<p>In this project the aim has been to study the model protein carbonic anhydrase in Neisseria gonorrhoeae, a bacterium whose carbonic anhydrase has great similarities both structurally and functionally with the human form. By measuring and comparing the wild type of NGCA with mutants lacking one of the four tryptophan residues it can be seen what effect these tryptophans has on stability and activity and then compare with the known data of HCA II to learn more about their differences and similarities. The results from the stability and activity measurements are that the wild type is by far the most stable protein with W141L mutant coming thereafter.</p><p>From Trp-fluorescence and CO2-hydration measurement a clear two-transition steps (N→ I→ U) can be seen. This differs from earlier data where it instead only was a one-transition step for the wild type (N→U). The data is also very reliable and gives in most cases a perfect fit to the line. We also see this two-transition step for the other mutants stable enough, strengthening the theory further.</p><p>One fact that could be drawn from all the measurements is that when an intermediate is formed the ability for the enzyme NGCA to perform it’s catalytically ability is disabled.</p><p>Another thing is that the purification scheme of HCA II is not optimal to be directly applied to NGCA, despite the similarity in secondary and tertiary structure.</p>

Intracellular pH was monitored at the cytosolic surface of plasma membrane solute transporters (Na+/H+/nucleoside co-transporters, or Cl-/HCO3- exchangers), using pH-sensitive fluorescent proteins (FPs), dual emission green FP (deGFP4) and a monomeric red FP Nectarine (mNect), whose development and characterization are also reported here. Human concentrative nucleoside transporter, hCNT3, mediates Na+/H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in HEK293 cells. pH changes at the intracellular surface of hCNT3 were monitored by fusing mNect to the cytoplasmic N-terminus of hCNT3 (mNect.hCNT3) or an inactive hCNT3 mutant (mNect.hCNT3-F563C). Cells were incubated at the permissive pH for H+-coupled nucleoside transport, pH 5.5, under both Na+-free and Na+-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mM uridine, providing the first direct evidence for H+-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H+ flux) in the presence or absence of Na+. This suggests that in acidic Na+-containing conditions, 1 Na+ and 1 H+ are transported/uridine molecule, while in acidic Na+-free conditions, 1 H+ alone is transported/uridine. In acid environments, including renal proximal tubule and intestine, H+/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Microdomains, discrete regions of altered cytosolic solute concentration, are enhanced by rapid solute transport and slow diffusion rates. pH-regulatory membrane transporters, like the Cl-/HCO3- exchanger AE1, could nucleate H+ microdomains, since AE1 has a rapid transport rate and cytosolic H+ diffusion is slow. As AE1 drives Cl-/HCO3- exchange, differences in pH, near and remote from AE1, were monitored simultaneously by deGFP4 fused to AE1 (deGFP4.AE1) and mNect.hCNT3-F563C. deGFP4.AE1-mNect.hCNT3-F563C distance was varied by co-expression of different amounts of the two proteins in HEK293 cells. As the deGFP4.AE1-mNect.hCNT3-F563C distance increased, mNect.hCNT3-F563C detected the cytosolic pH change with a time delay and reduced rate of pH change, compared to deGFP4.AE1. Carbonic anhydrase activity was essential for H+ microdomain formation. H+ diffusion along the plasma membrane was 60-fold slower than to the cytosolic ER-surface. During physiological HCO3- transport, a H+ microdomain 0.3 µm in diameter develops around AE1, which will affect nearby pH-sensitive processes.

