The UDP-glucuronosyltransferases (UGTs) are a superfamily of enzymes that glucuronidate small, lipophilic molecules, thereby altering their biological activity and excretion. In humans, important examples of UGT substrates include molecules of both endogenous and xenobiotic origin; thus, UGTs are considered essential contributors to homeostatic regulation and an important defence mechanism against chemical insult. In keeping with both roles, UGTs are most strongly expressed in the liver, a predominant organ involved in detoxification. Rates of glucuronidation in humans are neither uniform among individuals, nor constant in an individual over time. Genetic determinants and non-endogenous signals are both known to influence the expression of UGTs, which in turn may affect the efficacy of certain pharmaceutical treatments or alter long-term risk of developing disease. Thus, this thesis focuses on the transcriptional regulation of UGT genes in humans, particularly on mechanisms that are likely to be relevant to their expression and variation in the liver. Two major approaches were used: firstly, extensive studies of several UGT promoters were performed to identify and characterise transcriptional elements that are important for UGT expression; and secondly, important hepatic transcription factors were investigated as potential regulators of UGT genes. UGT1A3, UGT1A4 and UGT1A5 are a subset of highly related, but independently regulated, genes of the human UGT1 subfamily. UGT1A3 and UGT1A4 are expressed in the liver, whereas UGT1A5 is not. The presented analysis of the UGT1A3, UGT1A4 and UGT1A5 proximal promoters demonstrates that a hepatocyte nuclear factor (HNF)1-binding site common to all three promoters is important for UGT1A3 and UGT1A4 promoter activity in vitro, but is insufficient to drive UGT1A5 expression. Two additional elements required for the maximal activity of the UGT1A3 promoter were also identified that may distinguish this gene from UGT1A4. UGT1A3 was investigated further, focusing on mechanisms that may contribute to interindividual variation in UGT1A3 expression. Polymorphisms in the UGT1A3 proximal promoter were identified and their functional consequences tested. Known variants of HNF1alpha were also tested for altered activity towards the UGT1A3 gene. UGT1A9 is the only hepatic member of the UGT1A7-1A10 subgroup of UGT1 enzymes. Previous work had identified HNF1-binding sites in all four genes, and HNF4alpha as an UGT1A9-specific regulator. The work presented herein extends these findings to show that HNF1 factors and HNF4alpha synergistically regulate UGT1A9, and that HNF4alpha is not the only transcription factor responsible for the unique presence of UGT1A9 in the liver. Liver-enriched transcription factors screened as potential UGT regulators were chosen from the HNF1, HNF4, HNF6, FoxA and C/EBP protein families. Functional interactions newly identified by this work were HNF4alpha with UGT1A1 and UGT1A6, HNF6 with UGT1A4 and UGT2B11, FoxA1 and FoxA3 with UGT2B11, UGT2B15 and UGT2B28 and C/EBPalpha with UGT2B17. Observations were also made regarding different patterns of interaction between each UGT and the transcription factors tested, particularly HNF1alpha.

Based on reactions with two flavanones, three flavonols, and five flavones the regioselectivities of twelve human UDP-glucuronosyltransferase (UGT) isozymes were elucidated. The various flavonoid glucuronides were differentiated based on LC-MS/MS fragmentation patterns of [Co(II)(flavonoid – H)(4,7-diphenyl-1,10-phenanthroline)2]+ complexes generated upon post-column complexation. Glucuronide distributions were evaluated to allow a systematic assessment of the regioselectivity of each isozyme. The various UGT enzymes, including eight UGT1A and four UGT2B, displayed a remarkable range of selectivities, both in terms of the positions of glucuronidation and relative reactivity with flavanones, flavonols and flavones. The UGT1A enzyme selectivities are affected by the presence of a hydroxyl group at the 3, 6, 4’, or 3’ positions as well as by the presence of a methoxy at the 3’ position. The UGT2B enzymes show poor to no reactivity with the flavonols or flavones. This result implies that the greater planarity of the flavonols and flavones compared to structure of flavanones inhibits interaction with the UGT2 enzymes. For baicalein and scutellarein, three of the UGT1A isozymes (1A8, 1A9, and 1A10) resulted in the formation of 6-O glucuronides, enabling the fragmentation rules for the metal complexation/MS/MS strategy to be expanded. / text

Flavonoids are polyphenolics compounds that constitute a major group of plant specialized metabolites, biosynthesized via the phenylpropanoid/polymalonate pathways. The resulting specialized metabolites can be due to decoration of flavonoid compounds with sugars, usually glucose, by the action of regiospecific UDP-glycosyltransferase (UGT) enzymes. In some cases, glycosylation can involve enzymatic attachment of other sugar moieties, such as glucuronic acid, galactose, rhamnose or arabinose. These modifications facilitate or impact the bioactivity, stability, solubility, bioavailability and taste of the resulting flavonoid metabolites. The present work shows the limitations of utilizing mammalian UDP-glucuronosyltransferases (UGATs) for flavonoid glucuronidation, and then proceeds to investigate plant UG(A)T candidates from the model legume Medicago truncatula for glucuronidating brain-targeted flavonoid metabolites that have shown potential in neurological protection. We identified and characterized several UG(A)T candidates from M. truncatula which efficiently glycosylate various flavonoids compounds with different/multiple regiospecificities. Biochemical characterization identified one enzyme, UGT84F9, that efficiently glucuronidates a range of flavonoid compounds in vitro. In addition, examination of the ugt84f9 gene knock-out mutation in M. truncatula indicates that UGT84F9 is the major UG(A)T enzyme that is necessary and sufficient for attaching glucuronic acid to flavonoid aglycones, particularly flavones, in this species. Finally, the identified UG(A)T candidates were analyzed via homology modeling and site-directed mutagenesis towards increasing the repertoire of UG(A)Ts applicable for synthesis of flavonoid glucuronides with potential human health benefits in neurological protection.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12834号 / 論農博第2798号 / 新制||農||1026(附属図書館) / 学位論文||H26||N4857(農学部図書室) / 31372 / (主査)教授 栗原 達夫, 教授 植田 和光, 教授 平竹 潤 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

This thesis focuses on the importance of the heme catabolic pathway in chronic hepatitis C (HCV). The aim is mainly to investigate, whether expresion/activity of key enzymes of the heme catabolic pathway, heme oxygenase (HMOX) and biliverdin reductase (BLVRA) in the liver and blood (study A) or promoter variations of HMOX1 and UDP- glucuronosyltransferase (UGT1A1) (study B) may be associated with the progression of fibrosis and may also predict antiviral treatment outcome in patients chronically infected with HCV. We set up a new sensitive method to quantify HMOX activity by reduction gas chromatography. We developed and extensively validated RealTime PCR assay for HMOX and BLVRA expression in the liver and peripheral blood leucocytes (PBL). The (GT)n and (TA)n dinucleotide variations in HMOX1 and UGT1A1 gene promoters, respectively, were determined by fragment analysis. No association was detected between either expression of HMOX/BLVRA or the HMOX1/ UGT1A1 promoter variants and the individual histological stages of liver disease in the HCV positive patients. A marked difference in BLVRA expression in PBL between the sustained responders (SVR) and patients with treatment failure (NVR) was detected before antiviral treatment and during the follow-up. Our data suggests, that BLVRA basal expression...