Thesis (Ph. D.)--University of Wisconsin--Madison, 1955. / Typescript. Vita. Includes: (as Section I.A.): Studies of the enzyme fumarase : II. Isolation and physical properties of crystalline enzyme / By Carl Frieden, Robert M. Bock and Robert A. Alberty. Reprinted from Journal of the American Chemical Society, vol. 76 (1954), p. 2482-2484 -- (as Section II. A.): Studies of the enzyme fumarase : III. The dependence of the kinetic constants at 25⁰ upon the concentration and pH of phosphate buffers / By Robert A. Alberty, Vincent Massey, Carl Frieden, and Armin R. Fuhlbrigge. Reprinted from Journal of the American Chemical Society, vol. 76 (1954), p. 2485-2493 -- (as Section III. A.): The effect of pH on fumarase activity in acetate buffer / By Carl Frieden and Robert A. Alberty. Reprinted from Journal of biological chemistry, Vol. 212, no. 2 (Feb. 1955), p. 859-868. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 99-101).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 79-84).

The finely tuned regulation of haematopoiesis is crucial in order to maintain life-long haematopoiesis. The disruption of the balance among cell fates, can lead to malignant transformation. It has become increasingly evident that the metabolic regulation haematopoietic stem cells is critical for stem cell fate decisions. Haematopoietic stem cells reside in a hypoxic microenvironment within the bone marrow and are thought to mainly utilize glycolysis rather than oxidative phosphorylation in order to maintain their pool. However recent evidence suggests that oxidative phosphorylation is critical for quiescent HSCs and in several cases, for leukaemic stem cells (LSCs). One of the key parts of mitochondrial respiration is the tricarboxylic cycle (TCA), providing co-factors for its efficient activity. The TCA functions by catalysing the oxidation of pyruvate via key enzymatic activities. A key component of the TCA cycle is fumarate hydratase (Fh1) which catalyses the hydration of fumarate into malate within the mitochondria, but also catalyses the same reaction in the cytoplasm. FH is a tumour suppressor in human lyomeioma and renal kidney cancer (HLRCC). Previous work conducted by our team has shown that Fh1 is essential for foetal and adult haematopoiesis, as Fh1 deletion within the haematopoietic system is embryonic lethal. Furthermore, conditional deletion of Fh1 in donor cells of the Mx1-Cre system that were injected in lethally irradiated recipients, resulted in the complete reduction of their chimerism in the peripheral blood of recipient mice. Mechanistically, these phenotypes were mostly associated with supra-physiological levels of fumarate as a result of Fh1 deletion. Interestingly, by employing mice that ubiquitously express the human cytosolic isoform of FH (FHCyt, which lacks the mitochondrial targeting sequence and therefore is excluded from the mitochondria), we rescued the embryonic lethality that Fh1 causes, and reduced the levels of fumarate. Importantly, although FHCyt expression restored fumarate-associated lethality, it did not restore the mitochondrial defects, allowing us to study the importance of genetically intact TCA in the context of haematopoiesis. Here I investigated the impact that a genetic truncation of the TCA cycle (as a result of the lack of the mitochondrial isoform of Fh1) has on leukaemic transformation and on aged haematopoiesis. Fh1fl/fl; FHCyt; Vav-iCre mice of approximately 60 weeks old displayed and expansion in the pool of early stem and progenitor compartment (Lin- Sca-1+ c-Kit+), as well as in the early progenitors HPC-1 (LSK CD48+ CD150-) and HPC-2 (LSK CD48+ CD150+). Furthermore, the