Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Barbora Umanová Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Expression and function of placental drug transporters in health and disease There are many physiological changes during pregnancy. Placenta is a crucial organ which mediates exchange of nutrients, metabolites and respiratory gases, provides endocrine functions and fetal protection. A pregnant woman and her fetus may be exposed to various potentially harmful substances during pregnancy, including drugs that may endanger fetal health. Protection of the fetus from xenobiotics is enabled by drug transporters. Drug transporters are membrane proteins expressed in most tissues of the human body. In the placenta, they are localized in the placental syncytiotrophoblast and occur also in the endothelial cells of the fetal vessels. They belong into two large superfamilies of transporter proteins: ATB-binding cassette (ABC) and solute carrier (SLC). While ABC transporters mediate exclusively efflux of their substrates, SLC are predominantly influx transporters. Therefore, these transport proteins play a key role in the disposition of drugs, some of which facilitate drugs entry into a fetus, and others actively...

Organic cation (OC) secretion across renal proximal tubules (RPTs) involves basolateral OCT2- mediated uptake from the blood, followed by apical MATE1/2-mediated efflux into the tubule filtrate. Whereas OCT2 supports electrogenic OC uniport, MATE is an OC/H exchanger. OCs make up ~40% of all prescribed drugs and renal secretion plays a major role in clearing them. This study looked at two aims with the intent of resolving two outstanding issues dealing with the mechanism of MATE-mediated OC transport. First: Understanding the nature of intracellular sequestration of OC in cells that express hMATE1 as an integral part of characterizing 'the potential difference of substrate selectivity between the intracellular and extracellular face of MATE1.' Second: Testing whether structurally distinct MATE substrates can display different quantitative profiles of inhibition when interacting with structurally distinct ligands to determine 'the potential influence of the substrate on the profile of ligand interaction with MATE1.' All uptake experiments were realized with CHO cells that stably expressed hMATE1, hMATE2K or hOCT2. By epifluorescence microscopy cultured CHO-hMATE1 cells accumulated the fluorescent OC, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4- yl)amino]ethanaminium (NBD-MTMA) in the cytoplasm and in a smaller, punctate compartment; accumulation in hOCT2 expressing cells was restricted to the cytoplasm. A second intracellular compartment was also evident in the multicompartmental kinetics of efflux of the prototypic OC, 1-methyl-4-phenylpyridinium, [³H]MPP, from MATE1-expressing CHO cells. Punctate accumulation (20 min) of NBD-MTMA was markedly reduced by coexposure of MATE1-expressing cells with 5 μM bafilomycin (BAF), an inhibitor of the V-Type H-ATPase, and 20 min accumulations of [³H]MPP and [³H]NBD-MTMA were reduced by >30% by coexposure with 5 μM BAF. BAF had no effect on the initial rate of MATE1-mediated uptake of NBD-MTMA (10-300 sec) suggesting that the effect of BAF was a secondary effect involving inhibition of the V-type H-ATPase. The 15 min accumulation of [³H]MPP by isolated single non-perfused rabbit RPTs was also reduced >30% by coexposure to 5 μM BAF. Thus, the native expression in RPTs of MATE protein within endosomes can increase steady-state OC accumulation. However, the rate of [³H]MPP secretion by isolated single perfused rabbit RPTs was not affected by 5 μM BAF suggesting that vesicles loaded with OCs are not likely to recycle into the apical plasma membrane at sufficient rates to provide a parallel pathway for OC secretion. The uptake of three structurally distinct MATE substrates: MPP, triethylmethylammonium (TEMA) and NBD-MTMA into CHO-hMATE1 and CHO-hMATE2K cells was inhibited by three structurally similar cationic ionic liquids (ILs, salts in the liquid state: N-butylpyridinium, NBuPy; 1-methyl-3-butylimidazolium, Bmim; and N-butyl-N-methylpyrrolidinium, BmPy). The three ILs displayed a higher affinity for the pyridinium-based NBuPy (IC50 values, 2-4 μM) than for either the pyrrolidinium- (BmPy; 20-70 μM) or imidazolium-based ILs (Bmim; 15-60 μM). Inhibition of MPP, TEMA, and NBD-MTMA transport by NBuPy was competitive, with comparable Ki values against all substrates. Bmim also competitively blocked the three substrates but with Ki values that differed significantly (20 μM against