In this project the aim has been to study the model protein carbonic anhydrase in Neisseria gonorrhoeae, a bacterium whose carbonic anhydrase has great similarities both structurally and functionally with the human form. By measuring and comparing the wild type of NGCA with mutants lacking one of the four tryptophan residues it can be seen what effect these tryptophans has on stability and activity and then compare with the known data of HCA II to learn more about their differences and similarities. The results from the stability and activity measurements are that the wild type is by far the most stable protein with W141L mutant coming thereafter. From Trp-fluorescence and CO2-hydration measurement a clear two-transition steps (N→ I→ U) can be seen. This differs from earlier data where it instead only was a one-transition step for the wild type (N→U). The data is also very reliable and gives in most cases a perfect fit to the line. We also see this two-transition step for the other mutants stable enough, strengthening the theory further. One fact that could be drawn from all the measurements is that when an intermediate is formed the ability for the enzyme NGCA to perform it’s catalytically ability is disabled. Another thing is that the purification scheme of HCA II is not optimal to be directly applied to NGCA, despite the similarity in secondary and tertiary structure.

Analysis of the genome of Anabaena sp. PCC7120 reveals that it lacks the gene, ccaA, which encodes the bonafide carboxysomal, β-class carbonic anhydrase (CA) CcaA. However, the carboxysome enriched fraction of Anabaena PCC7120 exhibits CA activity. Bioinformatic analysis reveals that the N-terminal region of the carboxysome protein CcmM has high sequence and structural similarity to the γ-class CA of Methanosarcina thermophila. Recombinantly expressed CcmM is found to be inactive in in-vitro CA assays. E. coli cell extracts containing an overexpressed form of CcmM comprised of the N-terminal 209 amino acids (CcmM209) are also inactive. However, CcmM209 displays CA activity after incubation with the thiol oxidizing agent diamide or when bound to an affinity matrix. It appears that CcmM is indeed a functional γ-CA which is active under oxidizing condition. It is hypothesized that the C-terminal RbcS like domain in CcmM may regulate activity by allowing CcmM activation only when sequestered within the carboxysome.

Analysis of the genome of Anabaena sp. PCC7120 reveals that it lacks the gene, ccaA, which encodes the bonafide carboxysomal, β-class carbonic anhydrase (CA) CcaA. However, the carboxysome enriched fraction of Anabaena PCC7120 exhibits CA activity. Bioinformatic analysis reveals that the N-terminal region of the carboxysome protein CcmM has high sequence and structural similarity to the γ-class CA of Methanosarcina thermophila. Recombinantly expressed CcmM is found to be inactive in in-vitro CA assays. E. coli cell extracts containing an overexpressed form of CcmM comprised of the N-terminal 209 amino acids (CcmM209) are also inactive. However, CcmM209 displays CA activity after incubation with the thiol oxidizing agent diamide or when bound to an affinity matrix. It appears that CcmM is indeed a functional γ-CA which is active under oxidizing condition. It is hypothesized that the C-terminal RbcS like domain in CcmM may regulate activity by allowing CcmM activation only when sequestered within the carboxysome.

No description available.

I denna avhandling presenteras arbete utfört med enzymet humant karboanhydras II (HCAII). Enzymer är en typ av proteiner som accelererar (katalyserar) kemiska reaktioner, vilket är nödvändigt för allt levande. Den naturliga funktionen för HCAII är att katalysera omvandlingen av gasen koldioxid till vätekarbonat, som är löslig i vätska. Detta är viktigt bl.a. för att koldioxid som bildas i kroppen, och fraktas i blodet i form av vätekarbonat, skall hinna över till utandningsluften under den korta tid blodet är i lungorna. Proteiner består av aminosyror som länkats samman i en lång kedja, där varje aminosyra är en av de 20 naturliga aminosyratyperna. Ett proteins struktur och egenskaper bestäms av aminosyrasekvensen, som i sin tur bestäms av genen för just det proteinet. Med genteknik