Pyruvate kinase catalyses the final step in glycolysis and is responsible for net ATP production. There are four pyruvate kinase isoforms expressed in humans; LPYK, RPYK, M1PYK and M2PYK. The allosteric enzyme M2PYK plays an important role in cancer cell metabolism and is subject to complex regulation by numerous naturally occurring small-molecule metabolites. Post-translational modifications have also been found to play a key role in the regulation of M2PYK, among these cysteine oxidation. This thesis describes the production and characterisation of M2PYK cysteine point mutants in order to investigate the mechanism of regulation by cysteine modification. From a total of ten cysteines present in M2PYK, five were chosen for mutation based on a combination of the results from the cysteine oxidation prediction program (COPP) web interface and published experimental evidence for cysteine modification of M2PYK. Eight point mutants of these five cysteines were produced and characterised. Low resolution gel filtration of all the mutants shows that mutation of these cysteines has an effect on tetramer:dimer:monomer equilibrium of M2PYK suggesting that cysteine modifications could regulate M2PYK activity by affecting oligomeric state. Activity assays show that none of the cysteine point mutations are sufficient to protect M2PYK from oxidation by H2O2 indicating that more than one cysteine is involved in the regulation of M2PYK by oxidation. Nitric oxide (NO) imbalance has recently emerged as playing a key role in numerous diseases including cancer. NO regulates the function of target proteins through the addition of a nitroso moiety from NO-derived metabolites to a reactive cysteine, a process known as protein S-nitrosylation. M2PYK has been found to be S-nitrosylated in vivo. Using the biotin-switch assay in vitro combined with mass spectrometry I have shown that a likely candidate for the target of S-nitrosylation of M2PYK is C326. This thesis also describes the structures of two cysteine point mutants; M2PYK C424A and M2PYK C358S. The structures show that these mutations have very little effect on the overall conformation of M2PYK with only very subtle localised changes. The structure of the mutant M2PYK C358S shows some interesting features including varying occupation of the active site resulting in differing conformations of the B domains within the same tetramer, and an unusual B factor distribution which could be indicative of a perturbation in cooperativity within the tetramer caused by the mutation.

No description available.

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme expressed in highly proliferating cells. Studies of PKM2 have been focused on its function of promoting cell proliferation in cancer cells. Our laboratory previously discovered that extracellular PKM2 released from cancer cells promoted angiogenesis by activating endothelial cell proliferation and migration. PKM2 activated endothelial cells through integrin αvβ3. Angiogenesis and myofibroblast differentiation are key processes during wound healing. In this dissertation, I demonstrate that extracellular PKM2 released from activated neutrophils promotes angiogenesis and myofibroblast differentiation during wound healing. PKM2 activates dermal fibroblasts through integrin αvβ3 and PI3K signaling pathway. I also claim that extracellular PKM2 plays a role during liver fibrosis. PKM2 protects hepatic stellate cells from apoptosis by activating the survival signaling pathway.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: This study investigated the activities of several of the enzymes involved in the alternative route of PEP metabolism via PEPc (EC 4.1.1.31). This reaction circumvents the adenyl ate-controlled PK (EC 2.7.1.40) reaction of the conventional glycolytic network under conditions of P stress. It was hypothesized that the synthesis of pyruvate under Pi stress would induce the PEPc alternative route and that C for pyruvate synthesis would primarily be imported via this route. This was assessed by looking at how total enzyme activities are perturbed under P stress and also by following the route of radioactive labelled 14C02 under sufficient (2 mM) and deficient P (2 JlM) conditions in either roots or nodules. The significance of the pathway under P stress, was further assessed by determining pool sizes of pyruvate that was synthesized from PEPc-derived C. The experiments were conducted under glasshouse conditions, as two separate studies: one to investigate the phenomenon of Pi stress and its consequences for PEPc-derived C metabolism, and the other one to study the enzymes involved. Seeds of Lupinus angustifolius (cv. Wonga) were inoculated with Rhizobium sp. (Lupinus) bacteria and grown in hydroponic culture. Tanks were supplied with either 2 JlM P04 (LP) or 2 mM P04 (control) and air containing 