxylation of isocitrate by the soluble NADP+-linked isocitrate dehydrogenase (NADP-IDH, EC 1.1.1.42) from rainbow trout (Salmo gairdnerii) liver has been investigated. A particular interest was given those properties of the enzyme which might help to explain the temperature-independent function of the Krebs cycle and the large increase in fatty acid synthesis known to occur during low temperature acclimation. Within the thermal range experienced by rainbow trout, control of catalysis by this enzyme is temperature-independent. Acclimation to an altered thermal regime is accompanied by an increase in the relative proportion of the slowest migrating isozyme of liver NADP-IDH on starch-gel electrophoresis. These cold- and warm-isozyme variants display different and adaptive Km-temperature relationships, and allow for temperature-independent modulation of enzyme activity through the entire thermal range this species is likely to encounter in nature. Other trout species, including the brook (Salvelinus frontinalis), lake (Salmo namaycush) and their hybrid, the splake trout, were investigated for similar responses. The elaboration of enzyme variants in brook and splake trout are complexly regulated by temperature changes, but the lake trout genome contains a single gene coding for liver NADP-IDH which is not affected by temperature. Catalysis by the trout liver enzyme is modulated not only by temperature, but also ADP and ɣ-KGA. Both of these metabolites alter the Km of DL-isocitrate; at physiological ADP concentrations, the Km is reduced as it is with ɣ-KGA below 0.05 mM, but at higher ɣ-KGA concentrations it is markedly increased. These two controls suggest this enzyme may be important for the Krebs cycle oxidation of isocitrate. The availability of a purified NADP-IDH from pig heart allowed a study of the kinetic properties of homologous enzymes from both a poikilotherm and a homeotherm. Even though the molecular weights, Ea values, substrate, cofactor and inhibitor specificities are similar, subtle changes in enzyme structure and/or conformation as identified by electrophoresis, may result in the observed differences in temperature characteristics. These apparent adaptive enzyme responses are of importance to the rainbow trout which lives in a fluctuating thermal regime, but not the pig which does not experience these changes. In vivo, the response of enzymes to temperature fluctuations may be quite different to those seen in vitro. The locus(i) coding for rainbow trout liver NADP-IDH was found to contain a large amount of heterogeneity; in fact, seven distinct phenotypes were found to coexist in one hatchery population. The kinetics of three of these phenotypes were investigated and it was found that by increasing the number of slow moving isozymes, an increase in Km(DL-isocit) at high assay temperatures occurs. This suggests that irrespective of changes in the cellular milieu, isozymal content can determine the Km-temperature response. The data from this study suggest that changes in enzyme-substrate affinity with temperature as a result of either the temperature directed production of enzyme variants and/or their genetic expression, are important in controlling the catalytic activity of NADP-IDH from the eurythermal rainbow trout. Also, unlike the mammalian enzyme, the trout liver enzyme may be important in both fatty acid synthesis and the Krebs cycle oxidation of isocitrate. / Science, Faculty of / Zoology, Department of / Graduate