kan ett proteins gen ändras (muteras), så att aminosyrasekvensen ändras, och det har här utnyttjats för att förändra HCAIIs katalytiska egenskaper. Förutom dess naturliga funktion kan HCAII även klyva (hydrolysera) vissa estrar. Mutationer gjordes så att en ’ficka’ i HCAIIs struktur, där molekylerna (substraten) som skall klyvas binder, fick en större volym. På så sätt skapades varianter med en kraftigt ökad kapacitet för att hydrolysera långa estersubstrat jämfört med icke-muterat HCAII. Som en utveckling av detta projekt skapades en mutant av HCAII, som kan hydrolysera ett än mer skrymmande substrat. I ett annat projekt har en ny katalytisk aktivitet skapats i HCAII, som inte utnyttjar enzymets naturliga katalytiska förmåga. Ett nytt estersubstrat konstruerades, med en del som binder kraftigt till HCAII, så att en stark substratbindning erhölls. Sedan muterades vissa aminosyror till en reaktiv aminosyra som heter histidin. Valet av positioner för mutation baserades på en datormodell av enzymet med bundet substrat. Eftersom histidin kan delta i hydrolysreaktioner, får det muterade enzymet möjlighet att klyva substratet. Flera olika mutanter testades, och den effektivaste innehöll ett nära kopplat par av histidiner. Denna mutant undersöktes mer noggrannt, vilket gav viss information om den katalytiska mekanismen. Det långsiktiga målet med detta arbete är att konstruera muterade enzymer som kan klyva giftiga ämnen, eller användas vid framställning av kemikalier. Det finns behov av nya enzymer för olika typer av substrat, och att med rationella metoder skapa nya katalytiska aktiviteter i proteiner är ett svårt vetenskapligt problem som ännu är i ett tidigt utvecklingsskede. / The main part of this thesis describes results from protein engineering experiments, in which the catalytic activity of the enzyme human carbonic anhydrase II (HCAII) is engineered by mutagenesis. This enzyme, which catalyzes the interconversion between CO2 and HCO3- in the body, also has the ability to hydrolyze ester bonds. In one project, the specificity of HCAII towards a panel of para-nitrophenyl ester substrates, with acyl chain lengths ranging from one to five carbon atoms, was changed by enlarging the substrate binding hydrophobic pocket. A variant was identified that has highly increased specificity towards substrates with long acyl chains. The mutant V121A/V143A hydrolyzes pNPV, which has four carbon atoms in the acyl chain, with an efficiency that is increased by a factor of 3000 compared to HCAII. Further, transition state analogues (TSAs) were docked to HCAII and mutant variants, and the results were correlated to the results from kinetic measurements. This indicated that automated docking could be used to some extent to construct HCAII variants with a designed specificity. Using this approach, a HCAII mutant that can hydrolyze a model benzoate ester was created. Interestingly, the resulting variant V121A/V143A/T200A was found to be highly active with other ester substrates as well. For pNPA, a kcat/KM of 1*105 M-1s-1 was achieved, which is the highest efficiency for hydrolysis of carboxylic acid esters reported for any HCAII variant. In another project, the strong affinity between the active site zinc ion and sulfonamide was used to achieve binding of a designed substrate. Thus, the natural Zn-OH- site of HCAII was not used for catalysis, but for substrate binding. The substrate contains a benzenesulfonamide part in one end, with a para-nitrophenyl ester connected via a linker. The linker was chosen to ensure that the scissile bond is positioned close to His-64 and histidine residues introduced by mutagenesis in other positions. Using this approach, an enzyme was designed with a distinctly new two-histidine catalytic site for ester hydrolysis. The mutant, F131H/V135H, has a kcat/KM of approximately 14000 M-1s-1, which corresponds to a rate enhancement of 107 compared to a histidine mimic. Finally, results are reported on a project aimed at cloning and producing a putative carbonic anhydrase from the malaria parasite Plasmodium falciparum. The gene was cloned by PCR and the construct was overexpressed in E. coli. However, the resulting protein was not soluble, and initial attempts to refold it are also reported.

