Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: Advances and open questions

ABSTRACT:UDP-glucuronosyltransferases (UGTs) are major mediators in conjugative metabolism. Current data suggest that UGTs, which are anchored in the endoplasmic reticulum membrane, can oligomerize with each other and/or with other metabolic enzymes, a process that may influence their enzymatic activities. We demonstrated previously that the UGT1A locus encodes previously unknown isoforms (denoted "i2"), by alternative usage of the terminal exon 5. Although i2 proteins lack transferase activity, we showed that knockdown of endogenous i2 levels enhanced cellular UGT1A-i1 activity. In this study, we explored the potential of multiple active UGT1A_i1 proteins (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10) to interact with all spliced i2s by coimmunoprecipitation. We further studied the functional consequences of coexpressing various combinations of spliced i1s and i2s from highly similar UGTs, namely UGT1A7, UGT1A8, and UGT1A9, based on expression profiles observed in human tissues. The i1 isoform of each UGT1A coimmunoprecipitated its respective i2 homolog as well as all other i2s, indicating that they can form heteromeric complexes. Functional data further support the fact that i2 splice species alter glucuronidation activity of i1s independently of the identity of the i2, although the degree of inhibition varied, suggesting that this phenomenon may occur in tissues expressing such combinations of splice forms. These results provide biochemical evidence to support the inhibitory effect of i2s on multiple active UGT1As, probably through formation of inactive heteromeric assemblies of i1s and inactive i2s. The relative abundance of active/inactive oligomeric complexes may thus determine transferase activity.

Section: Introductionmentioning

confidence: 82%

Alternatively Spliced Products of theUGT1AGene Interact with the Enzymatically Active Proteins to Inhibit Glucuronosyltransferase Activity In Vitro

Bellemare

Rouleau²,

Girard³

et al. 2010

“…There is evidence that UGTs are functional as homodimers and heterodimers in monoglucuronide formation and they may form tetramers in diglucuronide formation (Bock and Köhle, 2009). As published by Gordon et al (1983), microsomal diglucuronide-forming activity differs characteristically from the monoglucuronide-forming activity, and there is evidence that microsomes are intensely sensitive to manipulation of its membrane environment.…”

Section: Trdan Luš In Et Almentioning

confidence: 96%

Raloxifene Glucuronidation in Human Intestine, Kidney, and Liver Microsomes and in Human Liver Microsomes Genotyped for theUGT1A1*28Polymorphism

Lušin

Trontelj²,

Mrhar³

2011

ABSTRACT:Raloxifene, a selective estrogen receptor modulator, exhibits quite large interindividual variability in pharmacokinetics and pharmacodynamics. In women, raloxifene is metabolized extensively by different isoforms of UDP-glucuronosyltransferase (UGT) to its glucuronides. To gain an insight into intestine, kidney, liver, and lung glucuronidation of raloxifene, human microsomes of all tested organs were used. Raloxifene-6-␤-glucuronide (M1) formation followed the Michaelis-Menten kinetics in intestinal, kidney, and liver microsomes; meanwhile, raloxifene-4-␤-glucuronide (M2) formation followed the substrate inhibition kinetics. Human lung microsomes did not show any glucuronidation activity. The tissue intrinsic clearances for kidney, intestine, and liver were 3.4, 28.1, and 39.6 ml ⅐ min ؊1 ⅐ kg ؊1, respectively. The aim of our in vitro study was to explain the mechanism behind the observed influence of UGT1A1*28 polymorphism on raloxifene pharmacokinetics in a small-sized in vivo study (Br J Clin Pharmacol 67:437-444, 2009). Incubation of raloxifene with human liver microsomes genotyped for UGT1A1*28 showed a significantly reduced metabolic clearance toward M1 in microsomes from donors with *28 allele. On the contrary, no significant genotype influence was observed on the formation of M2 because of the high variability in estimated apparent kinetic parameters, although a clear trend toward lower glucuronidation activities was observed when UGT1A1*28 polymorphism was present. The liver intrinsic clearances of both homozygotes differed significantly, whereas the clearance of heterozygotes did not differ from the wild-type and the mutated homozygotes. In conclusion, our results show the high importance of the liver and intestine in raloxifene glucuronidation. Moreover, the significant influence of UGT1A1*28 polymorphism on metabolism of raloxifene was confirmed.

“…Many UGTs are expressed in the liver, but other drug entry points such as epithelial surfaces of the nasal mucosa, gut, skin, brain, prostate, uterus, breast, placenta, and kidney also host UGT activity (Tukey and Strassburg, 2000). UGT enzymes are integrally associated with the membranes of the endoplasmic reticulum, possibly with most of the protein oriented toward the luminal side, and may form homo-and hetero-oligomeric structures (Bock and Köhle, 2009).…”

mentioning

confidence: 99%

Glucuronide Production by Whole-Cell Biotransformation Using Genetically Engineered Fission YeastSchizosaccharomyces pombe

Drăgan¹,

Buchheit²,

Bischoff³

et al. 2009

ABSTRACT:Drug metabolites generated by UDP glycosyltransferases (UGTs) are needed for drug development and toxicity studies, especially in the context of safety testing of metabolites during drug development. Because chemical metabolite synthesis can be arduous, various biological approaches have been developed; however, no whole-cell biotransformation with recombinant microbes that express human UGTs was yet achieved. In this study we expressed human UDP glucose-6-dehydrogenase together with several human or rat UGT isoforms in the fission yeast Schizosaccharomyces pombe and generated strains that catalyze the whole-cell glucuronidation of standard substrates. Moreover, we established two methods to obtain stable isotope-labeled glucuronide metabolites: the first uses a labeled aglycon, whereas the second uses 13 C 6 -glucose as a metabolic precursor of isotope-labeled UDP-glucuronic acid and yields a 6-fold labeled glucuronide. The system described here should lead to a significant facilitation in the production of both labeled and unlabeled drug glucuronides for industry and academia.