<p>Gliomas are the most common tumors of the central nervous system. Our lab recently identified mutations in <italic>IDH1</italic> and <italic>IDH2</italic> as occurring frequently in progressive gliomas. We applied a series of biochemical and genetic approaches to explore the roles of the mutations in tumors and generate models for study. </p><p><italic>IDH1/2</italic> mutations have the potential to impact a number of metabolic pathways. IDH1/2 convert isocitrate to &#945;-ketoglutarate while simultaneously converting NADP+ to NADPH. To assess changes in metabolism, we completed metabolic profiling and complementary studies in cell lines with and without mutant <italic>IDH1</italic> or mutant <italic>IDH2</italic>. We identified a decrease in hypoxia signaling and a decrease in global 5-hydroxymethylcytosine in cell lines with mutant <italic>IDH1/2</italic> .</p><p>Having observed mutations in <italic>IDH1/2</italic> in a large fraction of progressive gliomas, we asked if the mutations were either 1) advantageous for growth in brain parenchyma, or 2) advantageous in a particular cell-of-origin. Sequencing of a series of metastases to the brain from non-central nervous system tumors identified no mutations in <italic>IDH1/2</italic>, lending less credence to the first hypothesis. To elucidate whether mutations in <italic>IDH1/2</italic> can initiate glioma progression and explore the potential cell-of-origin for progressive gliomas, we generated mice in which we induced expression of mutant <italic>IDH2</italic> in different populations of cells in the brain, either alone or in combination with <italic>TP53</italic> deletion, another frequently altered gene in progressive gliomas. Mice with broad expression of mutant <italic>IDH2</italic> developed hydrocephalus and encephalomalacia early in life, but did not develop tumors. Therefore, we restricted expression, and two brain tumors were identified in mice with both <italic>IDH2</italic> mutation and <italic>TP53</italic> deletion. While this suggests that both mutations might be required for the development of tumors, this is too small a number to draw significant conclusions. Further research with an expanded cohort of mice, utilization of additional drivers of expression, and further characterization of identified tumors will help in elucidating the role of mutant <italic>IDH2</italic> and the cell-of-origin for progressive gliomas.</p> / Dissertation

Aberrant metabolism has become an increasingly interesting area of cancer biology. In many cancers including lower grade glioma, glioblastomas and some leukemias, a mutation in the metabolic enzyme Isocitrate Dehydrogenase (IDH), has been found in more than 70% of cases and has been shown to lead to a distinct hypermethylator phenotype. IDH commonly converts isocitrate to alpha-ketoglutarate in normal cell metabolism. Three isoforms of this enzyme are found in humans: IDH1, IDH2 and IDH3. Studies on IDH1, the cytosolic isoform, have revealed that mutations in the enzyme’s binding site lead to a novel gain of function: the synthesis of an oncogenic metabolite, 2-hydroxyglutarate (2HG). 2HG competitively inhibits alpha-ketoglutarate dependent enzymes such as the TET 5-methylcytosine (5mC) oxygenases and histone demethylases. These oxygenases are responsible for the hydroxymethylation (5hmC) of cytosine residues, ultimately leading to demethylation and gene re-expression. Thus, mutant IDH may lead to an elevation in 5mC levels producing the hypermethylator phenotype described. A similar gain-of-function mutation in IDH2, the mitochondrial isoform of IDH1, has been associated with leukemias and gliomas lacking IDH1 mutations. Mutations in IDH1, IDH2 and TET2 are mutually exclusive, and each yields a similar hypermethylator phenotype. IDH2, along with IDH3, is primarily involved in the TCA cycle and energy production for the cell. Recently, the Taylor lab has uncovered evidence of 5mC and 5hmC residues in mitochondrial DNA, established and maintained by mtDNMT1 and TET2. Changing levels of mtDNMT1 appears to alter the patterns and levels of mtDNA transcription from the mitochondrial genome. We hypothesized that mutant IDH would produce a similar effect on the mitochondrial genome as that found in the nuclear genome and result in a decrease in the level of 5-hydroxymethylcytosine, as well as a subsequent increase in the level of 5-methylcytosine caused by the competitive inhibition of the TET enzymes by 2-hydroxyglutarate accumulation. Using molecular biology techniques such as Western blots and MeDIP (methylated DNA immunoprecipitation) I aim to uncover the role of IDH mutation on mitochondrial DNA methylation and its effect on energy production in mammalian cells.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2006. / Vita. Includes bibliographical references.