A obesidade e a cárie dentária são problemas de saúde pública, que atingem a população infantil. O objetivo deste estudo foi identificar a prevalência de cárie dentária e relacioná-la com a concentração da enzima anidrase carbônica VI, do íon cálcio, fluxo salivar e quantidade de biofilme dentário em crianças com sobrepeso/obesidade. Foram avaliadas 112 crianças de 4 a 6 anos de idade, de ambos os gêneros. A análise antropométrica foi realizada (percentil do IMC) e através dessa análise as crianças foram divididas em dois grupos: G1 sobrepesos/obesos (n=41) e G2 normais (n=71). Os exames bucais realizados para a cárie dentária foram os índices ceo-s e ICDAS II, quantidade de biofilme dentário pelo Índice de Placa de Turesky e volume de fluxo salivar estimulado. A concentração do íon Cálcio na saliva foi analisada pelo kit colorimétrico e da enzima Anidrase Carbônica VI pelo kit ELISA. Na sequência, as crianças de cada grupo foram divididas em 3 subgrupos: LC (livres de cárie), LI (com lesões iniciais) e C (com cárie). Os testes Wilcoxon, Mann-Whitney, teste t e correlação de Spearman foram aplicados (p<0,05). Não houve diferença significativa no ceo-s entre os grupos. Houve maior concentração média de cálcio salivar no G1 (G1=2847,96mM; G2=1230,90mM;p=0,001) e maior concentração da Anidrase Carbônica VI no G2 (G1=3455,18 pg/mL; G2=442428,9pg/mL;p=0,000). No G1 houve correlação negativa entre o ceo-s e íon Cálcio (r=-0,444;p=0,010). Já no G2, houve correlação negativa entre placa e a Anidrase Carbônica VI (r=-0,551;p=0,014). Pode-se concluir que o íon cálcio é fator protetor para cárie dentária em crianças. Já a anidrase carbônica VI parece não ser biomarcador para a cárie dentária. / Obesity and dental caries are public health problems that affect the child population. The aim of this study was to identify the prevalence of dental caries and relate it to the concentration of the enzyme carbonic anhydrase VI, calcium ion, salivary flow, and dental plaque in overweight/obesity children. The study was conducted on 112 children aged 4-6, of both genders. Anthropometric analysis was performed (BMI percentile) and by this analysis the children were divided into two groups: G1 - overweight/obese (n=41) and G2 - normal (n=71). The oral examinations performed for dental caries were the dmfs and ICDAS II indexes, measurement of the amount of dental plaque by the Turesky Board Index and volume of stimulated salivary flow. The concentration of calcium ion in saliva was measured by a colorimetric kit and the enzyme carbonic anhydrase VI by an ELISA kit. Then, children from each group were divided into three subgroups: CF (caries-free), IL (initial lesions) and D (decayed teeth). The Wilcoxon test, Mann-Whitney, t test and Spearman correlation (p<0.05) were applied. There was no significant difference in the dmfs between groups. There was higher concentration of salivary calcium in G1 (G1=2847.96mM; G2=1230.90mM; p=0.001), and higher concentration of carbonic anhydrase VI in G2 (G1 = 3455.18 pg/ml; G2 = 442428.9pg/ml; p = 0.000). In G1, there was negative correlation between dmfs and salivary calcium (r = -0.444; p = 0.010). In G2, there was negative correlation between dental plaque and carbonic anhydrase VI (r=-0.551; p=0.014). It can be concluded that the calcium ion is a protective factor for dental caries in children. The carbonic anhydrase VI does not seem to be a biomaker of dental caries.

Exposing Amyloid Beta 1-42 to neurons causes cell death. When carbonic anhydrase inhibitors (e.g. methazolamide or acetazolamide) are introduced along with 1-42 in a similar experiment, cell apoptosis is disrupted. However, when non-CA inhibitors are tested, (e.g. the indole derivative melatonin), the same disruption occurs. Are these carbonic anhydrase inhibitors acting on the same or a different pathway? One way to study the molecular mechanisms of these small molecule inhibitors is to modify their chemical structure. In this sense, when acetazolamide is methylated, apoptosis is resumed (Fossati et al., 2016). Finding a way to create N-methyl acetazolamide and N-methyl methazolamide through methylation procedures will lead to a better understanding of the pathways involved in neuronal apoptosis triggered by the Abeta peptide.