MPP and 30 μM against NBD-MTMA versus 60 μM against TEMA). By trans-stimulation, all three ILs were transported by both MATE transporters. Together, these data indicate that renal secretion of ILs by the human kidney involves MATE transporters and suggest that the mechanism of transport inhibition is ligand-dependent, supporting the hypothesis that the binding of substrates to MATE transporters involves interaction with a binding surface with multiple binding sites. In order to further verify this hypothesis the uptake of four structurally distinct MATE substrates: MPP, NBD-MTMA, Cimetidine and Metformin into CHO-hMATE1 cells was characterized. Inhibition by ~400 drugs from the NIH clinical collection (NCC) was determined, and the rank order and level of inhibition seen were comparable against all substrates. IC₅₀ were measured for ~20 drugs selected from the NCC using principal component analysis (PCA); their IC₅₀ values were very similar against all four substrates, showing no systematic influence of substrate structure on inhibitory profile. The development and comparison of pharmacophores for each individual substrate revealed no substantial difference among them as proved by cluster analysis, leading to the conclusion that contrary to what was predicted based on the preliminary IL data, the substrates tested appear to have no influence on the inhibitory profile of ligands with hMATE1.

The majority of the mitochondrial proteins is synthetized on the cytosolic ribosomes in the form of the protein precursors bearing mitochondrion-targeting signal presequences. Once the protein precursor has reached the mitochondrial matrix the signal presequence is no longer necessary and is cleaved off by heterodimeric mitochondrial processing peptidase (MPP; α/β). Although the crystal structure of MPP is available, the MPP mechanism of function is still matter of discussion. An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the regulatory α-subunit of the yeast MPP. Wild-type and GRL-deleted MPP structures were studied both in the presence and absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the peptidase active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at...

Fatty acid (FA) transport proteins are important regulators of FA uptake at the cell surface and the mitochondria where they are oxidized. Tight regulation of this process is necessary in order to meet metabolic requirements, while preventing excess lipid accumulation. In an obese state, there is an increase in FA uptake and increased storage of lipids in skeletal muscle, including diacylglycerol (DAG) and ceramides, which interfere with insulin-stimulated glucose uptake. Leptin administration has been shown to reduce muscle triacylglycerol accumulation and restore insulin response in obese rodents. However, it is not known whether this is mediated through a redistribution of the FA transport proteins to the cell surface and mitochondria. In addition to hyperglycemia, post-prandial lipidemia is also observed in the obese state, suggesting a resistance to insulin-stimulated FA uptake. The possibility that insulin-stimulated FA transporter translocation is impaired has received little attention. Lastly, while recent studies have demonstrated that the transverse (t)-tubules may be an important site for glucose uptake in muscle, this has not yet been examined with regards to the FA transporters. In the first study of this thesis, the recovery of insulin response with short-term (2 week) chronic leptin administration in high-fat fed rats was associated with a decrease in muscle reactive lipid species (DAG, ceramide) and an increase in markers of oxidative capacity. Contrary to our expectations, this was not mirrored by an alteration in the distribution of FA transport proteins (FAT/CD36 or FABPpm) at the sarcolemma or the two major mitochondrial populations. To gain further insight into FA transporters and their localization at the cell surface, the second study of this thesis analyzed both the sarcolemma and t-tubules (constitute 40 and 60% of the cell surface, respectively). The novel observation was made that the t-tubules contain FA transport proteins (FAT/CD36, FABPpm, FATP1 and FATP4), and that the distribution and response of these transporters to acute metabolic stimuli (insulin and muscle contraction) was unique from that of the sarcolemma. The third study of this thesis characterized the translocation of FA transport proteins in response to insulin in the obese, insulin resistant Zucker rat. FA transport proteins were chronically increased on both membrane fractions