mice exhibited a drastic depletion of B cells (CD19+ B220+) and an expansion in the frequency of the myeloid compartment (Mac-1+ Gr1+). In order to assess the importance of the TCA cycle in malignant transformation, I isolated stem and progenitor cells from Fh1fl/fl; FHCyt; Vav-iCre (and control (Fh1fl/fl; FHCyt Vav-iCre negative or Fh1fl/fl Vav-iCre negative)) E 14.5 day old embryos and infected them with retroviruses expressing Meis1 and Hoxa9, and generated pre-leukaemic cells (pre-LCs). Genetically intact TCA was required for the efficient generation of leukaemia-initiating cells (LICs), as injection of pre-LCs lacking mitochondrial Fh1 into sub-lethally irradiated recipient mice, resulted in 76 % of leukaemia-free mice while injection of control pre-LCs resulted in 25 % of leukaemia-free mice. However, the genetic perturbation of the TCA did not exert and effect on the long-term self-renewal capacity of LICs. Inducible deletion of mitochondrial Fh1 in established LICs of the Mx1-Cre background using poly (I:C) did not affect their ability to generate AML in primary and secondary recipient mice. These data indicate that genetically intact TCA is required for the efficient generation of LICs in vivo but is dispensable for their long-term self-renewal capacity, highlighting the metabolic rewiring that occurs at different stages of leukaemic transformation. In an effort to understand whether, similarly to HLRCC, Fh1 plays a tumour-suppressive role in malignant haematopoiesis, I isolated LSK cells from the foetal liver of E 14.5 old embryos lacking both isoforms of Fh1. Fh1fl/fl; Vav-iCre cells transduced with Meis1/Hoxa9 or MLL-AF9, MLL-ENL, AML-ETO (chromosomal translocations involved in AML development) -expressing retroviruses, failed to generate colonies in methylcellulose, indicating that stem and progenitor cells require Fh1 to undergo in vitro transformation by these oncogenes. Furthermore, acute deletion of Fh1 (via the use of lentivirally-expressed Cre) in pre-LCs generated using the Meis1/Hoxa9 retroviruses, rendered them unable to generate colonies in methylcellulose, indicating that Fh1 is required for the self-renewal capacity of pre- LCs in vitro. Similarly, when LICs (Fh1fl/fl; Vav-iCre negative) isolated from primary recipient mice were infected with Cre to induce deletion of Fh1, they were unable to generate colonies indicating that Fh1 is required for the self-renewal capacity of LICs in vitro. Finally, in order to identify whether Fh1 is important for LIC self-renewal in vivo I generated Fh1fl/fl; Mx1-Cre pre-LCs by infecting stem and progenitor cells of E 14.5 embryos with Meis1/Hoxa9 retroviruses, and injected them into sub-lethally irradiated mice. After the mice developed AML, I induced the deletion of Fh1, by injecting the mice with poly (I:C). Interestingly, the percentage of LICs in the peripheral blood of recipient mice was drastically decreased, leaving recipient mice leukaemia-free for the remaining time they were monitored. Surprisingly however, approximately 50 % of the recipient mice exhibited a drastic increase in LIC chimerism after two weeks post poly (I:C). Assessment of LICs isolated from recipient mice indicated that Fh1 was fully deleted. These data indicate that while in some cases Fh1 is required for LIC self-renewal in vivo, in other cases it is dispensable. Therefore, the tumour-suppressive roles of Fh1 are likely tissue-specific and do not extend to haematopoietic cells. Overall, this study agrees with published work supporting the notion that intact mitochondrial respiration is important (in varying degrees), in both the contexts of normal and malignant haematopoiesis.