The main subject of my thesis is the investigation of mechanisms of glioma tumorigenesis associated with the recently identified mutations in isocitrate dehydrogenase. Gliomas account for 80% of primary brain cancers. They represent a diverse group of tumours, and are graded from I-IV based on histopathological features. Whilst grade I tumours may be curable with surgery alone, grade II and III gliomas inevitably progress to glioblastoma multiforme (GBM), which is highly resistant to current therapies and carries a very poor prognosis. Despite an improved understanding of the pathways and mechanisms involved in the development of glioma and its progression to grade IV disease, current and novel treatments have so far failed to significantly improve outcome. Isocitrate dehydrogenase (IDH) enzymes catalyse the oxidative decarboxylation of isocitrate to α-ketoglutarate (&alpha;-KG). Somatic mutations in genes encoding IDH1 and IDH2 were first identified in glioma and subsequently in acute myeloid leukemia and other solid tumours. These heterozygous point mutations occur at the arginine residue of the enzymes active site and cause both loss of normal enzyme function and gain-of-function, causing the reduction of &alpha;-KG to D-2-hydroxyglutarate (D-2HG), which accumulates. D-2HG may act as an oncometabolite through the inhibition of various &alpha;-KG dependent enzymes, stimulating angiogenesis, histone modifications and aberrant DNA methylation. Possibly, IDH1/2 mutations may also cause oncogenic effects through dysregulation of the tricarboxylic acid (TCA) cycle, or by increasing susceptibility to oxidative stress. The exact role of mutant IDH1/2 in tumorigenesis however remains unclear. In the work outlined in this thesis, I have demonstrated that the expression of mutant IDH1/2 in glioma cell lines leads to 2-HG accumultation and a reduction in &alpha;-KG production and results in HIF1&alpha; accumulation and a reduction in 5hmC production. Furthermore, the brain-specific expression of mutant Idh1 in mice also results in 2-HG accumulation and reduced &alpha;-KG production, whilst a reduction in 5hmC levels are also seen. This data appears to support the theory that IDH1/2 mutant activity results in the inhibition of &alpha;-KG dependent enzymes, either through the accumulation of 2-HG or due to a reduction in &alpha;-KG levels. The brain-specific expression of mutant Idh1 in mice also results in increased cellular proliferation and an increase in the expression of the neural stem cell marker, nestin. However gliomas do not develop, perhaps suggesting that additional mutations are required in conjunction with those occuring in IDH1/2 in order to initiate tumourigenesis. Clinically, IDH1/2 mutations may represent a novel therapeutic target in glioma and may also serve as useful diagnostic, prognostic and predictive biomarkers. However, a better understanding of the pathogenesis of mutant IDH is required, to enable effective IDH1/2 directed therapies to be developed in the future.

Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (<i>Nicotiana tabacum L.</i>), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, indicating the possibility of two tandem targeting signals. In this work the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) to generate a series of constructs and introduced into tobacco leaves by <i>Agrobacterium</i>-mediated transient transfection. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined under the confocal microscope. Constructs predicted to be targeted to the chlroplast all localized to the chloroplast. However, this was not the case for constructs that were predicted to be mitochondrial targeted. While some constructs localized to mitochondria, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein which was present in those constructs. In addition, during the process of generating these constructs our sequence analysis indicated a stop codon present at amino acid position 161 of the mature NADP+-ICDH protein from both Xanthi and Petit Havana cultivars of tobacco. This was confirmed by multiple sequencing reactions and created discrepancies with the reported sequence present in the database. The results of this study raise interesting questions with regard to the targeting and processing of NADP+-ICDH.

Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (<i>Nicotiana tabacum L.</i>), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, indicating the possibility of two tandem targeting signals. In this work the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) to generate a series of constructs and introduced into tobacco leaves by <i>Agrobacterium</i>-mediated transient transfection. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined under the confocal microscope. Constructs predicted to be targeted to the chlroplast all localized to the chloroplast. However, this was not the case for constructs that were predicted to be mitochondrial targeted. While some constructs localized to mitochondria, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein which was present in those constructs. In addition, during the process of generating these constructs our sequence analysis indicated a stop codon present at amino acid position 161 of the mature NADP+-ICDH protein from both Xanthi and Petit Havana cultivars of tobacco. This was confirmed by multiple sequencing reactions and created discrepancies with the reported sequence present in the database. The results of this study raise interesting questions with regard to the targeting and processing of NADP+-ICDH.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 6 uppsatser.