360 ppm CO2. Roots experienced pronounced P stress with a greater decline in Pi, compared to nodules. LP roots synthesized more pyruvate from malate than LP nodules, indicating the engagement of the PEPc route under Pi stress. In this regard, pyruvate is considered as a key metabolite under Pi stress. The role of pyruvate accumulation under Pi stress, was further highlighted by the metabolism of PEP via both the PK and PEPc routes. The enhanced PK activities supported these high pyruvate levels. Under P stress, PEPc activities increased in roots but not in nodules and these changes were not related to the expression of the enzyme. Root and nodular PEPc were not regulated by expression, but possibly by posttranslational control. The novelty of our results for symbiotic roots demonstrates that using metabolically available Pi is indeed a more sensitive indicator ofP stress. These results show that under Pi stress, nodules are able to maintain their Pi and adenylate levels, possibly at the expense of the root. It is suggested that nodules do not experience P stress to the same extent as roots or alternatively function optimally under conditions of low P availability. The increase in concentration of pyruvate synthesized from malate, indeed suggest that under LP conditions there is an increase requirement for pyruvate. It is clear from this data that the operation of bypass route in nodules should be investigated further. Nevertheless, this study provided incentives for understanding the role of C pathways in Ni-fixation, in particular under conditions ofP limitation. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die aktiwiteite van verskeie ensieme van die alternatiewe metaboliese roete via phosphoenolpirovaat karboksilase (pEPc, EC 4.1.1.31) te ondersoek. Dié reaksie omseil die adenilaat-beheerde pirovaatkinase (PK, EC 2.7.1.40) reaksie van die konvensionele glikolitiese weg onder toestande van fosfaat (P) stremming. Dit is gepostuleer dat die sintese van pirovaat onder toestande van Pstremming die alternatiewe roete via PEPc sou induseer en dat die koolstof (C) vir pirovaatsintese gevolglik hoofsaaklik vanaf hierdie roete sou kom. Dit is bepaal deur die veranderinge in die totale ensiemaktiwiteite wat sou plaasvind onder P-stremming te ondersoek. Daar is ook gekyk na die roete' wat radioaktiewe C C4C02) sou volg in wortles en wortelknoppies wat behandel is deur blootsteling aan eerder lae fosfaat (2 1lM) of genoegsame fosfaat (2 mM; kontrole), Die betekenis van die alternatiewe roete is ook ondersoek deur die poel-groottes van pirovaat, soos gesintetiseer via die PEPc reaksie, te bepaal. Twee eksperimente is in 'n glashuis uitgevoer. Eerstens is die verskynsel van Pstremming, asook die invloed daarvan op PEPc-afgeleide C-metabolisme, bepaal. Tweedens is die betrokke ensieme bestudeer. Sade van Lupinus angustifolius (cv. Wonga) is geïnokuleer met Rhizobium sp. (Lupinus) bakterieë en in 'n waterkultuur gekweek. Die houers is voorsien met óf2 IlM P04 (LP) óf 2 mM P04 (HP) en lug wat 360 ppm C02 bevat het. Wortels, anders as wortelknoppies, het 'n betekenisvolle afname in anorganiese P (Pi) ervaar. Onder P-stremming, het lae fosfaat wortels meer pirovaat vanaf malaat gesintetiseer as wortelknoppies, wat 'n definitiewe bydrae vanaf die PEPc roete impliseer. Hiervolgens is pirovaat 'n sleutel metaboliet onder P-stremming. Die belangrikheid van die akkumulering van pirovaat onder P-stremmende toestande is verder beklemtoon deur die toename in metabolisme van PEP via beide die PK- en die PEPcreaksies. Die toename in PK-aktiwiteite is goed gekorreleer met die verhoogde produksie van pirovaat. Onder toestande van P-stremming het die aktiwiteit van PEPc in wortels verhoog, maar nie in wortelknoppies nie. Dit was nie die gevolg van 'n verhoogde uitdrukking van die ensiem nie. Wortel- en wortelknoppie- uitdrukking van PEPc is derhalwe nie gereguleer deur die uitdrukking daarvan nie, maar eerder deur post-tranlasie kontrole. Hierdie resultate vir wortels met wortelknoppies demonstreer dat metaboliese Pi 'n beter maatstaf is om P-stres aan te dui. Hierdie resultate toon dat wortelknoppies beter daartoe instaat is om hul Pi-vlakke en adenilaatvlakke te reguleer, en dit mag ten koste van die gasheerwortel wees. Ons stel voor dat wortelknoppies nie P-stremming tot dieselfde mate ervaar as die gasheerwortel nie en dat dié knoppies optimaal funksioneer by lae Pi vlakke. Die verhoogde konsentrasie van pirovaat, wat vanaf malaat gesintetiseer is, impliseer dat daar 'n groter vereiste is vir dié metaboliet onder toestande van Pstremming. Hierdie studie het die rol van koolstofmetabolismein stikstofbindende organismes, spesifiek onder toestande van fosfaat-tekort, beklemtoon.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 107-118).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 97-104).