Hypoxia inducible factor-1α (HIF-1α) plays a critical role in the oxygen homeostasis of all metazoans. HIF-1α is a master transcriptional regulator which coordinates the adaptive response to low oxygen tension. Through activation of a plethora of downstream target genes, HIF-1α facilitates oxygenation by promoting angiogenesis and blood vessel dilation, in addition to modulating metabolic pathways to inhibit oxidative phosphorylation and promote glycolytic energy production. Given the critical roles of hypoxia, insufficient blood supply and perturbed energetics in the pathogenesis of cardiovascular disorders, notably ischaemic heart disease, therapeutic modulation of HIF-1α is of significant clinical interest. Previous studies have demonstrated an acute cardioprotective role for both endogenous and supraphysiological HIF-1α signalling in the context of myocardial ischaemia. In contrast, chronic supraphysiological HIF-1α activation in the unstressed heart has been shown to induce cardiac dysfunction. To address the effect of chronic endogenous HIF-1α activation post-myocardial infarction (MI), the present work employed a murine coronary artery ligation (CAL) model in conjunction with temporally-inducible, cardiac-specific deletion of Hif-1α. While CAL surgery successfully modelled myocardial infarction – eliciting substantial adverse cardiac remodelling and contractile dysfunction – there was no evidence of chronic HIF-1α activation by CAL in HIF knockout or control left ventricular samples. In keeping with this, chronic ablation of Hif-1α (from 2 weeks post-CAL) had no discernible additional effect upon cardiac function. Overall, these findings do not support a potential therapeutic role for inhibition of HIF-1α signalling in the chronic phase post-MI. The fundamental tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) converts fumarate to malate. FH deficiency is associated with smooth muscle and kidney tumours which exhibit normoxic HIF signalling due to fumarate accumulation. To investigate the potential for fumarate accumulation to elicit protective HIF signalling, a cardiac-specific Fh1 null mouse was developed through Cre-loxP recombination. Strikingly, despite interruption of the TCA cycle in a highly metabolically demanding organ, cardiac Fh1 null mice were viable, fertile and survived into adulthood, demonstrating the remarkable metabolic plasticity of the heart. However, by 3-4 months Fh1 null mice develop a lethal cardiomyopathy characterised by cardiac hypertrophy, ventricular dilatation and contractile dysfunction. Despite lack of a pseudohypoxic response, Fh1 null hearts did exhibit another phenomenon observed in FH-deficient cancers and also attributed to fumarate accumulation – activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) antioxidant pathway. Heterozygous, but not homozygous, somatic deletion of Nrf2 extended the life expectancy of cardiac Fh1 null mice. Exploration of redox status revealed a more reductive environment in Fh1 null hearts than controls. As a corollary, inhibition of the rate limiting enzyme of the pentose phosphate pathway – a major source of cellular reducing equivalents – with dehydroepiandrosterone conferred striking amelioration of the Fh1 null cardiomyopathy, suggesting a possible pathogenic role for reductive stress. While loss of mitochondrial Fh1 activity and subsequent TCA cycle dysfunction likely contribute to the Fh1 null phenotype, the importance of cytosolic FH was unclear. To clarify this, FH was expressed specifically in the cytosol in vivo. This was sufficient to substantially rescue the Fh1 null cardiomyopathy, supporting a role for cytosolic FH disruption in its pathogenesis. Taken together, these findings highlight the potential for reductive stress to contribute to cardiac dysfunction and suggest a function for cytosolic FH in cardiac metabolic homeostasis.

Fumarato hidratases ou fumarases (FH) catalisam a reação estereoespecífica reversível de hidratação do fumarato em L-malato. Essas enzimas se apresentam em todas as classes de organismos, desde procariotos a eucariotos, e podem ser encontradas nas formas mitocondrial e citosólica. A enzima tem papel importante na produção de energia pois participa do ciclo do ácido cítrico, na resposta ao dano do DNA e como supressor tumoral. A fumarase humana (HsFH), que pertence à classe II, é codificada pelo gene 1q42.1, possui 467 aminoácidos em cada monômero com peso molecular de 50,2 kDa cada. Estudos associaram mutações no gene da FH com diversas doenças humanas como acidúria fumárica, leiomiomatoses de útero e pele (MCUL), que quando associadas com um agressivo carcinoma múltiplo de células é conhecido como leiomiomatose hereditária e câncer renal (HLRCC). Apesar da grande importância da fumarase humana no metabolismo energético, ainda há