in muscle from the obese rats. Furthermore, a blunting of the insulin-induced translocation of FA transporters to both cell surface domains was observed, demonstrating that insulin resistance extends to the movement of FA as well as glucose transport proteins. The t-tubules appear to play an important role regarding substrate uptake. Together the data from this thesis suggests that a chronic elevation in FA transporters at both cell surface domains contributes to lipid accumulation in obese skeletal muscle, and that reduced sensitivity of both FA and glucose transport proteins to translocate in response to insulin may explain the lipidemia and hyperglycemia that often characterizes post-prandial situations in the obese condition. As the prevalence of obesity reaches epidemic proportions, research into the functional role of FA transport proteins in the progression of obesity related pathologies is warranted as we work to further our knowledge of this significant health issue. / Natural Sciences and Engineering Research Council, Canadian Institute of Health Research

[Truncated abstract] Nitrogen fixation is a feature of the symbiotic association between legumes and rhizobia, which occurs within the symbiosomes of root nodules and involves the conversion of atmospheric N2 to ammonia to be used by the plant in exchange for carbon compounds. Exchange of other nutrients is controlled by plant-synthesised proteins on the symbiosome membrane. Iron is a component of symbiotically important proteins, so is essential for nitrogen fixation. Low soil iron leads to decreased plant yields, whilst in other environments plants may accumulate iron to toxic levels. Knowledge of iron acquisition, transport and storage mechanisms is important to elucidate the role of iron transporters in the maintenance of iron homeostasis in the plant. This study provides evidence that iron has a profound effect in the Bradyrhizobium japonicum-Glycine max symbiosis on the development of the nodule, and on the development of the symbiotic soybean plant itself. cDNAs encoding four putative iron transporters in soybean; GmDmt1, GmYSL1, GmCCC1;1 and GmCCC1;2, were identified, isolated and characterised in this study. GmDmt1 is localised to the symbiosome membrane. Expression of GmDmt1 occurs in nodules, roots and leaves and increases in response to iron starvation. GmDmt1 rescues growth and enhances 55Fe(II) uptake in the iron transport deficient yeast strain fet3fet4, with uptake following Michaelis-Menten kinetics, resembling the situation in isolated symbiosomes. Competition experiments using fet3fet4 indicated that GmDmt1 is able to transport other divalent cations, including zinc, copper and manganese, and is also able to complement a zinc transport deficient yeast mutant. ... These results suggest the divalent metal transporter GmDmt1, the putative iron chelate transporter GmYSL1 and the putative vacuolar iron transporters GmCCC1;1 and GmCCC1;2 act together to maintain iron homeostasis in symbiotic soybeans. The possible interactions and regulation of these proteins and their roles in the acquisition, transport and utilisation of iron in symbiotic soybeans are discussed.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22117号 / 医博第4530号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 松原 和夫, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Orientador: Marcelo Menossi / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T00:11:19Z (GMT). No. of bitstreams: 1 Cancado_GeraldoMageladeAlmeida_D.pdf: 5945955 bytes, checksum: bf1d61ab84a403512834994e49e96bf1 (MD5) Previous issue date: 2006 / Resumo: Solos ácidos são encontrados em todas as regiões do planeta e em grandes proporções nas regiões tropicais e subtropicais. Como conseqüência da acidez do solo, o alumínio (Al), metal mais abundante da crosta terrestre, torna-se solúvel e atinge concentrações tóxicas para a maioria das espécies de plantas cultivadas. O primeiro dano provocado pelo Al iônico é a redução do desenvolvimento radicular, causando distúrbios fisiológicos que acarretam na redução da produção. Devido às limitações dos métodos convencionais de correção do pH do solo e a necessidade de longos períodos de tempo para o desenvolvimento de novas cultivares pelo melhoramento genético clássico, muita ênfase tem sido dada para a compreensão dos mecanismos de toxidez e de tolerância ao Al em plantas. Pois, com a aquisição destes conhecimentos, espera-se mais sucesso na obtenção de plantas tolerantes ao Al com o emprego da tecnologia de DNA recombinante e da seleção assistida por marcadores moleculares. Neste