L'hypoxie est une caractéristique saillante de nombreuses tumeurs solides et provient d'un apport vasculaire inadéquat et immature, entraînant une diminution l'apport d'oxygène et nutriments. Ces régions hypoxiques montrent une résistance aux modalités de traitement classiques telles que la radio et la chimiothérapie et sont associées à une survie insuffisante. Dans des conditions hypoxiques,le HIF-1α améliore l'expression de nombreux gènes cibles,l'un d'eux étant l'anhydrase carbonique IX (CAIX). La CAIX est une enzyme transmembranaire,impliquée dans l'hydratation réversible du dioxyde de carbone au bicarbonate et au proton. L'objectif de cette thèse était de cibler CAIX en utilisant divers médicaments à double cible combinés avec des radiosensibilisateurs, des médicaments cytotoxiques etdes médicaments bio-réductibles. Dans cette thèse,plusieurs approches de ciblage CAIX ont été étudiées.Étant donné que les nitroimidazoles sont de bons sensibilisants cellulaires hypoxiques, nous avons conçu plusieurs composés à double cible existant par combinaison d'un nitroimidazole et d'une fraction inhibitrice d'anhydrase carbonique IX (chapitre 3). Auparavant, notre groupe a montré que le dérivé à base de sulfamide 7 a amélioré l'efficacité thérapeutique de l'irradiation de manière dépendante la CAIX avec un rapport d'amélioration de la sensibilisation (SER) de 1,50, ce qui est plus élevé que plusieurs radiosensibilisateurs testés cliniquement tels que le misonidazole et le nimorazole. L'efficacité de la chimiosensibilisation a été observée lors de la combinaison de 7 avec doxorubicine chez des souris porteurs de tumeur HT29 (chapitre 3).Une approche à double cible similaire peut être exploitable pour délivrer des médicaments cytotoxiques vers des cellules exprimant CAIX, ce qui entraîne une administration ciblée spécifique de la tumeur et par conséquent réduit la toxicité normale des tissus. Le chapitre 4 décrit la conception et la synthèse de nouvelles séries de composés à double cible combinant plusieurs médicaments anti-cancéreux. Un seul composé,c'est-à-dire un dérivé ATRi, a montré une efficacité supérieure en association avec un rayonnement dans des cellules surexprimant CAIX par rapport aux cellules dépourvues d'expression CAIX.Le chapitre 5 décrit la conception et la synthèse de divers dérivés de nitroimidazole bio-réductibles,des agents alkylants de moutarde d'azote et des dérivés de N-oxyde combinés avec une fraction inhibitrice de l'anhydrase carbonique IX. Tous ces composés présentent des profils d'inhibition faibles à modérés vers plusieurs isoformes CA testées. Nous avons observé que différentes substitutions et liens dans la même famille de composés influencent la capacité de liaison à CAIX.Pour élargir notre étude sur les médicaments bio-réductibles, le chapitre 6 décrit la conception et la synthèse de 2, 5-nitroimdazole et moutarde d'azote combinées avec des inhibiteurs de l'anhydrase carbonique IX par un agent de liaison de carbamate. Le dérivé 2-nitroimidazole 1b a révélé une cytotoxicité dans les lignées cellulaires HT29 et HCT116 et pourrait être expliqué par le potentiel de réduction des 2-nitroimidazoles par rapport aux 5-nitroimidazoles, car nos résultats n'ont montré aucune cytotoxicité des dérivés du 5-nitroimidazole. Nos futures études visent à optimiser l'efficacité radiosensibilisante de 2b et 2c explorent davantage les propriétés cytotoxiques de 1b.En conclusion, cette thèse a montré que le double nitroimidazole cible combiné à la CAIXi augmente l'efficacité des modalités de traitement standard telles que la chimiothérapie et la radiothérapie. Le ciblage de la CAIX avec la combinaison de médicaments cytotoxiques continue d'être une approche intéressante pour les tumeurs hypoxiques cibles à l'avenir. Les médicaments bio-réductibles avec des potentiels de réduction plus élevés serviraient d'agents cytotoxiques potentiels aux tumeurs hypoxiques, ce qui réduirait la toxicité tissulaire