pouca informação em relação ao mecanismo catalítico adotado pela enzima e o efeito estrutural e cinético causado pelas mutações envolvidas com essas doenças. Diante disso, nosso trabalho utilizou uma abordagem híbrida que envolve a caracterização biofísica, bioquímica e estrutural da enzima HsFH, e seus mutantes: N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, G282VHsFH, E362QHsFH, S365GHsFH e N373DHsFH, identificados em pacientes. Estudos cinéticos foram realizados em sete diferentes pHs e, pela primeira vez para fumarases, o ensaio foi realizado com os dois substratos presentes na mesma mistura reacional, confirmando a contribuição da reação reversa para a velocidade global da enzima. De acordo com os estudos de termoflúor a proteína é estabilizada em pHs alcalinos e através da ligação de compostos no sítio ativo. A estrutura da enzima HsFH nativa foi resolvida a 1,8 Å e identificou a presença de moléculas de HEPES complexadas na região C-terminal da enzima. Os estudos cinéticos demonstraram um aumento da eficiência catalítica na presença do HEPES, sugerindo um possível papel alostérico de seu sítio de ligação para a atividade catalítica. Foram determinadas as estruturas para os mutantes N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, E362QHsFH, S365GHsFH e N373DHsFH. As mutações Q185R, E362Q, S365G e N373D foram identificadas no sítio ativo afetando diretamente a capacidade da proteína em ligar os substratos, enquanto que a mutação H180R foi localizada no sítio B, que conduz os substratos e produtos para dentro e fora do sítio ativo. Já a mutação K230R está localizada no domínio central, mas os resultados de termoflúor demonstram um efeito direto na capacidade da enzima em acomodar o substrato. A mutação N107T, localizada longe do sítio ativo foi a única que permaneceu ativa e teve seus parâmetros cinéticos residuais determinados. O presente trabalho contribui para o entendimento das bases estruturais que correlacionam mutações na HsFH, deficiência enzimática e patologia. / Fumarate hydratases or fumarases (FH) catalyze the reversible stereospecific hydration of fumarate to L-malate. They are present in all classes of organisms, from prokaryotes to eukaryotes, and can be found in the mitochondrial and cytosolic forms. The enzyme has an important role in energy production as part of the well-known Citric Acid Cycle, in DNA damage response and as tumor suppressor. Human fumarase (HsFH) belongs to class II and is encoded by 1q42.1 gene. HsFH is tetrameric and has 467 amino acids per monomer, with predicted molecular weight of 50.2 kDa. Several studies associated FH gene mutations with some human diseases such as fumaric aciduria, multiple cutaneous and uterine leiomyomatosis (MCUL), which when associated with an aggressive form of multiple cell carcinoma is known as hereditary leiomyomatosis and renal cancer (HLRCC) syndrome. Although the major role of HsFH in energetic metabolism, there are still little structural and kinetic information about the mutants involved in these diseases. Thus, this study aims, through a hybrid approach, composed by biophysics, biochemical and structural characterization of mutants N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, G282VHsFH, E362QHsFH, S365GHsFH and N373DHsFH identified from patients. Steady-state kinetics studies were performed in seven different pHs and, for the first time, the contribution of both substrates was analyzed simultaneously in a single kinetic assay and allowed to quantify the contribution of the reverse reaction for kinetics. According to thermofluor studies, structural stability can be achieved at alkaline pHs and suggests that ligand binding can modulate the protein stability. HsFH crystal structure was solved at 1.8 Å resolution and identified HEPES molecules complexed with the enzyme C-terminal region. Kinetics studies with HEPES showed an increase of the catalytic efficiency and suggests that HEPES binding site might have an allosteric role. Crystal structures for the mutants N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, E362QHsFH, S365GHsFH and N373DHsFH were determined. The mutations Q185R, E362Q, S365G and N373D were identified in the active site and affect the substrate binding capacity directly, while mutation H180R was localized in the B site, which conducts the substrates and products in and out the active site. The mutation K230R is localized in the central domain, but thermofluor results demonstrate a direct effect on the ability of the enzyme to accommodate the substrate. The N107T mutation located far from the active site was the only one that remained active and had its residual kinetic parameters determined. The present work contributes to the understanding of the structural bases that correlate mutations in HsFH, enzymatic deficiency and pathology.