Made available in DSpace on 2018-06-27T18:56:01Z (GMT). No. of bitstreams: 2 Amanda Lima de Oliveira.pdf: 513074 bytes, checksum: 393de3a3c8893cb2dad143281cc76ca4 (MD5) Amanda Lima de Oliveira.pdf.jpg: 3435 bytes, checksum: d98e4a9d95435991a70b3e949f124696 (MD5) Previous issue date: 2012-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cyclodextrins (CDs) are cyclic oligosaccharides formed by 6 (αCD), 7 (bCD) or 8 (γCD) glucose units with an internal hydrophobic cavity and outside surface hydrophilic. These three derivatives, the b-cyclodextrin (bCD) seems to be the most advantageous for pharmaceutical use for their availability, cavity size and low cost. The CDs have a future quite promising for their properties as greater absorption of drugs through the biological barriers and time of release, however, some types may not be considered non-toxic. The objective of this study was to investigate the intraperitoneal administration of βCD, M-β-CD and HP-ß-CD for 8 weeks with administered dose of 65.65 mg of CDs/kg rats/day, on parameters of biochemical analyzes, enzymes of energy metabolism, enzymes tiolicas sensitive to increase reactive oxygen species and to make this relationship, also evaluate parameters of oxidative stress in cerebral cortex, liver, kidneys and heart of wistar rats. The results showed that for the group treated with βCD there has been a significant increase in serum urea and creatinine levels, indicating nephrotoxicity, however not related to the other parameters. There was also a great reduction in serum levels of iron for the 3 CDs. The heart showed a reduction in the activity of CKmitocondrial and increase for AK by M-β-CD and reduction of CKmit by HP-ß-CD, but showed a reduction in the levels of diclorofluorceina (DCF) to the 3 CDs and protein carbonyl) by βCD. For the rim there was no significant change in comreducao activity of CKmit by HP-β-CD. In liver tissue, the βCD and M-β-CD reduced the activity of PK, but this is not reflected in blood glucose levels. In the cerebral cortex, the βCD reduced the activity of enzymes CK mitochondrial and PK, also reduced TBARS, but increased carbonyl protein. The indices lipidemic reduced reported by other researchers was not observed in this work, because the group of M-β- CD has a significant increase in serum levels of LDL cholesterol, in addition to aspartate aminostransferase AST, albumin, total protein, alkaline phosphatase, sodium, calcium, magnesium and phosphate. Our results indicate that some CDs alter enzymes crucial for energy metabolism, mainly of brain tissue with a reduction in activity and the PK by βCD. If changes in the activity of these enzymes occur in people who use drugs by intraperitoneal route, it is possible that the energy metabolism and brain functioning may be affected causing damage to the tissue. However more studies are needed to elucidate how there was a reduction of serum iron and as the cyclodextrins affect a structure so well protected by blood-brain barrier as the brain. / As ciclodextrinas (CDs) são oligossacarídeos cíclicos formados por 6 (αCD), 7 (bCD) ou 8 (γCD) unidades de glicose com uma cavidade interna hidrofóbica e superfície externa hidrofílica. Destes três derivados, a b-ciclodextrina (bCD) parece ser a mais vantajosa para utilização farmacêutica pela sua disponibilidade, tamanho da cavidade e baixo custo. O interesse pelas CDs se dá pelas suas propriedades como maior absorção dos fármacos através das barreiras biológicas e tempo de liberação, entretanto, alguns tipos não podem ser considerados atóxicas. O objetivo deste estudo foi investigar a administração intraperitoneal de βCD (Beta Ciclodextrina), M-β-CD (Metil Beta Ciclodextrina) e HP-β-CD (Hidroxypropil Beta Ciclodextrina) durante 8 semanas com dose administrada de 65,65 mg das CDs/kg rato/dia, sobre parâmetros de análises bioquímicas, de enzimas do metabolismo energético, enzimas tiólicas sensíveis ao aumento de espécies reativas e para fazer a relação, também avaliar parâmetros de estresse oxidativo em córtex cerebral, fígado, rins e coração de ratos wistar. Os resultados mostraram que para o grupo tratado com βCD houve um aumento significativo nos níveis séricos de uréia e creatinina, indicando nefrotoxidade, porém não relacionada com os demais parâmetros. Também houve uma grande redução nos níveis séricos de ferro para as 3 CDs. O coração apresentou redução na atividade da Creatinaquinase mitocondrial (CKmit) e aumento para Adenilatoquinase (AK) pela M- β-CD e redução da CKmit pela HP-β-CD, porém apresentou uma redução nos níveis de diclorofluoresceína (DCF) para as 3 CDs e carbonilas proteicas pela βCD. Para o rim houve alteração significativa com redução na atividade da CKmit pela HP-β-CD. No