trabalho utilizamos duas linhagens de milho contrastantes para tolerância ao Al: Cat100-6 (tolerante ao Al) e S1587-17 (sensível ao Al), com o objetivo de estudar as alterações na expressão gênica promovidas pelo estresse de Al no ápice radicular. A presente tese esta dividida em três linhas de pesquisa: i) identificação, clonagem e caracterização de um gene codificando uma enzima glutationa S-transferase e avaliação dos efeitos do Al na sua expressão gênica; ii) avaliação das alterações na expressão gênica em ápices de raízes de duas linhagens de milho quando submetidos ao estresse de Al, utilizando um sistema de hibridização heterólogo com ESTs (expressed sequence tags) de cana-de-açúcar; e iii) clonagem e caracterização em milho do gene ALMT1, pertencente a uma nova classe de proteína transportadora de moléculas orgânicas especificamente ativada pelo Al. No trabalho com o gene GST27.2 observamos que este gene foi induzido pelo estresse provocado por Al e por Cd (cadmio) e que mutações que provocavam alterações na composição de aminoácidos da proteína poderiam promover alterações vi na atividade e na especificidade desta enzima. Além disso, o gene GST27.2 parece ser um novo alelo do gene GST27 estando presente como cópia única no genoma das linhagens de milho estudadas. Já no trabalho de avaliação da expressão gênica em larga escala, foram identificados 85 genes nos ápices radiculares das duas linhagens de milho cuja expressão foi diferencialmente alterada pela presença do Al. Embora alguns dos genes já tivessem sido descritos como responsivos ao Al, para a maior parte dos genes identificados neste trabalho, este foi o primeiro relato descrevendo seu envolvimento com o estresse de Al. A clonagem em milho do gene homológo ao gene ALMT1, demonstrou que o milho também deve ter uma proteína de membrana presente em células do ápice radicular que pode estar envolvida com transporte de moléculas orgânicas. Embora a proteína não esteja envolvida com o transporte de malato, a mesma teve sua atividade melhorada pela presença do Al. Entretanto, o gene que codifica esta proteína é reprimido no tecido do ápice radicular das linhagens de milho tolerante e sensível ao Al, o que pode indicar a existência de regulação pós-transcricional. Os resultados obtidos a partir destas três abordagens contribuíram para a compreensão dos mecanismos de tolerância e toxidez ao Al em raízes de milho. Futuramente, essas informações auxiliarão na escolha de genes mais apropriados para a criação de plantas geneticamente alteradas mais adaptadas a presença do Al no solo / Abstract: Acid soils are found worldwide but most of them are located in tropical and subtropical regions. Aluminum (Al), the most abundant metal on the earth surface, becomes soluble in the soil solution as consequence of low pH in acid soils and achieves phytotoxic levels for most of the cultivated plant species. The first symptom of Al toxicity is the inhibition of the root growth that promote physiological disturbs reducing crop yield. Because of limitations of correcting soil pH by liming and the time-consuming process of traditional plant breeding, the elucidation of the mechanisms involved with plant Al-tolerance and Al-toxicity has received more attention, since the production of genetically altered plants has emerged as an effective and fast strategy to the production of improved cultivars. Two maize lines, Cat100-6 (Al-tolerant) and S1587-17 (Al-sensitive), were used in this study with the aim of understanding at the transcriptional level the alterations promoted by Al on the roots. The research was divided in three main sections: i) detection, cloning and characterization of a gene encoding a Glutathione S-transferase in maize and evaluation of Al effects on its expression; ii) Large-scale evaluation of gene expression in root tips of maize under Al stress using a heterologous system with Expressed Sequence Tags (ESTs) of sugarcane; and iii) cloning and characterization of the ALMT1 gene in maize and evaluation of Al-effects on its activity. In the first section was observed that Al and Cd-stress induced the GST27.2 gene. Two mutations present on the nucleotide chain of this gene promoted alteration on the amino acid compositions. These alterations might be responsible by alterations on the specificity and activity of the GST enzyme. Besides that, the GST27.2 is a single copy gene in maize and seem to be a new allele of GST27. In the section of large-scale gene expression evaluation were identified 85 genes in root tips of two Al-tolerant contrasting maize lines whose expression was