<p>In eukaryotic cells, DNA is packaged into a structure named chromatin which contains DNA and proteins. Nucleosomes are building blocks of chromatin and contain DNA wrapped around a histone octamer. Chromatin modifications (histone post-translational modifications and histone variants) play central roles in various cellular processes including gene expression and DNA damage response. Chromatin modifying enzymes use metabolites as co-substrates and co-factors, and changes in metabolic pathways and metabolite availability affects chromatin modifications and chromatin-associated functions. Moreover, recent studies have uncovered direct roles of metabolic enzymes in chromatin-associated functions. Fumarase, a TCA cycle enzyme that catalyzes the reversible conversion of fumarate to malate in mitochondria (a hydration reaction), is an example of an enzyme with dual functions in metabolism and genome integrity. Cytoplasmic fraction of yeast fumarase, Fum1p, localizes to the nucleus and promotes growth upon DNA damage. Fum1p promotes homologous recombination by enhancing DNA end resection. Human fumarase is involved in DNA repair by non-homologous end joining. Here, we provide evidence that yeast Fum1p and the histone variant Htz1p are also involved in DNA replication stress response and DNA repair by non-homologous end joining (NHEJ). Using mutants lacking the histone variant <i>HTZ1</i>, we show that high cellular levels of fumarate, by deletion of <i>FUM1</i> or addition of exogenous fumarate, suppressed the sensitivity to DNA replication stress by modulation of activity of Jhd2p. This suppression required sensors and mediators of the intra-S phase checkpoint, but not factors involved in the processing of replication intermediates. These results imply that high cellular levels of fumarate can confer resistance to DNA replication stress by bypassing or complementing the defects caused by loss of <i>HTZ1</i> and replication fork processing factors. We also show that upon induction of DSBs, exogenous fumarate conferred resistance to mutants with defects in NHEJ, early steps of homologous recombination (DNA end resection pathway) or late steps of homologous recombination (strand invasion and exchange). Taken together, these results link the metabolic enzyme fumarase and the metabolite fumarate to DNA damage response and show that modulation of DNA damage response by regulating activity of chromatin modifying enzymes is a plausible pathway linking metabolism and nutrient availability to chromatin-associated functions like genome integrity.<br><a></a></p>

Introduction: Uterine fibroids are the most common benign tumours of female genital tract with the peak incidence in the 4th and 5th decennium. The aetiology of uterine fibroids still remains poorly understood. Genetic factors play undisputed role in the onset of uterine fibroids. Up to date numerous gene mutations were identified in certain percentage of patients with uterine fibroids. One of the candidate genes is Fumarate hydratase gene (FH). Heterozygous germiline mutations of FH cause two hereditary syndromes: Multiple smooth muscle tumours of the skin and uterus (MCUL1)/ Hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC) characterised by leiomyomata of the skin, early onset uterine fibroids between 20-30 years of age and renal papillary carcinoma. The aim of our thesis was to identify the frequency of FH mutations in patients with early onset sporadic uterine fibroids. Methods: Patients with the diagnosis of uterine fibroids up to the age of 30 years were enrolled in the study. Control group consisted of patients with absence of uterine fibroids. Activities of Fumarate hydratase and control protein Citrate synthase were measured in lymphocytes and compared to the results obtained from the healthy controls. Mutation analysis of FH gene was performed. Activity of Fumarate...

Introduction: Uterine fibroids are the most common benign tumours of female genital tract with the peak incidence in the 4th and 5th decennium. The aetiology of uterine fibroids still remains poorly understood. Genetic factors play undisputed role in the onset of uterine fibroids. Up to date numerous gene mutations were identified in certain percentage of patients with uterine fibroids. One of the candidate genes is Fumarate hydratase gene (FH). Heterozygous germiline mutations of FH cause two hereditary syndromes: Multiple smooth muscle tumours of the skin and uterus (MCUL1)/ Hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC) characterised by leiomyomata of the skin, early onset uterine fibroids between 20-30 years of age and renal papillary carcinoma. The aim of our thesis was to identify the frequency of FH mutations in patients with early onset sporadic uterine fibroids. Methods: Patients with the diagnosis of uterine fibroids up to the age of 30 years were enrolled in the study. Control group consisted of patients with absence of uterine fibroids. Activities of Fumarate hydratase and control protein Citrate synthase were measured in lymphocytes and compared to the results obtained from the healthy controls. Mutation analysis of FH gene was performed. Activity of Fumarate...