tecido hepático, a βCD e M-β-CD reduziram a atividade da Piruvatoquinase (PK), porém isto não refletiu nos níveis glicêmicos. No córtex cerebral, a βCD reduziu a atividade das enzimas CK mitocondrial e PK, também reduziu TBARS (Espécies reativas ao ácido tiobarbitúrico), mas aumentou carbonilas proteicas. Os índices lipidêmicos reduzidos relatados por outros pesquisadores não foi observado neste trabalho, pois o grupo da M-β-CD apresentou um aumento significativo nos níveis séricos de LDL (lipoproteína de baixa densidade), além de AST (aspartato aminostransferase), albumina, proteínas totais, fosfatase alcalina, sódio, cálcio, magnésio e fosfato. Os resultados indicam que algumas CDs alteram enzimas cruciais do metabolismo energético, principalmente do tecido cerebral com redução na atividade da PK pela βCD. Possíveis alterações na atividade destas enzimas podem afetar o metabolismo energético e o funcionamento cerebral causando dano ao tecido. Entretanto mais estudos são necessários para elucidar de que forma ocorreu a redução sérica de ferro e como as ciclodextrinas afetaram uma estrutura tão bem protegida pela barreira hemato-encefálica como a cerebral.

Globally, methicillin resistant Staphylococcus aureus (MRSA) has become increasingly difficult to manage in the clinic and new antibiotics are required. The structure activity relationship (SAR) study presented in this thesis forms part of an international collaborative effort to identify potent and selective inhibitors of an MRSA pyruvate kinase (PK) enzyme target. In earlier work the known marine natural product bromodeoxytopsentin (1.6), isolated from a South African marine sponge Topsentia pachastrelloides, exhibited selective and significant inhibition of MRSA PK (IC₅₀ 60 nM). Accordingly bromodeoxytopsentin provided the initial chemical scaffold around which our SAR study was developed. Following a comprehensive introduction, providing the necessary background to the research described in subsequent Chapters, this thesis has been divided into three major parts. Part one (Chapter 2) documents the synthesis of two natural imidazole containing topsentin analogues 1.40, 1.46, five new synthetic analogues 1.58—1.61, 2.104. In the process we developed a new method for the synthesis of topsentin derivatives via selenium dioxide mediated oxidation of N-Boc protected 3-acetylindoles to yield glyoxal intermediates which were subsequently cyclized and deprotected to yield the desired products. Interestingly we were able to demonstrate a delicate relationship between the relative equivalents of selenium dioxide and water used during the oxidation step, careful manipulation of which was required to prevent the uncontrolled formation of side products. Synthetic compounds 1.40, 1.46, 1.58—1.61 were found to be potent inhibitors of MRSA PK (IC₅₀ 238, 2.1, 23, 1.4, 6.3 and 3.2 nM respectively) with 1000-10000 fold selectivity for MRSA PK over four human orthologs. In the second part of this thesis (Chapter 3) we report the successful synthesis of a cohort of previously unknown thiazole containing bisindole topsentin analogues 1.62—1.68 via a Hantzsch thiazole synthesis. Bioassay results revealed that these compounds were only moderate inhibitors of MRSA PK (IC₅₀ 5.1—20 μM) which suggested that inhibitory activity was significantly reduced upon substitution of the central imidazole ring of topsentin type analogues with a thiazole type ring. In addition in Chapter 3 we describe unsuccessful attempts to regiospecifically synthesize oxazole and imidazole topsentin analogues through a similar Hantzsch method. As a consequence of our efforts in this regard we investigated three key reactions in depth, namely the synthesis of 2.2, 3.38, 3.40, 3.41 via α-bromination of 3-acetylindole and the synthesis of indolyl-3-carbonylnitriles 2.13, 3.45—3.47 and α-oxo-1H-indole-3-thioacetamides 3.48—3.51. The investigation of the latter led to the isolation and elucidation of two anomalous N,N-dimethyl-1H-indole-3-carboxamides 3.52 and 3.53. Finally the third part of this thesis (Chapter 4) deals with in silico assessment of the binding of both the imidazole and thiazole containing bisindole alkaloids to the MRSA PK protein which initially guided our SAR studies. In this chapter we reveal that there appears to be no correlation between in silico binding predictions and in vitro MRSA PK inhibitory bioassay data. Superficially it seems that binding energy as determined by the docking program used for these studies correlated with the size of the indole substituents and did not reflect IC₅₀ MRSA PK inhibitory data. Although this led us to computationally explore possible alternative binding sites no clear alternative has been identified.

