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.

Le processus de calcification chez Corallium rubrum conduit à la formation de deux structures squelettiques composées de CaCO3, l’axe squelettique et les sclérites, de taille et de forme différentes. Comme chez de nombreuses espèces calcifiantes, la calcification se fait sous contrôle biologique impliquant notamment des enzymes et des transporteurs ioniques. Une question centrale est d’identifier les mécanismes communs ou propres à chaque espèce qui sous-tendent leur convergence fonctionnelle envers ce processus. Deux approches ont été utilisées pour caractériser ces mécanismes chez C. rubrum: 1) Une approche physiologique avec le développement d’une technique de culture de microcolonies sur lamelles permettant d’observer différents stades de calcification, et de mesurer le pH aux sites de calcification par imagerie confocale ; 2) Une approche moléculaire afin de caractériser une famille d’enzymes, les anhydrases carboniques (ACs), qui jouent un rôle clef dans la calcification.Nous avons réalisé une cartographie du pH en effectuant des mesures dans différents compartiments intra- et extracellulaires. Nos résultats montrent notamment que le pH aux sites de calcification est supérieur à celui du milieu circulant dans les canaux gastrodermiques et non à celui l’eau de mer. Les mesures d’expression différentielle des ACs dans différents tissus mettent en évidence une isozyme préférentiellement exprimée dans les cellules calcifiantes.Ces résultats intégrés dans un contexte de calcification comparée pointent sur la convergence fonctionnelle des ACs et de la régulation du pH par les cellules calcifiantes, tout en soulignant des divergences évolutives. / The calcification process in Corallium rubrum leads to the formation of two skeletal structures made of calcium carbonate, the skeletal axis and sclerites, of different size and shape. As in many calcifying species, calcification occurs under a biological control that involves enzymes and ion transporters. A central issue is to determine the common and the species-specific mechanisms of calcification in order to identify functional convergences in this process. Two approaches were used to characterize these mechanisms in C. rubrum: 1) A physiological approach involving the development of a microcolony culture technique on glass coverslips, allowing the observation of the different stages of calcification, and the measurement of pH at the sites of calcification by the use of confocal microscopy; 2) A molecular approach to characterize an enzyme family, the carbonic anhydrases, which play a key role in calcification.We performed pH mapping by making measurements in different intra- and extracellular compartments. Our results show higher pH values at the sites of calcification compared with the fluid circulating in the gastrodermal canals, but not with the seawater surrounding the microcolony. Measurements of differential expression of carbonic anhydrases in different tissue fractions highlight an isozyme preferentially expressed in the calcifying cells.Within comparative calcification perspectives, these results point towards the functional convergence of carbonic anhydrases and pH regulation by the calcifying cells, while highlighting evolutionary divergences.

Small cell lung cancer (SCLC) is an extremely aggressive disease characterized by early metastasis and acquired resistance to therapy. SCLC is distinguished by its neuroendocrine (NE) component; the role of which is not fully understood in metastasis and response to therapy. Patients respond exceptionally well to first round chemotherapy; however, relapse with therapy-resistant tumours is virtually inevitable. Hypoxic regions within tumours can contribute towards metastasis and therapy resistance, highlighting hypoxia-targeted therapy as a novel approach for improving treatment for SCLC patients. Tumours are highly phenotypically heterogeneous, raising debate over the roles played by each cell type. Analysis of NE and epithelial markers in SCLC cell lines highlighted this inter-tumour heterogeneity. Further heterogeneity is displayed in SCLC xenograft tumours that show areas of dual epithelial and NE marker expression as well as regions negative for both markers. Irradiating xenograft tumours enhanced heterogeneity of the NE marker, pro-opiomelanocortin (POMC), which is ectopically secreted by a subset of SCLC tumours. Examining changes in marker expression post-therapy could provide vital information regarding transitions that can serve to guide therapy. SCLC is a highly metastatic disease. The role of the NE phenotype in human SCLC is not fully understood, but is considered essential for metastasis in murine models. Sub-cutaneous, intravenous and intra-splenic injection were carried out and resulted in no metastasis, spontaneous tumour generation and peripheral liver tumour growth, respectively. POMC expression was present and extremely heterogeneous within the liver, suggesting that NE properties are maintained in metastases; however, further work is necessary to develop a more consistent metastatic model that can be used to assess responses to therapy in a more clinically relevant setting. SCLC tumours proliferate rapidly and outgrow their nutrient and oxygen supplies, resulting in hypoxic conditions. Here, carbonic anhydrase IX (CA IX) becomes up-regulated in order to maintain pH levels suitable for survival. The specific CA IX inhibitor, S4, induces hypoxia-specific cell death in vitro and impairs tumour growth in vivo. This response is further accentuated by combining S4 with single or repeated cisplatin doses. Combination treatment reduced gene expression of S-phase kinase-associated protein (Skp2), associated with cisplatin resistance. CA IX inhibition combined with cisplatin chemotherapy therefore presents a novel treatment for SCLC tumours that could reduce therapy resistance. In summary, heterogeneity is extremely important when choosing treatment options for SCLC and must be considered when basing treatment on single biopsies. NE and epithelial markers are present within sub-cutaneous and liver tumours; however, a reliable multi-organ metastatic model is necessary to fully appreciate the role of these markers in the spread of SCLC. Hypoxic regions within sub-cutaneous xenograft tumours upregulate CA IX. Inhibition of this enzyme resulted in impaired tumour growth, particularly when used together with cisplatin. Combining CA IX inhibition with cisplatin presents a much-needed novel therapy for SCLC.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第22087号 / エネ博第395号 / 新制||エネ||76(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 木下 正弘, 教授 片平 正人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

This work deals with carborane and metallacarborane clusters, in terms of their fundamental chemistry and complexation with cyclodextrins, and in the context of emerging pharmacophores applicable in medicinal chemistry. Arguably, the most important part of this work is the preparation of cobalt bis(dicarbollide) sulfamide derivatives. The sulfamido group is attached to the metallacarborane carbon vertex by an alkyl chain that may be modified in its length. This was accomplished by, firstly, the abstraction of the acidic hydrogen, located on the {CH}-vertex from the metallacarborane, by reaction with lithium base, followed by, secondly, reaction with electrophilic agents (PFA, oxirane and oxetane), which leads to a cascade of reactions to give the desired sulfamide derivatives. These compounds were then tested by collaborators in other institutes for in vitro and in vivo activity towards Carbonic Anhydrase IX (CA IX), which is an enzyme associated with tumour growth. In vivo tests on mice have shown that these types of substances are able to effectively reduce tumour size by 30%. The synthetic research continued with the preparation of sulfonamide compounds of the isomers of the carborane series. The reactions began exclusively with propylhydroxy carborane starting materials, which provide optimum...