normale. / Hypoxia is a salient feature in many solid tumors and arises due to an inadequate and immature vascular supply resulting in a decreased delivery of oxygen and nutrients. These hypoxic regions show resistance towards conventional treatment modalities such as radio- and chemotherapy and are associated with poor survival. Under hypoxic conditions HIF-1α enhances the expression of many target genes, one of them being carbonic anhydrase IX (CAIX).CAIX is a transmembrane enzyme, which is involved in reversible hydration of carbon dioxide to bicarbonate and a proton.The aim of this thesis was to target CAIX using various dual target drugs combined with radiosensitizers, cytotoxic drugs and bio-reducible drugs.In this thesis several CAIX targeting approaches have been investigated.Since nitroimidazoles are good hypoxic cell sensitizers,we have designed several dual target compounds existing out of a combination of a nitroimidazole and a carbonic anhydrase IX inhibitory moiety (Chapter 3). It has been shown that extracellular acidity limits the uptake of weak basic chemotherapeutic drugs, such as doxorubicin, and thereby decreases its efficacy. We hypothesized that combining these nitroimidazole moieties with a sulfonamide/sulfamide/sulfamate to target CAIX results in a decrease in extracellular acidosis and sensitizes hypoxic tumors to chemo- and radiotherapy. Previously, our group has shown that the sulfamide-based derivative 7 enhanced the therapeutic efficacy of irradiation in a CAIX dependent manner with a sensitization enhancement ratio of 1.50,which is higher than several clinically tested radiosensitizers such as misonidazole and nimorazole. Chemosensitization efficacy was observed upon combination of 7 with doxorubicin in HT29 tumor-bearing mice (Chapter 3).A similar dual target approach may be exploitable to deliver cytotoxic drugs towards CAIX expressing cells, resulting in a specific tumor targeted delivery and consequently reduced normal tissue toxicity. Chapter 4 describes the design and synthesis of new series of dual target compounds combining several anti-cancer drugs, Only one compound, i.e. an ATRi derivative, showed a higher efficacy in combination with radiation in CAIX overexpressing cells as compared to cells lacking CAIX expression.Chapter 5 describes the design and synthesis of various bio-reducible nitroimidazole derivatives, nitrogen mustard alkylating agents and N-oxide derivatives combined with a carbonic anhydrase IX inhibiting moiety. All these compounds showed weak to moderate inhibition profiles towards several tested CA isoforms. We have observed that different substitutions and linkers within the same family of compounds influence the binding capacity to CAIX. For example derivatives 17 and 20 belong to same family, but have a different linker and substitution of the aromatic ring, leading to a different binding capacity towards CAIX.To expand our study on bio-reducible drugs, Chapter 6 describes the design and synthesis of 2-, 5-nitroimdazole and nitrogen mustards combined with carbonic anhydrase IX inhibitors by a carbamate linker. The 2-nitroimidazole derivative 1b revealed cytotoxicity in HT29 and HCT116 cell lines and might be explain by the higher reduction potential of 2-nitroimidazoles compared to 5-nitroimidazoles, since our results did show no cytotoxicity of the 5-nitroimidazole derivatives. Our future studies aim to optimize the radiosensitizing efficacy of 2b and 2c and further explore the cytotoxic properties of 1b.In conclusion, this thesis showed that the dual target nitroimidazole combined with CAIXi increases the efficacy of standard treatment modalities such as chemo and radiotherapy. Targeting CAIX with combination of cytotoxic drugs continues to be an interesting approach to target hypoxic tumors in future. Bio-reducible drugs with higher reduction potentials would serve as potential cytotoxic agents to hypoxic tumors thereby decreasing the normal tissue toxicity.