La protéine kinase activée par AMP (AMPK) est un senseur et régulateur central de l'état énergétique cellulaire, mais ces voies de signalisation ne sont pour le moment que partiellement comprises. Deux criblages non-biaisés pour la recherche de partenaires d'interaction et de substrats d'AMPK ont précédemment été réalisés dans le laboratoire. Ces derniers ont permis l'identification de plusieurs candidats (protéines), mais leur rôle fonctionnel et physiologique n'était pas encore établi. Ici nous avons caractérisé la fonction de la relation entre AMPK et quatre partenaires d'interaction : gluthation S-transferases (GSTP1 and GSTM1), fumarate hydratase (FH), l'E3 ubiquitine-ligase (NRDP1), et les protéines associées à la membrane (VAMP2 and VAMP3). Chacune de ces interactions parait avoir un rôle différent dans la signalisation AMPK, agissant en amont ou en aval de la protéine AMPK. GSTP1 et GSTM1 contribueraient à l'activation d'AMPK en facilitant la S-glutathionylation d'AMPK en conditions oxydatives moyennes. Cette régulation non-canonique suggère que l'AMPK peut être un senseur de l'état redox cellulaire. FH mitochondrial est l'unique substrat AMPK clairement identifié. Etonnamment le site de phosphorylation se trouve dans le peptide signal mitochondrial, ce qui pourrait affecter l'import mitochondrial. NRDP1, protéine pour laquelle nous avons pour la première fois développé un protocole de production de la protéine soluble, est faiblement phosphorylée par l'AMPK. L'interaction ne sert pas à l'ubiquitination d'AMPK, mais affecte le renouvellement de NRDP1. Finalement, l'interaction de VAMP2/3 avec AMPK n'implique pas d'évènement de phosphorylation ou d'activation d'un des partenaires. Nous proposons un mécanisme de recrutement d'AMPK par VAMP2/3 (" scaffold ") au niveau des vésicules en exocytose. Ce recrutement favoriserait la phosphorylation de substrats de l'AMPK à la surface des vésicules en exocytoses. Une fois mis en commun, nos résultats enrichissent les connaissances sur les voies de signalisation AMPK, et suggèrent une grande complexité de ces dernières. Plus que les kinases en amont et des substrats en aval, la régulation de la signalisation d'AMPK se fait via des modifications secondaires autres que la phosphorylation, via des effets sur le renouvellement de protéines, et probablement via un recrutement spécifique de l'AMPK dans certains compartiments cellulaires. / AMP-activated protein kinase (AMPK) is a central energy sensor and regulator of cellular energy state, but the AMPK signaling network is still incompletely understood. Two earlier non-biased screens for AMPK interaction partners and substrates performed in the laboratory identified several candidate proteins, but functional and physiological roles remained unclear. Here we characterized the functional relationship of AMPK with four different protein interaction partners: gluthatione S-transferases (GSTP1 and GSTM1), fumarate hydratase (FH), an E3 ubiquitin-ligase (NRDP1), and vesicle-associated membrane proteins (VAMP2 and VAMP3). Each of these interaction partners seems to have a different function in AMPK signaling, either acting up- or down-stream of AMPK. GSTP1 and GSTM1 can contribute to AMPK activation by facilitating S-glutathionylation of AMPK under mildly oxidative conditions. This non-canonical regulation suggests AMPK as a sensor of cellular redox state. Mitochondrial FH was identified as the only clear AMPK downstream substrate, but surprisingly the phosphorylation site is present in the mitochondrial targeting prepeptide, possibly affecting mitochondrial import. NRDP1, whose expression as a full-length soluble protein was achieved here for the first time, is phosphorylated by AMPK only at low levels. The interaction does neither serve for AMPK ubiquitinylation, but rather affects NRDP1 turnover. Finally, interaction of VAMP2/3 with AMPK does not involve phosphorylation or activation events of one of the partners. Instead, we propose VAMP2/3 as scaffolding proteins that recruit AMPK to exocytotic vesicles which could favor phosphorylation of vesicular AMPK substrates for exocytosis. Collectively, our results add some new elements to the AMPK signaling network, suggesting that it is much more complex than anticipated. In addition to upstream kinases and downstream substrates, regulation of AMPK signaling occurs by second