altered by Al stress. Although several of the genes identified here were previously described as Al responsive in other works, to most of them this study is the first report about the involvement of these genes with Al stress. The cloning of the ALMT1 homologue in tissue from the root apex of maize shown that maize has a gene encoding a membrane protein that might be involved with organic molecules transport. Although the protein encoded by the maize homologue gene was not associated with malate transport the activity of this protein was stimulated by the presence of Al. Interestingly, the gene expression of the this gene was repressed by Al in the Al-tolerant and Al-sensitive genotype. This result might be an indicative of existence of posttranscriptional regulation. The results accomplished with the experiments described here launched new light into the understanding of the Al-tolerance and Al-toxicity mechanisms in maize roots. Furthermore, the information presented here will contribute to a more accurate selection of genes that will be used to produce transgenic plants better adapted to soils with high Al concentration / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

The expression of polysaccharide capsules is common in bacteria and associated with virulence in some pathogenic strains. Strains of the Gram-negative bacterium Escherichia coli express a structurally diverse range of capsular polysaccharides. E. coli strains expressing group 2 capsules are associated with a number of extra-intestinal infections, including sepsis, urinary tract infections, and neonatal meningitis. Group 2 capsular polysaccharides are synthesised on the cytoplasmic face of the inner membrane. Evidence from previous work suggests that export of polysaccharides across the Gram-negative membranes involves four transport proteins which interact to form a continuous membrane-spanning translocation complex (the KpsMTED translocon). Polysaccharide translocation across the inner membrane requires the ABC transporter KpsMT, in which KpsM is the integral inner membrane component and KpsT is the ATPase. Transport across the periplasmic space and outer membrane involves the integral inner membrane protein KpsE and the outer membrane protein KpsD, respectively. This thesis addressed some of the key areas in the study of group 2 polysaccharide transport by employing the K5 capsule as a model system. Using biochemical and molecular genetics approaches, the study focused on establishing functional and structural characteristics of the translocon members and analysing protein-protein interactions within the complex. This study demonstrated that KpsE can self-associate as dimers, tetramers and possibly higher order oligomers in the absence of other capsule gene products and the K5 substrate. A mutagenesis study of KpsE revealed that the periplasmic, membrane-associated C-terminus is essential for correct protein function. Work presented here confirmed previous data, which suggested a direct interaction between KpsE and KpsM, by alternative methods, and demonstrated that the C-terminal domain of KpsE is required for this interaction. Further experiments suggested that KpsE and KpsM can both form higher order oligomers when interacting as a complex. The C-terminus of KpsE is not required for an interaction between KpsE and KpsD, and the two proteins are thus more likely to interact via their respective periplasmic domains. Generation of a theoretical model of the secondary structure and topology of KpsD predicted that KpsD is made primarily of β-sheets with some interspersed α-helices, including a larger coiled coil region. The theoretical topology model proposed an N-terminal transmembrane domain made of eight membrane-spanning regions, and a large periplasmic domain. Substituted-cysteine accessibility method and myc-epitope insertion analysis were both assessed for their suitability for topology analysis of KpsD. Myc-epitope insertion was identified as the recommended approach for future topology study. Myc-epitope tagging of the periplasmic C-terminus of KpsD revealed that a native C-terminus is essential for correct KpsD function.In conclusion, this thesis contributes to the model of group 2 polysaccharide export in E. coli, and, more generally, provides clues about the transport of high-molecular weight molecules across Gram-negative membranes. It is hoped that a thorough understanding of polysaccharide transport might reveal therapeutic targets to block capsule export in pathogenic E. coli in the future.

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