RATIONALE - Metabolic enzymes, like pyruvate kinase M2 (PKM2), play an essential role in altering endothelial cell (EC) phenotypes and behavior. Extensive research has elucidated the function of PKM2, a rate-limiting glycolytic enzyme, in the context of cancer cells and in activated immune cells, but its role in EC biology is only newly emerging. Recent findings show PKM2 acts as a key regulator of angiogenesis. Where exogenous circulating PKM2 induces EC cell proliferation leading to increased tumor angiogenesis and growth. Also, PKM2 deficient ECs exhibit decreased proliferation and migration. The relevance of PKM2 in modulating vascular barrier function is yet to be defined. OBJECTIVE -This study attempts to elucidate the role of PKM2 in regulating vascular barrier function. METHODS AND RESULTS - In vivo, EC specific deletion of PKM2 promotes increased vascular permeability in pulmonary capillary vessels and increased VEGF-induced acute vessel permeability in mouse dermal vessels. Similarly, in vitro, PKM2 deficient ECs exhibit decreased electrical resistance, disrupted VE-cadherin junctions and gap formations (illustrated via florescent VE-cadherin staining and phosphorylation of VE- cadherin protein at tyrosine residue Y658). Mechanistically, the deletion of PKM2 in ECs leads to increased angiopoietin-2 (Ang-2) expression, a well-known modulator of vascular permeability. Also, deletion of Ang-2 was sufficient to attenuate vascular leakage in PKM2 deficient endothelium, indicating that vascular leaky phenotype observed in PKM2 deficient endothelium is mediated by increased Ang-2 expression. CONCLUSIONS - PKM2, by modulating Ang-2 expression, plays a vital role in maintaining vascular barrier function. / 2019-07-11T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / Regulation of the pyruvate dehydrogenase complex (PDC) is important for glucose homeostasis and control of fuel selection by tissues. Knocking out pyruvate dehydrogenase kinase 4 (PDK4), one of four kinases responsible for regulation of PDC activity, lowers blood glucose levels by limiting the supply of three carbon compounds for gluconeogenesis. Down regulation of PDK4 expression is also important for control of blood glucose by insulin. The primary goal was to determine whether PDK4 should be considered a target for the treatment of diabetes. A major concern is that inhibition of fatty acid oxidation by PDK4 deficiency may promote fat accumulation in tissues and worsen insulin sensitivity. This was examined by feeding wild type and PDK4 knockout mice a diet rich in saturated fat. Fasting blood glucose levels were lower, glucose tolerance was better, insulin sensitivity was greater, and liver fat was reduced in PDK4 knockout mice. The reduction in liver fat is contradictory to the finding that fibrate drugs increase PDK4 expression but ameliorate hepatic steatosis in rodents. To investigate this phenomenon, wild type and PDK4 knockout mice were fed the high saturated fat diet with and without clofibric acid. The beneficial effect of clofibric acid on hepatic steatosis was greater in the PDK4 knockout mice, indicating up regulation of PDK4 is not necessary and likely opposes the effect of clofibric acid on hepatic steatosis. Clofibric acid dramatically lowered the amount of hepatic CD36, a plasma membrane translocase required for fatty acid import, suggesting a novel mechanism for prevention of hepatic steatosis by fibrates. PDK4 deficiency had no effect on CD36 expression but reduced the enzymatic capacity for fatty acid synthesis, suggesting clofibric acid and PDK4 deficiency ameliorate hepatic steatosis by independent mechanisms. Investigation of the mechanism by which insulin regulates PDK4 expression revealed a novel binding site for hepatic nuclear factor 4α (HNF4α) in the PDK4 promoter. The stimulatory effect of HNF4α was sensitive to inhibition by Akt which is activated by insulin. The findings suggest PDK4 is a viable target for the treatment of hepatic steatosis and type 2 diabetes.